"TRANSITION METAL COMPLEXES FOR PHARMACEUTICAL

APPLICATIONS"

Technical field of the invention

It has been reported in the literature that ruthenium-based compounds can play a significant role as antitumoral agents, and may become an important alternative to chemotherapy overcoming part (if not most) of the problems related with the treatment with cisplatin and alike drugs approved for clinical use worldwide. In effect, the most important difficulties associated with platinum-based drugs in chemotherapy are their inactivity against many cancer conditions and against tumor metastasis, the development of resistance to treatment and many undesirable severe side- effects. The urgency to overcome these many drawbacks stimulated the search for new effective agents.

Background and prior art

One main challenge in the field of chemotherapy is the finding of alternative pharmaceu- ticals to cisplatin drugs, which have many drawbacks that urge to overcome despite their being the most effective anticancer pharmaceuticals in clinical use.

In fact, the undesirable side-effects of cisplatin drugs, its inactivity against many cancer cell lines and tumors metastasis (T.M. Klapotke et al . , Organometallics , 13 (1994) 3628-3633) and the problem derived of drug resistance, have been stimulating the search for new effective drugs in the recent years, in the field of non- platinum-based anticancer drugs (M.J. Clarke et al . , Chem. Rev. 99 (1999) 2511-2534; C.X. Zhang et al . , Curr. Opin. Chem. Biol. 7 (2003) 481-489; M. Galanski, et al . ,Curr. Pharm. Des. 9 (2003) 2078-2089). Research in the field of ruthenium based complexes revealed promising results for potential use as anticancer drugs.

Optically resolved enantiomers of ruthenium ( I I ) and cobalt (II) and 1 , 10 -phenanthroline derivatives, with the structure R ₃ M, are disclosed in US 4980473 (1990) to be useful for the treatment of tumor cells in a subject.

Antitumor activity has been reported for some other ruthenium ( I I ) and ruthenium ( I I I ) complexes such as trans-

[RuCl ₂ (DMSO) ₄ ] , [ImH] [trans-RuCl ₄ (DMSO) Im] , [ImH] [trans- RuCl ₄ lm ₂ ] (Im = imidazole) and (Hind) [ trans-RuCl ₄ (ind) ₂ I ,

(ind=indazole) (C.G. Hartinger et al . , J. Inorg. Biochem. 100 (2006) 891-904; S. Kapitza et al . , J. Cancer Res. Clin. Oncol. 131 (2005) 101-110; M.A. Jakupec et al . , Int. J. Clin. Pharm. Ther. 43 (2005) 595-602) .

U.S. Pat. No. 4,980,473 discloses 1 , 10 -phenanthroline complexes of ruthenium ( I I ) and cobalt (II) which are reported to be useful for the treatment of tumour cells in a subject.

Some other ruthenium ( I I ) and ruthenium ( I I I ) complexes which have been shown to exhibit antitumour activity are mentioned in Guo et al . , Inorganica Chimica Acta, 273 (1998), 1-7, specifically trans- [RuCl ₂ (DMSO) 4] , trans- [RUCI4 (imidazole) 2] and trans- [RUCI4 (indazole) 2] ·

Clarke et al . have reviewed the anticancer, and in particular the antimetastatic, activity of ruthenium complexes: Chem. Rev., 99 (1999) 251-253. Also, Sava has reviewed the antimetastatic activity in "Metal Compounds in Cancer Therapy" Ed by S P Fricker, Chapman and Hall, London 1994, p. 65-91.

Dale et al . , Anti-Cancer Drug Design, 7 (1992), 3-14, describes a metronidazole complex of ruthenium ( 11 ) i.e., [ (C ₆ H ₆ ) RuCl ₂ (metronidazole) ] and its effect on DNA and on E. coli growth rates. Metronidazole sensitizes hypoxic tumour cells to radiation and appears to be an essential element of the complexes of Dale et al.. There is no indication that the complexes would be at all effective in the absence of the metronidazole ligand.

Other ruthenium r| ⁶ -arene derivatives revealed very active in vitro against: i) hypotoxic tumor cells (C.S. Allardyce et al., Chem. Commun. (2001) 1396-1397; C.S. Allardyce et al . , J. Organometal . Chem. 668 (2003) 35-42); ii) breast and colon carcinoma cells (Y.N.V. Gopal et al . , Biochemistry 38

(1999) 4382-4388; Y.N.V. Gopal et al . , Arch. Biochem. Biophys. 401 (2002) 53-62); iii) inhibition of growth of both human ovarian cancer cells line A2780 (R.E. Morris et al., J. Med. Chem. 44 (2001) 3616-3621) and iv) mammary cancer cell line (L.A. Huxham et. al . , Inorg. Chim. Acta 352

(2003) 238-246) .

