GB2483253A

GB2483253A - Cytotoxic luminescent mono-nuclear metal complexes

Info

Publication number: GB2483253A
Application number: GB1014500.1A
Authority: GB
Inventors: Jim Thomas; Carl Smythe
Original assignee: University of Sheffield
Current assignee: University of Sheffield
Priority date: 2010-09-01
Filing date: 2010-09-01
Publication date: 2012-03-07
Also published as: GB201014500D0; WO2012028874A1

Abstract

Novel antineoplastic agents comprising mono-nuclear transition metal complexes. The said complexes contain a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2â ,3--j] phenazine (tpphz) ligand and have dual functioning imaging/therapeutic properties. Also described is their use in photodynamic therapy and as chemotherapeutic agents. The cytotoxicity of these complexes is comparable to that of platinum-based chemotherapeutics, especially for platinum-resistant tumour cells. Preferred transition metals include Ru (II), Os (II) or Ir (III), preferably Ru (II).

Description

I

CYTOTOXIC LUMINESCENT METAL COMPLEXES

This invention relates to mono-nuclear complexes containing a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"-j] phenazine, tpphz, ligand for use as antineoplastic agents. The invention includes inter a/ia such complexes having dual functioning imaging/therapeutic properties, their use in photodynamic therapy and as chemotherapeutic agents having cytotoxicity comparable to the platinum-based chemotherapeutics and especially for platinum-resistant tumour cells.

BACKGROUND

Cisplatin, cisplatinum, or cis-diamminedichloroplatinum(ll) (CDDP) is a platinum-based chemotherapy drug used to treat various types of cancers and was the first member of a class of anti-cancer drugs which now also includes carboplatin and oxaliplatin. Cisplatin remains the predominant treatment for breast and ovarian tumours and like other members of its class achieves its toxicity by targeting nuclear DNA by irreversibly creating intra-strand DNA cross links, the complex provokes a cellular DNA damage response, triggering apoptosis leading to programmed cell death or cell cycle arrest. However, a major limitation of platinum-based chemotherapy arises from drug resistance, which may occur via a number of mechanisms for example a reduction in drug uptake, an increase in platinum-DNA adduct repair and/or increased drug inactivation. Thus, there remains a significant need for therapeutic strategies that address the emergence of platinum-resistance in tumours.

Osmium and Ruthenium (II) and (Ill) complexes have also been investigated as potential anti-cancer therapeutics. Initial work centered on Ru(lll)-based coordination complexes and two such systems, NAMI-A (H21m [trans-RuCI4 (DMSO)(Him)], where Him = imidazole)} and KPIOI9 (H2lnd[trans-RuCI4(Hlnd)2]; where Hind = indazole), have reached clinical trial stages. Subsequent studies on organometallic Ru'Lcomplexes have led to a number of promising leads. In particular, "piano-stool" ruthenium(ll) arene complexes with the general formula [(ii6-arene)Ru(en)(X)] (en = ethylenediamine; X = a leaving group such as CL) are cytotoxic towards cancer cells, even when the cells are cisplatin resistant (Dougan et al; Proc. NatI. Acad. Sci. USA, 2008, 105, 11628-11633).

It is known from the prior art that that certain ruthenium complexes, for example [Ru(bpy)2(dppz)]2 (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine), function as a "light switch" upon reversibly binding to DNA, displaying quenched luminescence in aqueous media but emission from an MLCT (metal to ligand charge transfer) excited state as a result of intercalation. However, many of the Ru(ll) complexes studied to date, have shown that they are unable to penetrate across cell membranes into the cells themselves unless either the complexes are modified with hydrophobic residues or unless the cell itself is treated to make the cell plasma membranes more permeable which in turn has detrimental effects on the cell. Accordingly, not all Ru (II) or Ru (Ill) complexes are able to penetrate into the cell making their utility as cellular imaging agents unpredictable.

There is therefore a need for dual functioning imaging/therapeutic agents especially ones that display cytotoxicity comparable to the platinum-based chemotherapeutics even for platinum-resistant tumor cells.

BRIEF SUMMARY OF THE DISCLOSURE

According to a first aspect of the invention there is provided a mono-nuclear complex containing a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"-j] phenazine, tpphz, ligand for use as a medicament.

Preferably, the medicament is an antineoplastic agent.

In an embodiment, the mono-nuclear complex has the structure: (N N N N ::::1c:' XI t x N N' f' N' wherein: M is a transition metal;

CN

N and N each independently represent a bidentate chelating ligand; and n is2or3.

Preferably, the metal, M, of the mono-nuclear complex is selected from the group comprising Ru (II), Os (II) and Ir (Ill). More preferably the metal, M, is Ru (II).

In an embodiment, each bidentate chelating ligand independently has the structure: R2b *ia R R,wherein: each Rio, R2a, R3a, R1, R2' and R3D is independently selected from the group consisting of: hydrogen, -F, -Cl, -Br, -I, -OR, NRxR/, -ON, -NO2, -SO3R, -000R, 01-06 alkyl and substituted 01-06 alkyl; wherein R, Rx and R each independently represent hydrogen or a 01-04 alkyl group; and each R4 and R4 is independently selected from the group consisting of: hydrogen, -F, -01, -Br, -I, -OR, NRxRY, -ON, -NO2, -SO3R, -000R, 01-06 alkyl and substituted 01-06 alkyl, or each R4 and R4b of each bipyridyl together form OR5Et=OR5l), wherein each R5 and R5' is independently selected from the group consisting of: hydrogen, -F, -01, -Br, -I, -OR, NRXRY, -ON, -NO2, -SO3R, -000R, 01-06 alkyl and substituted 01-06 alkyl; and wherein each substituted Oi06 alkyl is substituted with up to 5 substituents, where chemically possible, independently selected from the group consisting of: -F, -Cl, -Br, -I, -OR, NRxR, -ON, -NO2, -SO3R and -000R, wherein R, Rxand R' each independently represent hydrogen or a 01-04 alkyl group.

