RUTHENIUM (II) COMPLEXES WITH HIGH ANTITUMORAL AND ANTIMETASTATIC ACTIVITIES

Abstract: The present invention is related to Ruthenium derived antitumoral compounds with high antimetastatic activity and with low toxicity for the host. It is described the use of inorganic Ruthenium complexes in the therapy of solid tumors, in particular of those tumors characterized by high metastatizing ability. Pharmaceutical compositions containing Ruthenium complexes in formulations suitable for the administration and a kit for the preparation of such complexes at the moment of use or immediately before use, are moreover described.

Description: RUTHENIUM (II) COMPLEXES WITH HIGH ANTITUMORAL AND ANTΪMETASTATIC ACTIVITIES Field of the invention The present invention relates to the use in therapy of a class of Ruthenium (II) complexes, specifically for the treatment of tumours characterised by high metastasizing ability. Background Art The search for new antitumoural compounds is in continuous development and is mainly aimed at the identification of new compounds with high selectivity, with high antimetastatic activity and with reduced toxicity for the host. Compounds based on transition metals, such as cisplatin for example, have been used for many years in the chemotherapy of tumours; these products, although pharmacologically active, have a reduced antimetastatic activity and show heavy side-effects caused by the elevated systemic toxicity. Recently, the applicative potentialities of some ruthenium complexes (II) with cytotoxic activity and their possible use in the therapy of neoplastic diseases has been considered. Unfortunately, even if these compounds show a higher tropism for the tumours than the cisplatin derivatives (Sava et al, Anticancer Res. 11 , 1103, 1991 ), they show also toxic effects for the organism. In order to improve the problems related to the activity and toxicity, new Ruthenium (III) complexes have been proposed in the form of more active and less toxic pro-drugs (Sava et al., Anticancer Res. 11 , 1103, 1991 , Sava et al. in Topics in Biological Inorganic Chemistry, 143, 1999) for which an "in vivo" activation mechanism by reduction of the Ruthenium (III) complexes to the corresponding Ruthenium (II) reactive species has been devised. This mechanism has been hypothesized to be more efficient in the hypoxic and reducing environment of the tumour tissue, and this would allow for the high selectivity and activity of the Ruthenium (III) complexes towards solid tumours (Sava et al., Anticancer Res. 11 , 1103, 1991 , Sava et al. in Topics in Biological Inorganic Chemistry, 143, 1999). The reduction process would occur to a lesser extent in healthy tissues which are normally vascula zed and where the partial pressure of oxygen is higher (40 mm Hg) than in the tumour tissue (5 mm Hg). Higher amounts of the more active and toxic species of Ruthenium (II) are formed in the tumour tissue with respect to the healthy tissues, with an accumulation effect resulting in a selective cytotoxicity towards the solid tumour cells. Among the above mentioned Ruthenium (III) complexes, the lmH[trans-RuCI 4 lm 2 ], (B.K. Keppler et al., J. Cancer Res. Clin. Oncol., 111 : 166-168, 1986), the complexes of formula Na[trans-RuCI 4 (Me 2 SO)(L)], (WO 90/13553) proved to be effective in slowing down the growth of the primary tumour and the most recent complexes of formula (LH) [trans-RuCI 4 (Me 2 SO)(L)] (WO98/00431 ) that show a marked antimetastatic activity. However, despite the positive results obtained in clinical applications in chemotherapy, the search in the field of ruthenium derivatives is still open to the identification of new compounds with optimal features of cytotoxicity and selectivity toward the tumour and with a reduced systemic toxicity in order to render the use of these compounds safer and more effective. Summary of the invention The present invention refers to the use in therapy of Ruthenium (II) anionic complexes with antitumour and antimetastatic activity having formula I: wherein, R 1f R 2 , R 3 either the same or different from one another, are selected in the group consisting of: H, C r C 6 alkyl, either linear or branched, saturated or unsaturated, C 3 -C 7 cycloalkyl, aryl; or NR 1 R 2 R 3 is a 5-7-membered nitrogen heterocycle, either saturated or unsaturated, possibly containing one or more O.S, N atoms or N substituted with an alkyl, aryl or benzyl residue; said nitrogen heterocycle can be possibly benzocondensed and/or substituted with C C 4 alkyl, C r C 4 alkoxy, C C 4 alkylthio, aryl or benzyl; the counterion Q + is represented by "NHR^Ra, wherein R 1 R 2 R 3 maintain the aforementioned meanings; R 4 ed R 5 , either the same or different from one another are selected in the group consisting of: H, C C 6 alkyl, C 3 -C 7 cycloalkyl , aryl; or R 4 and R 5 form together with the S atom to which they are bound, a 4-7-membered heterocycle. The Applicant has surprisingly found that the Ruthenium (II) complexes according to the invention, show unexpected antitumour activities in terms of activity and therapeutical index. A further object of the present invention is represented by pharmaceutical compositions containing the Ruthenium (II) complexes of formula I as the active compound, in association with suitable excipients and/or diluents