Also studies in vivo for several members of this family revealed high activity in models of human ovarian cells (C. Scolaro et al., J. Med. Chem. 48 (2005) 4161-4171) and reduction of the growth of lung metastases in CBA mice bearing the MCa mammary carcinoma (R. E. Aird et al . , British J. Cancer 86 (2002) 1652-1657) . WO 01/130790 discloses ruthenium ( I I ) compounds and their use as anticancer agents. The compounds have neutral N- donor ligands and the resulting ruthenium complexes are generally positively charged.

WO 02/102572 also discloses ruthenium ( I I ) compounds, generally positively charged, containing a bidentate neutral diamine ligand that have activity against cancer cell lines.

WO 06/018649, US 2006/0058270, US 2005/0239765, US 2009/0312301 Al relates to organometallic compounds useful in the treatment of metastasis, comprising a ligand that is covalently bound to a bioactive compound. These compounds are inhibitors of a resistance pathway.

The authors of the present patent found excellent results of cytotoxicity against several tumor cells, for a new family of ruthenium η ⁵ - cyclopentadienyl derivatives (see figures 1-3) when compared to the published results in the specialized literature concerning ruthenium compounds. Some of the present complexes revealed inhibition of the growth of LoVo human colon adenocarcinoma, MiaPaCa pancreatic cancer cell lines (M.H. Garcia et al . , J. Inorg. Biochem., 103 (2009) 354-361) and human leukemia cancer cells (HL-60 cells) with IC ₅₀ values in the range of nanomolar amounts (much higher cytotoxicity than the cisplatin) .

Apoptotic death percentage was similar to that of cisplatin (V. Moreno et al., Bioinorg. Chem. Appl . (2010); M.H. Garcia et al . , Inorg. Chim. Acta 363 (2010) 3765-3775; V. Moreno et al . , J. Inorg. Biochem. 105 (2011) 241-249). Very few, if any, of the compounds and complexes of the prior art cited above have resulted in clinical phase studies, not to mention actual therapies. Reasons for the poor performance of these principles are manifold and may be linked to toxicity problems or to insufficient efficiency in treatment.

It is thus an objective of the present invention to use compounds which structure is based on piano stool Μ(η ⁵ - ciclopentadienyl ) (M=Ru, Fe) possessing compulsory sigma bonded heteroaromatic ligands to treat tumor and /or metastasis .

Summary of the invention

The present invention describes an organometallic compounds comprising a half sandwich "piano-stool" Μ(η ⁵ - ciclopentadienyl ) (M=Ru, Fe) structure, with a sigma bond heteroaromatic ligands to the metals Ru or Fe .

A preferred embodiment of the present invention provides organometallic compounds that present cytotoxicity activity, after 24h incubation, not superior to 3.30 μΜ.

In another embodiment of the present invention, the organometallic compounds that present cytotoxicity activity, after 72h incubation, not superior to 25.60 μΜ.

A preferred embodiment of the present invention provides organometallic compounds which comprise the following compounds :

[RuCp(PPh ₃ ) ₂ (l-BI) ] [CF3SO3] ;

[RuCp(PPh ₃ ) (2,2 ^' -bipy) ] [CF3SO3] ;

[RuCp (PPh ₃ ) 2 (4-Mpy) ] [CF3SO3] ; [RuCp (DPPE) (4-Mpy) ] [CF ₃ SO ₃ ] ;

[RuCp(PPh ₃ ) (2,2 ^' -bipy) ] [CF3SO3] ;

[RuCp(PPh ₃ ) ₂ (l-BuIm) ] [CF ₃ SO ₃ ] ;

[RuCp(PPh ₃ ) ₂ (l-BuIm) ] [PF ₆ ] ;

[RuCp (PPh ₃ ) 2 (1-BuIm) ] [BPh ₄ ] ;

[RuCp (DPPE) (1-BuIm) ] [CF ₃ SO ₃ ] ;

[RuCp (DPPE) (1-BuIm) ] [PF ₆ ] ;

[RuCp (DPPE) (1-BuIm) ] [BPh ₄ ] .

In another embodiment of the present invention, the organometallic compounds bond do DNA, after 30 minutes of incubation, where the DNA is a plasmid pBR322 DNA.

A preferred embodiment of the present invention provides organometallic compounds bind to ubiquitin and cytochrome-C proteins .

In another embodiment of the present invention, the organometallic compounds inhibit Poly- (adenosine diphosphate (ADP) -ribose) polymerase.

A preferred embodiment of the present invention provides organometallic compounds bind to blood serum proteins forming a [Ru-protein] complex (adduct) .

In another embodiment of the present invention, the organometallic compounds bind to site I of the albumin protein, with a binding constant of Log Ks = 4.02.