In an embodiment, each Ria is 0i6 alkyl and optionally each Ria is 0i-03 alkyl. Preferably R' is methyl or ethyl, more preferably methyl.

In an embodiment, each Rib is 0i6 alkyl and optionally each Rib is 01-03 alkyl. Preferably Rib is methyl or ethyl, more preferably methyl.

In an embodiment, each R' and R are °i6 alkyl and optionally each R and R are 0- 03 alkyl. Preferably Ria and Rib are methyl or ethyl, more preferably methyl.

In an embodiment, R2a, R, R3, R3, R48 and R4' are each independently defined as per RTh and R" above.

In an embodiment, each R4 and R4D of each bipyridyl together form CR5E1CR5b. In an embodiment, each R5 is independently defined as per R and R above.

In an embodiment, the complex of the invention includes two identical bidentate chelating ligands. In an embodiment, the complex of the invention includes two different bidentate chelating ligands.

In an embodiment, each R, R2, R3, Rfl', R2b and R3' is H, each R4a and R4b of each bipyridyl together form CR5a=CR5b and each R5 and R51' is H. In this embodiment, the compound is referred to as [(phen)2Ru(tpphz)]2.

In an embodiment, each R, R2, R3, R4, R, R2b, R3b and R4b is H. In this embodiment, the compound is referred to as [(bpy)2Ru(tpphz)]2.

The above-illustrated complexes may be shown in association with appropriate counterion(s). Appropriate counterion(s) include halide counterion(s), e.g. chloride counterion(s).

The complexes of the present invention may be in the form of a water soluble salt, such as a water soluble chloride salt. The complexes may be in the form of a solution comprising the complex and one or more counterions.

The complexes are suitably in the form of physiologically acceptable salts, or solutions of such salts. Physiologically acceptable salts include, but are not limited to, inorganic acid salts such as the chloride, bromide, sulphate and phosphate salts; organic acid salts such as trifluoroacetate and maleate salts; sulfonates such as methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphor sulfonate and naphthalenesulfonate; amino acid salts such as arginate, alaninate, asparginate and glutamate; and carbohydrate salts such as gluconate and galacturonate (see, for example, Berge, eta!. "Pharmaceutical Salts," J Pharm. Sd. 1977;66:1).

Where a structural formula herein comprising a chiral centre does not indicate chirality (e.g. where all bonds are shown as dotted lines and there is no "wedge" bond), then, unless the context otherwise requires, the structure refers to all corresponding compounds or moieties irrespective of chirality and includes reference to individual compounds or moieties in which the chiral centre is of (R)-configuration, individual compounds or moieties in which the chiral centre is of (S)-configuration and mixtures of (R)-and (S)-isomers as, for example, in the case of racemic mixtures, amongst others. The skilled person will understand that the complexes of the present invention are chiral complexes. Accordingly, the present invention includes both of the following isomers as well as all other compositions falling within the above formula, e.g. mixtures: 7 N and ?T Ii,, *s Disclosed herein are embodiments in which certain one or more pendent moieties (R groups) of the complexes are defined as a moiety other than H. In one class of these embodiments, all other R groups are H. Preferably, the complex has the structure: I I [1 I*l r

C

Reference herein to complexes 2 and 3 respectively refer to (tpphz = tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"-j]phenazine, 2 I = bpy; 3 I = 1,10-phenanthroline [phen]).

Preferably, the complex has a structure selected from the group comprising:

N

ofT?> N According to a second aspect of the invention there is provided a pharmaceutical composition comprising a complex as defined in the first aspect above and a pharmaceutically acceptable excipient, diluent or carrier.

By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.According to a third aspect of the invention there is provided a mono-nuclear complex containing a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"-j] phenazine, tpphz, ligand for use in treatment of a neoplastic disease.

Preferably, the neoplastic disease is benign, pre-malignant or malignant. The neoplastic disease may be a tumour or cancer of any organ or tissue or cell type.

According to a third aspect of the invention there is provided a mono-nuclear complex containing a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"-j] phenazine, tpphz, ligand that binds to or intercalates with nuclear DNA in living cells.

According to a fourth aspect of the invention there is provided a mono-nuclear complex containing a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2" ,3"-j] phenazine, tpphz, ligand that binds to or intercalates with nuclear DNA in living cells and displays an in cellulo light-switch effect.

The complexes of the third and fourth aspects of the invention have a cytotoxic effect.

Complexes of the third aspect of the invention are cytotoxic in light or dark conditions whereas complexes of the fourth aspect of the invention are substantially more toxic in light conditions as opposed to dark conditions.

According to a fifth aspect of the invention there is provided a mono-nuclear complex containing a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"-j] phenazine, tpphz, ligand for use in photodynamic therapy.

According to a sixth aspect of the invention there is provided a method of treating a neoplastic disease comprising administering a therapeutically effective amount of an intercalating mono-nuclear complex containing a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"'-j] phenazine, tpphz, ligand to a patient suffering from a neoplastic disease.

According to a seventh aspect of the invention there is provided a method of treating a neoplastic disease in an individual who displays resistance to platinum-based chemotherapeutics, the method comprising administering a therapeutically effective amount of an intercalating mono-nuclear complex containing a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"-j] phenazine, tpphz, ligand to a patient suffering from a neoplastic disease.

It will be appreciated that all of the features ascribed to the first aspect of the invention apply mutatis mutandis to each and every other aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 shows details of the hypochromicity observed in the absorption spectral spectrum of complex 3 [3]C12 in buffer (25 mM NaCI, 5 mM Iris, pH 7.0) on progressive addition of CT-DNA.

Figure 2 shows changes in the emission spectrum of complex 3 [3]C12 in aqueous buffer solutions (25 mM NaCI, 5 mM Tris, pH 7.0) on addition of CT-DNA. Inset: relative viscosity changes for aqueous buffered DNA solution on addition of [3]C12 confirming this effect is due to intercalation.

Figure 3a shows an image of MCF-7 cells incubated with complex 3, cell death is indicated by positive staining by P1 and shows the toxic effects of cellular uptake of the complex.

Figure 3b shows MCF-7 cells incubated with complex 3 display nuclear uptake and staining but at a reduced intensity in comparison to Figure 3a.

Figure 4a shows MCF-7 cells incubated with complex 2 with poor nuclear staining, Figure 4b shows cells display clear nuclear staining by complex 2 when a higher incubation concentration is used.

Figure 5 shows cellular internalization of complexes 2 and 3 confirming cellular DNA-binding, Figure 5a shows live MCF-7 cells incubated with complex 3, Figure Sb shows MCF-7 cells co-stained with complex 3 and DAPI; Figure 5 shows lambda stacking analysis of cell stained with complex 3 and Figure Sd shows staining of fixed cells with complex 2.

Figure 6a shows fixed and membrane-permeablized MCF-7 cells stained by complexes 2 and 3 and co-stained with P1, Figure 6b shows fixed MCF-7 cells solely stained with complex 3.

Figure 7 shows TEM micrographs of MCF-7 cells incubated with complex 3. Figure 7a shows an image showing even distribution of the stain throughout the cytosol; Figure 7b shows a detailed image of nucleus revealing clear heterochromatin staining (ringed); Figure 7c shows a detailed image revealing distinctive granular patterning of the distribution of complex 3 within the cytosol.

Figure 8 shows TEM micrographs of fixed MCF-7 cells stained with complex 3. Figure 8a shows the cytosol exhibiting strong intracellular contrast and the nucleoli of the cell are clearly observable. Figure 8b shows the localization within heterochromatin within the nucleus and Figure 8c shows a distinctive granular pattern due to complex 3, where the complex is possibly protein-bound.

Figure 9a shows TEM micrographs of MCF-7 cells fixed before incubation with complex 7: Figure 9b shows CLSM of fixed and permeablized MCF-7 cells revealing no Ru(dppz)-based MLCT emission from the nucleus (left), same sample stained with positive control propidium iodide (right).

Figure 10 shows mechanism of uptake studies of Ru(ll)tpphz complexes. Figure ba and lOb show MCF-7 cells incubated with complexes 2 or 3 and Figure lOc shows cells co-incubated with endocytosis inhibitor chloroquine.

Figure II shows cytotoxicity of complexes towards MCF-7 breast cancer cells. Figure II a shows cytotoxicity of Ru(ll)tpphz complexes and cisplatin towards MCF-7 cells and Figure lib shows cytotoxicity of Ru(ll)dppz complexes towards MCF-7 cells. Cell viability determined by MTT assay.

Figure l2a shows the cytotoxicity of cisplatin and complex 2, Figure 12b shows the cytotoxicity of cisplatin and complex 3 and Figure 12c shows the the cytotoxicity of cisplatin towards A2780 (black) and A2780-CP7O (white) ovarian cancer cell lines.

Figure 13 shows the cytotoxicity of mononuclear Ru(ll)tpphz complexes towards selected cancer cells

DETAILED DESCRIPTION

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

The term "C1-C6 alkyl" means alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms. The term "C1-C4 alkyl" means alkyl having 1, 2, 3 or 4 carbon atoms. Alkyl groups may be linear or branched, e.g. linear.

The term each independently" means that the moieties referred to may be the same or different. In embodiments, such moieties qualified by the term "each independently" are all the same.

The term "complex" means a molecular entity structure having one or more ligands and, in the case of the present invention at least three ligands, loosely associated with a metal coordination centre and may be used synonymously with the term "coordination entity".

The present invention provides ruthenium(ll) complexes which contain the tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"-j]phenazine, tpphz, ligand. The complexes of the present invention reveal that they are internalized by cell lines where they function as imaging contrast agents for both confocal laser scanning microscopy (CLSM) and transition electron microscopy (TEM). The present invention provides evidence for the successful cellular uptake of [Ru(L)2tpphz]2 intercalating systems into live cancer cells, specifically for complexes 2 and 3. These complexes are multifunctional in ce/lu/a imaging probes and also display cytotoxicity. Furthermore, binding studies show that the complexes bind to DNA via intercalation and display an in vitro DNA light-switch effect. A combination of CLSM and TEM studies confirm that complexes bind to nuclear DNA in living cells where they display an in cellulo light-switch effect. It was also found that IC50 values for the complexes towards MCF-7 breast cancer and A2780 ovarian cancer cells are similar in magnitude to those of cisplatin. Strikingly, this potent toxicity is retained even with cisplatin-resistant A2780-CP7O ovarian cancer cells. Cell viability studies reveal that the complexes display cytotoxicity comparable to cisplatin, which they retain even for cisplatin-resistant tumour cells, signifying the potential of these complexes advantageously as dual functioning imaging/therapeutic agents.