It is also an objective of the present invention to describe the use of the compounds described previously for the treatment of human tumorigenic cell lines. It is also an objective of the present invention to describe the use of the compounds described in any of the previous claims for the treatment of promyelocytic leukemia, breast adenocarcinoma (hormone independent) , colon adenocarcinoma, prostate cancer, breast adenocarcinoma (hormone dependent, ER positive) , ovarian carcinoma, ovarian carcinoma.

Description of the invention

The present patent refers to the use of a family of organometallic compounds presenting half sandwich "piano- stool" structure as anticancer agents for which our in vitro studies revealed an unusual high efficiency against several cancer cell lines.

In fact, results found for most of the present compounds showed more effectiveness than cisplatin (cisPt) against a significant number of cancer cell lines. Moreover, many of the present compounds also presented high effectiveness against cell lines resistant to cisPt treatment.

Importantly, the present patent describes activity values (reported by IC ₅₀ values, see Tables I, II and III) for some of the compounds up to ten times higher than the ones reported in the scientific literature for other ruthenium compounds including the recent USPTO Patent Application 20090312301.

Examples

Hereinafter, the present invention is described in more detail and specifically with reference to the Examples, which however are not intended to limit the present invention . Cell culture

The human cancer cell lines used were A2780 and A2780cisR (cisPt sensitive and resistant, respectively) human ovarian cancer, PC3 human prostate cancer, MCF7 human breast cancer and one non-tumorigenic cell line V79 Chinese hamster lung fibroblast and were obtained from ATCC.

Table I - Human tumor cell lines used in our in vitro.

The cells were maintained in DMEM (Dulbecco's Modified Eagle's Medium) containing GlutaMax 1 (MCF7) or RPMI 1640 (A2780/A2780cisR, PC3 and V79) supplemented with 10% FBS and 1% penicillin/streptomycin.

All cell lines were kept in a CO ₂ incubator with 5% CO ₂ at 37°C in a humidified atmosphere. Cells from a confluent monolayer were removed from flasks by a trypsin-EDTA solution. Cell viability was determined by the trypan blue dye exclusion test. For experimental purposes cells were seeded in multi-well culture plates. Growth inhibition

Example 1 : Cytotoxicity

The cytotoxic activity of [RuCp (PPh ₃ ) (2, 2' - bipy) ] [CF ₃ S0 ₃ ] was screened against all cell lines, and the results are summarized in Figure 4, Table II and Table III.

Cells were seeded in 200 yL of complete medium in 96-well plates. The plates were incubated at 37°C for 24 h prior to complex testing to allow cell adhesion. The stock solution (20 mM) in DMSO of the complex was freshly prepared and used for sequential dilutions in complete medium. The final concentration of DMSO in cell culture medium did not exceed 0.5%. Control groups without or with DMSO (0.5~6 ) were included in the assays. The anticancer drug cisplatin was included in this study as a positive control.

Analysis of cell survival was performed at the end of 24h or 72 h cell exposure by the MTT [ 3- ( 4 , 5-dimethylthiazol-2- yl ) -2 , 5-diphenyltetrazolium bromide] colorimetric assay which is based on the reduction of MTT by viable cells following exposure to cytotoxic drugs. Briefly, a solution of MTT dissolved in PBS (0.5 mg/mL) was added to each well (200 yL) and the plates were incubated at 37°C for 3 - 4 h. After this time, the medium was discarded and 200 yL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm with a plate spectrophotometer (Power Wave Xs, Bio-TeK) . Each experiment was repeated at least three times and each concentration was tested in at least six replicates. Results are expressed as a percentage of survival with respect to control cells in the absence of the compound. IC5 ₀ values (i.e., the drug concentration that induces 50% of cell death) were calculated from dose-response curves obtained by plotting cell survival (%) versus compound concentration (M) .

Table II - IC5 ₀ values against HL-60 cells of some ruthenium compounds compared to cisplatin (CisPt) .

Table III - IC5 ₀ values against several cancer cell lines after a 72 h incubation period of some ruthenium compounds compared to cisplatin (CisPt) .

*n.a. (CisPt is not efficient against these cell lines.) **according to Gama, S. et al . , J. Inorg. Biochem. 105 (2011) 637-644.