The complexes of the present invention are also useful in photodynamic therapy, it is postulated that the complexes of the present invention exert their photodynamic effect by a "two-pronged" cytotoxic attack. Firstly, as they generate singlet oxygen, which is known to be highly reactive and cause damage to biomolecules and cellular structures, and secondly by DNA intercalation and hence are multifunctional and able to be cytotoxic to, for example, cisplatin resistant cells. Complexes that are useful in photodynamic therapy are more cytotoxic in light conditions than in dark conditions as they may exert their cytotoxic effect by two different mechanisms. tlt is also envisaged that for photodynamic therapy applications that Os and lr(lll) complexes might prove suitable candidates for phototherapy activation as they can be excited in the 600nm light region.

Reference herein to neoplastic disease includes benign, pre-malignant and malignant tumours or cancers and can be in any organ, tissue or cell type, the complexes of the present invention are particularly suited to any of the conditions which are treatable by platinum-based chemotherapeutics.

It will be appreciated that platinum-based chemotherapeutics include cisplatin, carboplatin and oxaliplatin and that the terms are used interchangeably.

Materials Solvents were dried and purified using standard literature methods, while other commercially available materials were used as received. Complexes 2 -5 were prepared as described previously.2223 The buffer used for DNA titrations consisted of 25 mM NaCI and 5 mM tris (pH 7.0) made with doubly distilled water (Millipore). Calf thymus DNA (CT-DNA) was purchased from Sigma and was purified until A260/A280> 1.9. Concentrations of CT-DNA solutions were determined spectroscopically using the extinction coefficient of CT-DNA (c = 6600 dm3 moL1 cm1 at 260 nm).

Instrumentation.

Standard 1H NMR spectra were recorded on a Bruker AM250 machine. FAB mass spectra were obtained on a Kratos M580 machine working in positive ion mode, with m-nitrobenzyl alcohol matrix. UV-visible spectra were recorded on a Unicam UV2 spectrometer or Cary spectrometer in twin beam mode. Spectra were recorded in matched quartz cells and were baseline corrected. Steady-state luminescence emission spectra were recorded either in aerated acetonitrile or tris buffer solutions using a HORIBA Jobin Yvon FluoroMax 3 spectrometer.

DNA titration protocols.

Absorption titrations: complexes were converted to their chloride salts by reaction with [nBu4N]Cl in acetone. 100 pL of a 100 pM complex stock solution in tris buffer was then diluted into tris buffer (3 cm3) (in a 1 cm path length optical glass cuvette maintained at 25 °C) to give a final drug complex concentration of ca. 15 pM. Tris buffer (3 cm3) was loaded into another identical cuvette and placed in the reference cell of the spectrometer. Both the sample and the reference cells were mixed 30 times with a Gilson P1000 pipette. After 30 mm to allow the cells to equilibrate, the first spectrum was recorded between 700 and 200 nm. 2-5 pL of DNA solution was then added to both the sample and reference cell, followed by a further 30 times mixing. The spectrum was taken again, this time showing the hypochromic shift indicating the formation of a drug-DNA complex. The titration process was repeated until there was no change in the spectrum for at least four titrations indicating binding saturation had been achieved.

Luminescence titrations were carried using a procedure similar to the UV-Vis titrations.

Both buffer and complex solutions show insignificant emission, so no reference cell was used. 3 mL of buffer were loaded in a 1 cm path length luminescence cuvette; a volume of buffer was removed and replaced with the same volume of a stock solution of complex, to give a final concentration of 50 mM. The cuvette was loaded into the spectrophotometer and kept at 25 °C. After equilibration, the emission spectrum of the solution was recorded using the excitation wavelength characteristic of the complex. 2 mL of a concentrated stock CT-DNA solution were added to the cuvette and mixed, and then the emission spectrum was recorded, showing an enhancement in emission. This procedure was continued until the emission became constant.

Viscosity measurements were carried out in a Cannon-Manning semi-micro viscometer (size 50) immersed in a thermostat bath maintained at 27 °C. The CT-DNA was broken into an average of 150-200 base pair (bp) by sonication, after that it was dialysed (dialysis tubing with a MWCO of 3,500 Daltons) in 2 L of the Tris buffer for 24 h. The concentration of CT-DNA was kept constant at 0.5 mM (bp), and samples were prepared by adding ligand to the DNA solution to give an increase in the ligand/bp ratios. The flow times were measured after thermal equilibration of at least 20 minutes. Each sample was measured three times and the averaged time was used in calculations.

Cell culture.

MCF-7, A2780 and A2780-CP7O cells were cultured in RMPI 1640 medium supplemented with 2 mM L-glutamine, 100 IU mV1 penicillin, 100 mg m11 streptomycin and 10% fetal bovine serum at 37°C in a 5% CO2 atmosphere. Where stated, cells were fixed and permeablized using chilled 70% ethanol. For uptake studies, temperature-dependence studies used cells that had been cooled at 4°C for 30 minutes then incubated with 200 tM of complexes 2 and 3 (10% PBS: 90% serum-free media) at 4°C for 1 hour. Chloroquine-treated cells were treated with the inhibitor (50 jiM) for 30 minutes then with the inhibitor plus 200 jiM complex 3 for 1 hour.

Microscopy.

CLSM: Cell cultures were grown on microdishes (Thistle Scientific) and incubated with solutions of complexes 2 and 3 (200 jiM, 1 hour) in serum-free media. Costaining was performed using 10 jiM P1 for 10 minutes or 500 nM DAPI for 2 minutes before the cells were washed with PBS and imaged. Cultures were luminescently imaged on a Zeiss LSM 510 META inverted confocal laser scanning microscope using 40x and 63x oil-immersion lenses. Complexes 2 and 3 were excited with an Ar-ion laser at 458 nm and emission monitored using META detection at 600-640 nm (red) wavelengths. P1 was excited using 543 nm (He-Ne laser) and emission collected using a 565-615 bandfilter. DAPI was excited using a 405 nm diode laser and emission detected using a 420 long-pass bandfilter. TEM: MCF-7 cells were incubated with complex 3 (200 iiM, I hr) then fixed using 3% glutaraldehyde and dehydrated using ethanol. For staining of cells after fixation, complex 3 was added (100 iiM, I hr) after the fixation and dehydration steps. TEM samples were sectioned in Araldite resin by microtome and examined on a FF1 Tecnai instrument operating at 80 kV equipped with a Gatan I k CCD Camera.

Cytotoxicity.

Cell cultures were seeded in 24 or 48 well plates and allowed to grow for 24 hours. Cell cultures were then treated with solutions of complexes 2 and 3 for 24 hours (in triplicate) before incubation with 0.5 mg m11 MTT (MTT = (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) for 30-40 minutes. The formazan product was eluted using acidified isopropanol and the absorbance at 540 nm quantified by spectrophotometer. Cell viability was determined as a percentage of untreated control and IC50 values calculated using Sigmaplot 11.0 software.

EXAMPLE I

Both complexes were synthesized, isolated, and fully characterized as hexafluorophosphate salts using reported methods. They were converted into water soluble chloride salts by counter-ion metathesis using acetone solutions of tetrabuylammonium chloride. Although, the in vitro DNA binding properties of complex 2 have been previously studied (Tysoe et al, lnorg. Chem., 1999, 38, 5196-5197 and Liu et al J. Am. Chem. Soc. 2005, 127, 10796-10797) corresponding data on complex 3 have not been reported. Consequently, prior to performing any in cellulo studies it was necessary to confirm that complex 3 had similar binding studies to complex 2 accordingly the optical absorption and emission response to the addition of calf-thymus DNA (CT-DNA) was investigated. In titrations with CT-DNA, the absorption bands of complex 3 displayed changes that are characteristic of an interaction with DNA; most notably the MLCT (metal to ligand charge transfer) bands of the complex show large hypochromicity of up to 48%, Figure 1 shows what was typically seen for the interaction of the complexes with duplex DNA. Figure 1 shows details of the hypochromicity observed in the absorption spectral spectrum of complex 3 [3]C12 in buffer (25 mM NaCI, 5 mM Tris, pH 7.0) on progressive addition of CT-DNA.

The luminescence properties of complex 3 are also modulated through interaction with DNA. As expected, the Ru->tpphz 3MLCT-based emission of the complex is greatly suppressed in water (unlike Ru(dppz) systems, it is not completely quenched) but, upon addition of CT-DNA, large-scale enhancements in luminescence are observed (Figure 2), behaviour that is typical of a DNA light switch system. Figure 2 shows the changes in the emission spectrum of complex 3 [3]C12 in aqueous buffer solutions (25 mM NaCI, 5 mM Tris, pH 7.0) on addition of CT-DNA. The inset shows relative viscosity changes for aqueous buffered DNA solution on addition of [3]C12 confirming this effect is due to intercalation. Although titrations consistently produced saturation binding curves, numerous attempts to analyze the absorption data using the McGhee and von Hippel model for binding to an isotropic lattice were unsuccessful resulting in poor data fits. In contrast, emission titrations were successfully fitted (R2 »= 95%) to the model and resulted in binding parameters estimates for complex 3 ( Kb = 3.0 x iO M1; n = 1.6) that are comparable to those previously reported for complex 2. Although these figures are consistent with intercalation into the DNA duplex; to confirm this hypothesis viscosity studies were also carried out. Classical intercalation results in a lengthening of DNA, thus producing a concomitant increase in the relative specific viscosity of aqueous solutions of short rigid DNA sequences. As shown in Figure 2, the relative specific viscosity of previously sonicated CT-DNA solutions significantly increased on progressive addition of complex 3, confirming that this complex is indeed a metallointercalator.

EXAMPLE 2

Cellular uptake and in cellulo DNA binding studies were undertaken. Since both complexes 2 and 3 bind to duplex DNA with high affinity and display light switching effects their cellular uptake and resultant DNA binding can, potentially, be directly monitored. This effect has led to several recent studies into the cellular internalization of structurally related compounds. Indeed, in recently reported work on related din uclear complexes it has been demonstrated that, in contrast to [{Ru(bpy)2}2(tpphz)]4, complex 4, the non-intercalating dinuclear complex [{Ru(phen)2}2(tpphz)]4' complex 5, is successfully internalized by live cells and targets the nucleus, where it functions as a non-cytotoxic structure-sensitive probe of DNA (Gill, M. R et al Nat Chem 2009, 1, 662-667). v