Example 2: Effect of HSA on cytotoxity

The effect of albumin (HSA) on cell viability, either alone or in combination with [RuCp (PPh ₃ ) (2, 2' - bipy) ] [CF ₃ S0 ₃ ] or any other of the present complexes, was evaluated using 1 and 5 μΜ of the compound. HSA and Ruthenium complexes concentrations were combined in order to obtain the following molar [compound] : [HSA] molar ratios: 1:1, 1:5, 1:10. A2780 cells were seeded on 96-well plates 24 h before incubation with the compound in complete medium containing 5% of FBS (A. Bergamo et al . , J. Inorg. Biochem., 104

(2010) 79-86) . After a 24 h incubation period the treatment solution was removed and cell viability was measured by the MTT assay as described above. In the case of

[RuCp(PPh ₃ ) (2,2'- bipy)] [CF ₃ S0 ₃ ] cell viability was found to be little affected by the presence of HSA (Figure 5) , especially at low molar excess.

Biological activity screened by in vitro essays

"Classical" biological targets: the role of DNA

Our approach to the study of DNA binding comprised the several different techniques described below in order to characterize both the strength of the interaction and the mechanism involved.

Example 3 : Atomic Force Microscopy

Different types of images were found as a consequence of interaction of the present compounds with the DNA chains, leading to quite different modifications. Some images reveal supercoiling and kink forms of DNA, while others show stacking of bases of DNA, intercalation, compactation or even small broken pieces of DNA. Below, on Figure 6 is exemplified the effect on plasmid pBR322 DNA after 30 minutes of incubation with one of the present compounds. Experiment: Atomic Force Microscopy (AFM) samples were prepared by casting a 3-μ1 drop of test solution onto freshly cleaved Muscovite green mica disks as support. The drop was allowed to stand undisturbed for 3 min to favour the adsorbate-substrate interaction. Each DNA-laden disk was rinsed with Milli-Q water and was blown dry with clean compressed argon gas directed normal to the disk surface. Samples were stored over silica prior to AFM imaging. All AFM observations were made with a Nanoscope III Multimode AFM (Digital Instrumentals , Santa Barbara, CA) . Nano- crystalline Si cantilevers of 125-nm length with a spring constant of 50 N/m average ended with conical-shaped Si probe tips of 10-nm apical radius and cone angle of 35° were utilized. High-resolution topographic AFM images were performed in air at room temperature (relative humidity < 40%) on different specimen areas of 2x2 um operating in intermittent contact mode at a rate of 1-3 Hz.

Example 4 : UV-Visible Absorption

Different mechanisms of interaction of compounds with DNA can be presumed by titration studies using the UV-vis technique. Studies carried out so far, for some compounds of the present patent revealed the presence of a non classic mechanism, albeit proving that intercalation is not the case (also found by viscosity) . In most of the cases there is evidence for more than one mechanism of interaction depending on the ratio compound/DNA. Experiment: Calf thymus DNA (ctDNA, highly polymerized), Hepes and Trizma used in UV-Visible absorption titrations were from Sigma-Aldrich . Ultrapure MilliCj water (18.2 πιΩ) was used in all experiments to prepare aqueous buffer solutions. Buffer media used were 10 mM Hepes (pH7.4), 50 mM NaCl/10 mM Hepes (pH7.4), 5 mM Tris/HCl (pH7.4) and 50 mM NaCl/5 mM Tris/HCl (pH7.4). Solutions of ctDNA were prepared in the desired buffer medium by slow dissolution at 4°C with gentle orbital strirring (typically over two days), stored at 4°C and used within four days. The DNA stock solution concentration (per nucleotide) was determined by absorption spectroscopy using the molar absorption coefficient at 260 nm (6600 M ^~1 cm ^~1 nuc ^~1 ) (M.E. Reichmann et al ., .Am. Chem. Soc . , 76 (1954) 3047-3053) and converted to base pairs (1 M.nuc ^-1 = 0.5 M.bp ^-1 ) . Absorbance at 280 nm and 260 nm gave a ratio of -1.9, indicating that the DNA was sufficiently free from protein (J. Marmur, J. Mol. Biol., 3 (1961) 208-211). Stock solutions of each complex were prepared in DMSO immediately prior the incubation with DNA under minimum light, and discarded after 30 min. An adequate concentration was used in each case to ensure the same % of DMSO (max. 5%) in the final sample. All samples were prepared individually. Complex concentration was kept constant (between 20 and 100 μΜ, depending on the complex) , and R (ctDNA-to-complex molar ratio) ranged from 0 to 25. ctDNA was incubated with complexes for 30 min prior to absorbance reading in the range 300-500 nm. For each titration point, the same amount of ctDNA was used in the reference cell.

Example 5 : Agarose Gel Electrophoresis

The behavior of the gel electrophoretic mobility of pBR322 plasmid DNA and of the plasmid DNA incubated with cisplatin (used as negative and positive controls, respectively) compared to the Ru compounds-DNA adducts show the presence of new DNA forms with different mobility in agarose gel as the result of modifications on the DNA tertiary structure due to the interaction with the Ru compound (Figure 7) . These results are compatible with the AFM images.