321cNxkflXV cTtNçNçLf33 ILtNtN1JN9 To probe the potential of complexes 2 and 3 for such a role, MCF-7 human breast cancer cells were incubated with solutions of the complexes and the cellular uptake and in cellulo DNA binding was examined using CLSM. In each individual case, the Ru(ll) complex was excited at 458 nm and luminescence at 600-640 nm was observed, these wavelengths correspond to the expected MLCT excitation and resultant MLCT emission from the complexes. Figure 3 shows the cellular internalization of complexes 2 and 3 confirming cellular DNA-binding: (a) live MCF-7 cells incubated with complex 3. Lack of emission from dead cell stain P1 indicates cell viability; (b) MCF-7 cells co-stained with 3 and DAPI; (c) lambda stacking analysis of cell stained with 3; (d) staining of fixed cells with complex 2. As Figure 3a shows, incubation of MCF-7 cells with complex 3 clearly results in uptake and nuclear emission from viable cells (dead cell staining using propidium iodide (P1) is included to indicate cell viability). Co-staining with the commonly used DNA marker stain DAPI (Figure 3b) clearly suggests that it is DNA being imaged in the nucleus. In order to confirm that complex 3 targets cellular DNA in live cells a Lambda stack experiment, which collects the emission intensity across a range of wavelengths, was conducted. A representative experiment shows the emission profile of complex 3 in the nuclei of cells is relatively broad, with the maximum emission located at 620-630 nm (Figure 3c), in good agreement with in vitro DNA binding luminescence titration.

CLSM studies also reveal the toxicity of the complex, with cellular uptake and DNA staining by complex 3 being accompanied by an increase in cell death (Figure 4). Figure 4 shows a) image of MCF-7 cells incubated with complex 3 (200 pM, lhr) 20 minutes after removal of the complex. Cell death is indicated by positive staining by P1 and shows the toxic effects of cellular uptake of the complex. (b) MCF-7 cells incubated with complex 3 (100 pM, 3 hrs) display nuclear uptake and staining but at a reduced intensity in comparison to incubation of 200 pM for 1 hr. In contrast to complex 3, while cells incubated with complex 2 show nuclear staing, a much lower intensity luminescence is observed relative to complex 3 (Figure 4). Figure 4 shows (a) MCF-7 cells incubated with complex 2 (200 pM, lhr) showing poor nuclear staining. Figure 4 (b)shows when a higher incubation concentration is used (500 pM, lhr), MCF-7 cells display clear nuclear staining by complex 2. This is most likely due to poor cellular uptake of the complex and is consistent with previous studies comparing the stainin capabilities of complexes 4 and 5.

Fixing cells before DNA staining is a common preparation technique that allows membrane-permeability problems to be overcome. To examine the ability of complexes 2 and 3 to function as DNA stains in such circumstances, MCF-7 cells were fixed and permeablized using ethanol, exposed to each of the complexes and imaged using CLSM.

As shown by Figure Sd and Figure 6, in such conditions, both complexes 2 and 3 function as excellent DNA stains in fixed and permeablized cells while colocalization with P1 confirms that DNA targeting is occurring. This experiment demonstrates that complexes 2 and 3 are both able to intercalate into nuclear DNA and, upon binding, their MLCT "light switch" is activated. This offers further evidence that it is the poor cellular uptake of complex 2 that prevents its luminescence from being observed in live cells.

EXAMPLE 3

As previously demonstrated, ruthenium complexes can also be used for TEM imaging as the electron-dense metal centre acts as a contrast agent for this technique (Gill, M. R et al Nat Chem 2009, 1, 662-667). TEM experiments provide more accurate information on cellular distribution of the complex as they are not reliant on intercalation -and subsequent activation of the "light switch" effect -for imaging. Therefore, to further probe the intracellular location of complex 3, cells were incubated as for CLSM studies, and then fixed and examined using TEM. Figure 7 shows TEM micrographs of MCF-7 cells incubated with complex 3: (a) image showing even distribution of the stain throughout the cytosol; (b) detailed image of nucleus reveals clear heterochromatin staining (ringed); (c) detailed image revealing distinctive granular patterning of the distribution of complex 3 within the cytosol. The results of Figure 7a shows a MCF-7 cell strongly stained with complex 3, revealing that the complex is evenly distributed throughout the cytosol of the cell. Notably, as the nucleus shows strong heterochromatin staining (Figure 7b), the greatest accumulation of the complex is in these regions, revealing that complex 3 possesses a high affinity for densely-packed DNA. The TEM micrographs show limited contrast signal from mitochondrial regions, suggesting that complex 3 is not internalized by these DNA-containing organelles and is nuclear DNA-specific. Interestingly, at high magnification, the location of complex 3 in the cytosol appears to be from distinct regions, where it is possibly protein-or RNA-bound (Figure 7c). Cells fixed and permeablized before incubation with complex 3 show a similar distribution to that observed in live cells, although with less well-defined heterochromatin aggregation. Again, at high magnification, complex 3 can be seen to occupy distinct locations within the cytosol (Figure 8). Figure 8 shows TEM micrographs of fixed MCF-7 cells stained with complex 3. Figure 8a shows the cytosol with strong intracellular contrast and the nucleoli of the cell are clearly observable. Figure 8b shows complex 3 localized within heterochromatin within the nucleus and Figure 8c shows that a distinctive granular pattern is observed due to complex 3, where the complex is possibly protein-bound. These results make complexes 2 and 3 of interest as DNA stains for use with both the techniques of CLSM and TEM, a unique property of DNA-binding metal complexes.