Experiment: pBR322 DNA aliquots (0.25 μg/mL) were incubated in TE buffer (lOmM Tris.HCl, ImM EDTA, pH = 7,5) at molar ratio ri = 0.50 for electrophoresis study. Incubation was carried out in the dark at 37 °C for 24 hours. 4 uL of charge marker were added to aliquots parts of 20 uL of the compound-DNA complex. The mixture was electrophoresed in agarose gel (1% in TBE buffer, Tris-Borate-EDTA) for 5 hours at 1.5V/cm. Afterwards, the DNA was dyed with ethydium bromide solution (0.75 μg/mL in TBE) for 6 hours. A sample of free DNA was used as control. The experiment was carried out in an ECOGEN horizontal tank connected to a PHARMACIA GPS 200/400 variable potential power supply and the gel was photographed with an image Master VDS, Pharmacia Biotech.

Example 6: Viscosity measuremets

Viscosity studies are a very efficient process to get information about the possibility of interaction between the target compounds and DNA, and are particularly useful to discriminate between mechanisms of DNA binding such as intercalation from other non-covalent interactions. Our studies carried out for some of the compounds of this family, revealed that intercalation might not be the preferential mode of interaction.

Experiment: Viscosity measurements were carried out with a Ostwald viscometer associated to a ViscoClock (SCHOTT) in a Julabo 18V thermostatized water bath maintaining a constant temperature of 25,15°C (±0,01). Kinematic viscosity was obtained by measuring the flow time for each sample and applying

v = kt (1)

where k is the calibrating constant determined for each essay using the flow time of pure MilliQ water. Stock solutions of ctDNA were prepared in the desired buffer medium (10 mM Hepes pH 7.4 or 5 mM Tris/HCl pH 7.4) by slow dissolution at 4°C with gentle orbital strirring (typically over two days), stored at 4°C and used within four days. The DNA stock solution concentration (per nucleotide) was determined by absorption spectroscopy using the molar absorption coefficient at 260 nm (6600 M ^~1 cm ^~1 nuc ^~1 ) (M.E. Reichmann et.al., J. Am. Chem. Soc . , 76 (1954) 3047-3053). All samples were prepared individually. An adequate dissolution of the ct-DNA stock solution was used to a final concentration of 200 μΜ.ηυο ^-1 . Samples were prepared by varying the complex-to-ctDNA molar ratio between 0 : 1 and 10:1. Adequate concentrations of the ruthenium compound in DMSO were used to maintain the final DMSO content constant at 5% (v/v) , and the same amount of DMSO was used on the ctDNA sample with no complex ( Do ) to cancel the effect of the organic solvent. Samples were incubated for 30 min at 37°C prior to measurement. Changes in the relative kinematic viscosity were observed on plots of ( Do /D ) versus compound concentration.

Example 7 : Circular Dichroism

Although DNA might not be the preferential target for ruthenium compounds in general, we found either by Circular Dichroism (CD) or the several techniques described above that significant interaction can exist. Our CD studies indicate modifications on the secondary structure of DNA as consequence of the interaction of the present complexes (Figure 8) . An induced CD signal that would be compatible with an intercalative binding mechanism is not observed, supporting the results already described with indicate other mechanisms as the preferential mode (s) of interaction .

Experiment: All compounds were dissolved in an aqueous solution (prepared with milli-Q water) of 4% DMSO. The stock solutions were freshly prepared before use. The samples were prepared by adition of aliquots of these stock solutions to the appropriate volume of Cal Thymus DNA in a TE buffer solution (50mM NaCl, lOmM tris-

(hydroxymethyl ) aminomethane hydrochloride (Tris-HCl) , 0. ImM H4edta, pH 7.4) (5mL) . The amount of complex added to the DNA solution was designated as ri (the input molar ratio of Ru to nucleotide and it is calculated with formula

) where m = mass of the compound ^g) ; Mnucl = mediumnnuclear mass per nucleotide (330 g/mol) ; C = concentration of the DNA solution ( q/ml) ; Mr = molecular mass of each compound (g/mol) ; V = total volume of each sample (5mL) . As a blank, a solution in TE of free native DNA was used. The CD spectra of DNA in the presence or absence of complexes (DNA concentration 20 μg/mL, molar ratios ri = 0.10, 0.30, 0.50) were recorded at room temperature, after 24 hours incubation at 37 °C, on a JASCO J-720 spectropolarimeter with a 450W xenon lamp using a computer for spectral subtraction and noise reduction. Each sample was scanned twice in a range of wavelengths between 220 and 330 nm. The CD spectra drawn are the average of three independent scans. Data are expressed as average residue molecular ellipticity (Θ) in degrees -cm ² -dmol ^-1 .