H N') 2 NtRLrjOJ 6,L = CH3CN, 7, L = pyridine By using TEM it has also been discovered that not every in vitro light-switching complex displays the effect in cellulo. If two previously reported Ru(dppz)-based complexes, 6 and 7 (see above) -which are known to display light-switching when intercalated into DNA -are incubated with MCF-7 cells and then prepared for TEM imaging, no contrast effects are observed confirming that the complexes are not internalized. However, if the cells are fixed with ethanol before incubation with complexes 6 or 7, TEM imaging clearly reveals nuclear staining by these complexes, despite the fact that CLSM studies reveal no luminescence from the nucleus (Figure 9). Figure 9 shows (a) TEM micrographs of MCF-7 cells fixed before incubation with complex 7: (b) CLSM of fixed and permeablized MCF-7 cells reveals no Ru(dppz)-based MLCT emission from the nucleus (left), same sample stained with positive control propidium iodide (right). These observation shed light on a recent study by Puckett et a!. Biochemistry 2008, 47, 11711-11716 in which related membrane permeable complexes still display no nuclear fluorescence and indicate that Ru(dppz) systems may bind to cellular DNA through a non-intercalative mode and/or target another nuclear binding site, such as histone proteins.

EXAMPLE 4

As complexes 2 and 3 are polar, positively charged and hydrophilic molecules (log P values of -2.08 and -1.24 respectively), they would not be predicted to permeate cell membranes. This is also suggested by the relatively high concentrations required for nuclear staining in live cells (200 pM), compared to membrane-permeable dyes such as DAPI, which are employed on the nanomolar scale. Therefore, the mechanism of uptake was investigated. MCF-7 cells incubated with either complex 2 or 3 at 4°C resulted in no observable in cellulo luminescence, indicating active transport as the mechanism of cellular uptake for each and confirming neither molecule is membrane-permeable. As one of the most common methods by which cells internalize membrane-impermeable molecules is endocytosis, this was briefly examined. Coincubation with the endocytosis inhibitor chloroquine, which prevents endosomal release, showed no reduction of nuclear staining due to complex 3, suggesting that uptake does not occur by an endocytotic pathway (Figure 10). Figure 10 shows mechanism of uptake studies of Ru(ll)tpphz complexes. Figures lOa and lOb show MCF-7 cells incubated with complexes 2 or 3 (200 pM, I hr) at 4°C and show no staining of live cells, indicating neither complex is membrane-permeable. Figure lOc shows cells co-incubated with endocytosis inhibitor chloroquine (50 pM) and complex 3 (200 pM, lhr) display uptake and nuclear staining by the complex. For all experiments, propidium iodide staining is included to indicate that the cells remain viable. More evidence for this hypothesis is supplied by the TEM micrographs of incubated cells (Figure 7), which show no contrast within intracellular vesicles -an observation that was also made on the dinuclear analogue of complex 3, complex 5. A further parallel between mono-and dinuclear complexes is the fact that the phen analogues display greater uptake than the bpy analogues. This is consistent with structure-specific protein binding occurring in the uptake pathway -most likely at the cell membrane -and highlights the importance of the ancillary ligand(s) for cellular uptake.

EXAMPLE 5

As both complexes 2 and 3 are taken up by cells they may affect numerous cellular processes such as DNA replication and translation. We therefore examined the cytotoxicity of these complexes towards selected cancer cell lines. Cell viabilities were assessed by MTT assay and 1050 values (the concentration that would induce 50% cell death) were thus determined, with cisplatin being employed as a positive control.

As would be expected for a non-cell permeable systems, mononuclear dppz-based systems complexes 6 and 7 each display low cytotoxicity (>200 pM and 190 pM, respectively). Contrastingly, complexes 2 and 3 show lC values that are more comparable to cisplatin, with complex 3 displaying a higher toxicity towards MCF-7 cells than 2 (Table 1, Figure 11). Figure 11 shows cytotoxicity of complexes towards MCF-7 breast cancer cells (24 hour incubation time) (a) Cytotoxicity of Ru(ll)tpphz complexes and cisplatin towards MCF-7 cells. (b) Cytotoxicity of Ru(ll)dppz complexes towards MCF-7 cells. Cell viability determined by MTT assay. Thus, toxicity correlates better with the relative uptake of the two complexes into cells and not their overall binding affinity to DNA.

It is also interesting to note that complexes 2 and 3 are significantly more cytotoxic than their non-intercalative dinuclear complexes, 4 and 5.

Table I lC values of Ru(ll)tpphz complexes towards cancer cells compared to cisplatin COMPLEX MCFJ (lC pM) A2780 (1050 pM) A2780-CP7O (1050 pM) cisplatin 12 --I-2 12 +1-2 55 +1-8 2 53+/-7 36+/-6 47+1-6 3 36+1-3 11+1-6 20+1-5 While cisplatin remains the first step in treating a variety of cancers, including ovarian cancers, acquired drug resistance represents a key challenge in cancer treatment. As the mechanism of DNA-binding and DNA-damage by complex 3 is radically different to cisplatin, this complex offers potential as a new class of therapeutics. With this in mind, the toxicity of complexes 2 and 3 towards A2780 human ovarian cancer cells and the derived cisplatin-resistant A2780-0P70 daughter cell line was investigated. It was found that both complexes demonstrate a higher toxicity towards A2780 cells than that observed for MCF-7s, again with complex 3 demonstrating a great toxicity than complex 2 (Figure 12), and the IC50 value of complex 3 being equal to that of cisplatin (Table 1). Figure 12a shows the cytotoxicity of cisplatin of complex 2 and Figure 12b with complex 3 and Figure 12 c towards A2780 (black) and A2780-CP7O (white) ovarian cancer cell lines. Cell viability determined by MTT assay.