Other Biological targets

Example 8 : Interactions in vitro with cell regulatory proteins

Interaction with ubiquitin and cytochrome-C monitored by ESI-MS

Studies by ESI-MS of samples of ubiquitin (Figure 9) and cytochrome-C (Figure 10) incubated with the present ruthenium compounds revealed the presence of new mass fragments compatible with the formation of Ru-protein adducts, indicating the binding of the ruthenium complexes to these proteins.

Experiment: Samples were prepared in aqueous or buffered (pH = 7.4) medium, with a protein concentration (horse heart cytochrome c, cyt c, or bovine ubiquitin, ubq) of 10 ^{~ 4} M, and a ruthenium compound to protein ratio of 3. The reaction mixtures were incubated for 24 h, at 37°C. After a 20-fold dilution with a mixture of methanol and 0.1% formic acid, mass spectra were recorded by direct infusion into an electrospray ionization mass spectrometer (ion trap or FTICR) . The nebulizing conditions, ion optics voltages and analyzer parameters were optimized to ensure a proper signal-to-noise ratio (S/N) . Collision induced dissociation experiments were performed to characterize the structure of the [protein-Ru] complex formed during incubation.

Example 9: Enzyme inhibition studies:

Poly- (adenosine diphosphate (ADP) -ribose) polymerase inhibition PARP-1 activity assay

Results on the PARP-1 inhibition the for the ruthenium complex [RuCp(PPh ₃ ) (2,2'-bipy)] [CF ₃ SO ₃ ] are presented in Figure 11. An IC ₅₀ value of (1.0+0.3) μΜ was determined for both a 2h and a 24 h incubation period, indicating that the equilibrium is reached quite fast. The IC ₅₀ obtained for

[RuCp (PPh ₃ ) (2, 2' -bipy) ] [CF3SO3] is lower than the one for the classical inhibitor 3-Aminobenzamide (ICso=33 μΜ) (N. J. Curtin, Expert Rev. Mol. Med. 2005, 7) or for cisplatin

(IC ₅₀ =12.3 μΜ) (F. Mendes et al . , J. Med. Chem. 2011, 54, 2196-2206), and to the best of our knowledge it is the lowest found in the literature for ruthenium complexes as well .

Experiment: PARP-1 inhibition was determined on the purified human enzyme. Recombinant human PARP-1 (High Specific Activity) was used as the enzyme source. The enzyme was incubated with each compound at various concentrations for 2h or 24h at room temperature before assessing its activity spectrophotometrically by measuring the incorporation of biotinylated poly (ADP-ribose) onto histone proteins. PARP-1 activity was determined using Trevigen's HT Universal Colorimetric PARP Assay which measures the incorporation of biotinylated poly (ADP-ribose) onto histone proteins in a 96 microtiter strip well format. 3-Aminobenzamide (3-AB) was used as control inhibitor. The final reaction mixture (50 \\L) was treated with TACS- Sapphire™, a horseradish peroxidase colorimetric substrate, and incubated in the dark for 30 minutes. Absorbance at 630 nm was read after 30 minutes. The results presented are the average data of at least three independent experiments done in triplicate ± SD (Standard Deviation) . Transport in the blood plasma - in vitro interaction with serum proteins

The possibility of distribution by the blood plasma and the eventual role of blood serum proteins as its possible biological targets was investigated through human serum albumin (HSA) binding. Serum protein binding yields crucial information for the selection of drug candidates, and albumin in particular plays a central role in the transport and bioavailability of metallodrugs.

[RuCp(PPh ₃ ) (2,2'-bipy)] [CF3SO3] interacted with the protein, equilibrium being reached in less than one hour. CD spectroscopy indicated that the chiral protein and the achiral Ru-complex are in close proximity (Figure 12) . Given the extent of quenching observed and the unchanged fluorescence lifetimes measured, the results for this complex are consistent with the formation of a ground-state non fluorescent [Ru-protein] complex (see Figure 13) with a binding constant (log Ks = 4.02) of the same magnitude as ruthenium anticancer agent KP1019 undergoing clinical trials ( Polec-Pawlak, K. et al . , Electrophoresis, 2006, 27, 1128-1135) .

Structural information on albumin binding is obtained by competitive displacement of well-established site-marker drugs (warfarin, benzylpenicillin, drug binding site I; Ibuprofen, Aspirin, drug binding site II) whenever meaningful. Our fluorimetric data indicate that binding of [RuCp (PPh ₃ ) (2, 2' -bipy) ] [CF3SO3] to HSA slightly disturbs the warfarin binding site indicating that the Ru-compound is possibly accommodated in the drug biding site I (Figure 14) .

The formation of the complex [Ru-protein] did not affect appreciably the overall cytotoxicity in the A2780 cell line, suggesting that the Ru ¹¹ compound can be effectively transported in the blood stream without loss of activity (see Figure 5) .

Experiment: Solutions of albumin from human serum (and the same protein in the fatty acid free form) were prepared in 10 mM Hepes buffer pH 7.4 containing 0.15 M NaCl . The protein concentration was obtained from the UV absorption spectrum (s ₂ 8o(HSA) = 36 850 M^cirf ¹ ) (G. H. Beaven, et al . , Eur. J. Biochem., 41 (1974) 539-546). All titration samples were prepared individually. Stock solutions of the ruthenium complexes were prepared in DMSO immediately prior to the incubation with the protein. An adequate concentration was used for the stock solution in each case to ensure the same amount of DMSO (2%) all final samples. For UV-Visible and CD absorption titrations the complex concentration was kept constant (10 - to 100 μΜ, depending on the complex) , and the protein-to-complex molar ratio ranged from 0 to 10. Static fluorescence emission quenching was monitored on independent samples with a constant protein concentration, e.g. for albumin binding, samples contained 1 μΜ HSA and varying HSA-to-ligand ratios (1:0 to 1:15 or 1:30, depending on the complex) . For steady-state measurements the excitation wavelength was 295 nm to excite selectively Trp214 and exclude the contribution from Tyr residues. Additional information on the specific binding to HSA (drug binding site I or II) was monitored by competitive displacement of a site-marker (e.g. warfarin, drug binding site I) pre-incubated with albumin for 30 min. For the site marker displacement experiments, the albumin to site-marker molar ratio was 1:1, and the complex concentration increased until a 15-fold excess; the excitation wavelength was 310 nm and emission was recorded in the range 320-650 nm. Albumin fluorescence quenching by the complex was measured at room temperature after an incubation of 3 and 24h in 10 mM Hepes (pH 7.4) /0.15 M NaCl/2% DMSO. Emission data recorded in the range 310-650 nm was corrected for inner filter effects (A. Coutinho et al . , J. Chem. Ed. (1993) 425-428) . Fluorescence measurements were carried out on a Horiba Jobin Yvon FL-1057 Tau 3 spectrofluorometer at 24.0°C. For time-resolved measurements using the single photon counting technique nanoLED N-280 (Horiba Jobin Yvon) was used for the excitation of albumin, and emission wavelength was 340 nm. LUDOX was used as the scatterer to obtain the instrumental response function. The program TRFA Data Processor v.1.4 (Minsk, Belarus) was used for the analysis of the experimental fluorescence decays. Fluorescence lifetimes were measured for the fluorophore (albumin Trp214) in the presence and absence of the complexes. The fluorescence intensity decays were analyzed by fitting a sum of exponentials,

t

i(t) = a _i cxp

(2)

where a± and τ± are the normalized amplitude and lifetime of component i, respectively. The intensity weighted and amplitude weighted mean fluorescence lifetimes are calculated by (3) and (4), respectively.

¹ (4)

The quality of the fit was evaluated by a reduced-χ value close to 1 and random distribution of weighted residuals and residuals autocorrelation. Description of the drawings

The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of invention.

Figure 1- Shows a single crystal X-ray structure of the cation of compound [RuCp(Dppe) ( 1 , 3, 5-triazine) ] [CF ₃ S0 ₃ ] which is an example of an organometallic compound of the invention showing Li and L2 as a one bidentate phosphane ligand and L3 as an N heteroaromatic possessing 3 N atoms, sigma bonded by one N.

Figure 2 - Shows a single crystal X-ray structure of the cation of compound [RuCp (PPhs) 2 (pyridazine) ] [ CF3 S O3 ] showing Li and L2 as a monodentate phosphane ligands and L3 an N heteroaromatic possessing 3 N atoms, sigma bonded by one N.

Figure 3 - Shows a single crystal X-ray structure of the cation of compound [RuCp(PPh3) (2, 2' -bipy)] [ CF3 S O3 ] where LI is a monodentate phosphane ligand and the two remaining coordination positions occupied by a N heteroaromatic bidentate sigma bonded ligand.

Figure 4 - Cytotoxicity of [RuCp (PPh ₃ ) (2, 2' - bipy) ] [ CF3 S O3 ] on the human tumorigenic cell lines (see Table I), breast cancer MCF-7 (·) , ovarian cancer A2780 (■) and A2780cisR

(A) , prostate cancer PC-3 (T) and on the non-tumorigenic Chinese hamster lung fibroblast V79 (♦) cell lines. Cells were treated with increasing concentrations of

[RuCp (PPh ₃ ) (2, 2' - bipy) ] [ CF3 S O3 ] in the range 10 ^"10 -10 ^"4 M, and the cellular viability was determined after 72 h by the MTT assay. Figure 5 - Effect of HSA on the cytotoxicy of [RuCp(PPh3) (2,2'-bipy)] [CF ₃ SO ₃ ] on the human tumorigenic

A2780 (left) and A2780 cisR (right) ovarian cancer cell lines after a 72 h incubation period at 1 μΜ concentration (the IC5 ₀ value) . Cell viability is little affected by the presence of HSA, especially at low molar excess.