Significantly, both complexes 2 and 3 retain their cytotoxicity towards the cisplatin-resistant A2780-CP7U cell line while, in contrast, cisplatin experiences a decrease in toxicity compared to the cisplatin-sensitive parental A2780 cell line, Table I and Figures 12 and 13. (Figure 13 shows the cytotoxicity of mononuclear Ru(ll)tpphz complexes towards selected cancer cells). The values obtained for cisplatin are in agreement with the 1050 values reported in the literature. Clearly the cytotoxicity properties of complex 3 are of great interest and this complex, and further derivatives based on the tpphz intercalating ligand, offer great potential as leads for the development of a new class of multifunctional anti-cancer chemotherapy drug.

Claims

CLAIMS1. A mono-nuclear complex containing a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"-j] phenazine, tpphz, ligand for use as a medicament.
2. A medicament according to claim I that is an antineoplastic agent.
3. A medicament according to either claim I or 2 wherein, the mono-nuclear complex has the structure: (N N N N N:!#" T' T1' %tT!=##Nwherein: M is a transition metal;N (NN and N each independently represent a bidentate chelating ligand; and n is2or3.
4. A medicament according to claim 3 wherein the metal, M, of the mono-nuclear complex is selected from the group comprising Ru (II), Os (II) and Ir (Ill).
5. A med icament according to either claim 3 or 4 wherein the metal, M, is Ru (II).
6. A medicament according to any one of claims 3 to 5 wherein each bidentate chelating ligand independently has the structure: R2b Rib R1a, wherein: each Ria, R2a, R3a, R, R2' and R3b is independently selected from the group consisting of: hydrogen, -F, -Cl, -Br, -I, -OR, NRxR/, -ON, -NO2, -SO3R, -000R, 0i06 alkyl and substituted 0iC6 alkyl; wherein R, Rx and R each independently represent hydrogen or a 0i04 alkyl group; and each R4 and R4b is independently selected from the group consisting of: hydrogen, -F, -01, -Br, -I, -OR, NRxR/, -ON, -NO2, -SO3R, -000R, 0i06 alkyl and substituted 0i06 alkyl, or each R4 and R4b of each bipyridyl together form OR5a=OR5l), wherein each R5 and R5b is independently selected from the group consisting of: hydrogen, -F, -01, -Br, -I, -OR, NRXRY, -ON, -NO2, -SO3R, -000R, 0i06 alkyl and substituted Oi06 alkyl; and wherein each substituted Oi.-O alkyl is substituted with up to 5 substituents, where chemically possible, independently selected from the group consisting of: -F, -Cl, -Br, -I, -OR, NRxR, -ON, -NO2, -SO3R and -000R, wherein R, Rxand R' each independently represent hydrogen or a 01-04 alkyl group.
7. A med icament according to claim 6 wherein R R, R2, R2', R3, R3', R4 and R4' are each independently selected from the group comprising: H and alkyl.
8. A med icament according to claim 6 wherein each R4 and R4' of each bipyridyl together form OR5a=OR5l).
9. A medicament according to any one of claims 3 to 8 which includes two identical or different bidentate chelating ligands.
10. A medicament according to any one of claims 3 to 9 wherein each Rfl', R2, R3, R, R2' and R3' is H, each R4E and R4b of each bipyridyl together form OR5OR5b and each R5 and R5b is H.
II. A medicament according to any one of claims 3 to 9 wherein each Ri6, R2a, R3a, R4, R, R2b, Rab and R4' is H.
12. A medicament according to any preceding claim wherein the complex is in association with a counterion(s).
13. A medicament according to any preceding claim wherein the complexes are suitably in the form of physiologically acceptable salts, or salt solutions of such salts.
14. A medicament according to any preceding claim in which the chiral centre is of (R)- configuration, individual compounds or moieties in which the chiral centre is of (S)-configu ration and mixtures of (R)-and (S)-isomers as a racemic mixture.
15. A medicament according to any preceding claim wherein the complex has a structure selected from the group comprising: r I I Ct j; 1cx1j [)] 2
16. A medicament according to any preceding claim wherein the complex has a structure selected from the group comprising: N N16. A pharmaceutical composition comprising a complex according to any preceding claim and a pharmaceutically acceptable excipient, diluent or carrier.
17. A cytotoxic mono-nuclear complex containing a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"-j] phenazine, tpphz, ligand that binds to or intercalates with nuclear DNA in living cells.
18. A complex according to claim 17 further including any one or more of the features recited in any one of claims 2 to 15.
19. A cytotoxic mono-nuclear complex containing a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"-j] phenazine, tpphz, ligand that binds to or intercalates with nuclear DNA in living cells and displays an in cellulo light-switch effect.
20. A complex according to claim 19 further including any one or more of the features recited in any one of claims 2 to 15.
21. A mono-nuclear complex containing a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"-j] phenazine, tpphz, ligand for use in photodynamic therapy.
22. A complex according to claim 21 further including any one or more of the features recited in any one of claims 2 to 15.
23. A method of treating a neoplastic disease comprising administering a therapeutically effective amount of an intercalating mono-nuclear complex containing a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"-j] phenazine, tpphz, ligand to a patient suffering from a neoplastic disease.
24. A method of treating a neoplastic disease in an individual who displays resistance to platinum-based chemotherapeutics, the method comprising administering a therapeutically effective amount of an intercalating mono-nuclear complex containing a tetrapyrido[3,2-a:2',3'-c:3",2"-h:2",3"-j] phenazine, tpphz, ligand to a patient suffering from a neoplastic disease.