Figure 6 - Atomic force microscopy image of the a) free plasmid pBR322 DNA, b) plasmid pBR322 DNA incubated with the ruthenium complex after 1 min and c) after 30 min.

Figure 7 - Example of a Agarose gel electrophoretic mobility of DNA pBR322 treated with some ruthenium compounds (lane 1-4) ; cisplatin (lane 5) and DNA pBR322 (lane 6) .

Figure 8 - Example of Circular Dichroism spectra of ct-DNA incubated with ruthenium complexes at molar ratios 0.1, 0.3, and 0.5, at 37°C for 24 hours. After 24 hours of incubation at 37°C, changes in molar ellipticity can be observed for all the complexes. These changes in the wavelength and the ellipticity of free DNA indicate modifications on the secondary structure of DNA as consequence of the interaction of the complexes with DNA.

Figure 9 - Interaction with ubiquitin: ESI mass spectra of ubiquitin in the presence of a Ru compound in HEPES lOmM pH7.4, over time. The new m/z signals observed and highlighted in orange indicate an interaction between the Ru and the protein. Figure 10 - Interaction with Cytochrome-C : FT-ICR mass spectra of: a) cytochrome-C in aqueous solution, b) cytochrome-C and Ru compound in aqueous solution after 3h incubation at 37°C, c) cytochrome-C and Ru compound in aqueous solution after 24h incubation at 37°C. An interaction between the Ru and the protein is clearly observed after 24 h (c) , on the left side) .

Figure 11 - PARP-1 inhibition: enzyme inhibition (%) determined for the ruthenium complex [RuCp(PPh3) (2,2'- bipy) ] [CF3S03] on the purified human enzyme incubated for 2 and 24 h with varying Ru-complex concentrations. No difference in the results were observed between the long and short incubation periods, with a IC50= (1.0+0.3) μΜ, which is lower than the one for the classical inhibitor 3- Aminobenzamide (IC50=33 μΜ) (N. J. Curtin, Expert Rev. Mol. Med. 2005, 7) .

Figure 12 - Interaction of [RuCp (PPh3) (2, 2' -bipy) ] [CF3S03] with HSA (fatty acid free form) : Circular Dichroism spectra of individual HSA (fatty acid free) samples in 2% DMSO/10 mM Hepes (pH 7.4) /0.15 M NaCl incubated with increasing Ru- complex:HSA molar ratios (1:1, 1:3, 1:4) for 3h at room temperature. The development of an induced CD signal consisting of two bands with positive Cotton effect in the 310 - 420 nm range indicates that the chiral protein and the achiral complex are in close proximity.

Figure 13 - Interaction of [RuCp (PPh3) (2, 2' -bipy) ] [CF3S03] with HSA: A) Fluorescence emission spectra (Dexc=295 nm) and fluorescence emission intensity (insert) at 380 nm of individual HSA samples in 2% DMSO/10 mM Hepes (pH 7.4) /0.15 M NaCl incubated with increasing Ru-complex : HSA ratios (1:0 to 1:27, 3h, room temperature, CHSA=1.2 μΜ) ; B) Static quenching observed in Fluorescence emission spectra at 380 nm of HSA samples incubated with increasing Ru-complex : HSA ratios (3h in 2% DMSO/ 10 mM Hepes pH 7.4/0.15 M NaCl) and linear fit to experimental data obeying IF0/IF = 1 +KS [Q], where [Q] is [Ru Complex] with Ks = (10.510.5) 103 (R2=0.9856) . Given the extent of quenching observed and the unchanged fluorescence lifetimes measured, fluorescence data for the Ru compound is consistent with the formation of a ground-state non fluorescent [Ru compound-protein] complex with log Ks = 4.02.

Figure 14 - HSA pre-incubated with warfarin - interaction of [RuCp (PPh3) (2, 2' -bipy) ] [CF3S03] : Fluorescence spectral changes and (insert) relative fluorescence emission at 380 nm of HSA-Warfarin incubated with increasing Ru- compound: [HSA-Warf ] ratios for lh (dark blue, 6h) in 2%DMSO/10 mM Hepes (pH 7.4) . Samples were prepared individually, CHSA=1.4 μΜ. Binding of the Ru-compound to HSA slightly disturbs the Warfarin binding site, suggesting that the Ru-compound is interacting with the protein in the drug binding site I.