and/or stabilizers. In a further embodiment, the invention provides a kit for the preparation of the Ruthenium (II) compounds having formula I. In a further embodiment the invention provides for the use of the formula (I) Ruthenium (II) complexes to prepare a medicament for the treatment of solid tumors in particular those characterized by high metastatizing properties. Detailed description of the invention Object of the present invention is the use of Ruthenium (II) complexes of formula I for therapeutic purposes: wherein the meanings of R^ R 2 , R 3 , R 4 , R 5 and Q + are hereinafter reported. The R 1; R 2 , R 3 groups, either different or the same, are selected in the group consisting of: H, C C 6 alkyl, either linear or branched, saturated or unsaturated, C 3 -C 7 cycloalkyl, aryl; or NR 1 R 2 R 3 is a 5-7-membered nitrogen heterocycle, either saturated or unsaturated, possibly containing one or more O, S, N atoms, N-substituted with an alkyl, aryl or benzyl residue; said nitrogen heterocycle can possibly be benzocondensed and/or substituted with C r C 4 alkyl, C,-C 4 alkoxy, C C 4 alkylthio, aryl or benzyl. When NR.,R 2 R 3 is a 5-membered nitrogen heterocycle, it is preferably selected in the group consisting of: imidazole, N-methylimidazole, pyrazole and oxazole; more preferably said nitrogen heterocycle is imidazole. When NR.,R 2 R 3 is a 6-membered heterocycle, it is selected preferably in the group consisting of : pyridine, 3,5-lutidine and 4-methylpyridine. When NR^Ra is a 7-membered heterocycle, it is selected preferably in the group consisting of: azepine, diazepine and oxazepine. Finally, when said heterocycle is benzocondensed, it is selected preferably in the group consisting of: indazole, isoquinoline, benzimidazole and 1 ,5,6-trimethyl- benzimidazole. In the complexes according to the invention, Q + represents a NH + R 1 R 2 R 3 nitrogen group wherein R.,, R 2 and R 3 maintain the meanings assigned above. In the complex according to the present invention, the sulfoxide ligand R 4 -SO-R 5 has R 4 and R 5 , either the same or different from one another, selected in the group consisting of: H, C r C 6 alkyl, C 3 -C 7 cycloalkyl, aryl; or R 4 and R 5 form, together with the S atom to which they are bound, a 4-7 membered heterocycle. In the complexes according to the present invention, the sulfoxide ligand R 4 -SO-R 5 is preferably dimethylsulfoxide (R 4 =R 5 =methyl) or diethylsulfoxide (R 4 =R 5 =ethyl). These products can be obtained by reduction of the corresponding Ruthenium (III) complexes, wherein with a corresponding Ruthenium (III) complex, the following formula II compound is meant: Wherein said R 17 R 2 , R 3 , R 4 , R 5 and Q + groups maintain the above mentioned meanings. Formula II compounds are obtainable for example as described in WO 98/00431 herein incorporated as a reference. The Ruthenium (II) complexes according to the invention are surprisingly more active than the known Ruthenium (II) complexes and than the corresponding Ruthenium (III) compounds of formula II, and unexpectedly less toxic than both complexes. In a further embodiment, the present invention provides pharmaceutical compositions containing, as the active component, the Ruthenium (II) complex of formula I, in combination with suitable excipients, diluents or pharmacologically acceptable stabilizers. The pharmaceutical compositions are in the form of solution or suspension, or else in the form of gel, grain powder, tablets, pills, capsules or inserts. The pharmaceutical compositions according to the invention may contain, besides the compounds of formula I, one or more antitumour drugs, such as cisplatin for example, vincristine, vinblastin, 5-fluoruracyl, cyclophosphamide, bleomycin, anthracyclin, taxole or other ruthenium complexes. In a preferred embodiment the pharmaceutical compositions of the invention are prepared at the moment of use or immediately before use by reduction of the corresponding Ruthenium (III) complexes of formula II with reducing agents, selected preferably in the group of physiologically compatible reducing agents. These reducing agents are selected among those with a redox potential wherein the sum of the two semipotentials (the sum of the reduction potential of the complex and the potential of the reducing agent with opposite sign) is positive so as to ensure an effective reduction. Potential measurements are performed as reported in Alessio et al. Inorganica Chimica Acta, 1993, 203: 205-217. In a preferred embodiment the physiologically compatible reducing agents are selected in the group consisting of: ascorbic acid, cysteine and glutathione. Reduction is performed by using a ratio of equivalents between the Ruthenium (III) complex and the reducing agent ranging from 5:1 to 1 :2 (the number of equivalents of a substance participating into a redox reaction has been obtained by dividing the grams of said substance for its equivalent weight. In turn, its equivalent weight has been obtained by dividing the molecular weight of the substance for the variation of the oxidation number). The ratio of equivalents between the Ruthenium (III) complex and the reducing agent is preferably 1. Under these conditions, the Ruthenium (III) reduction to Ruthenium (II) is complete and immediate. As an alternative, the reduction of the Ruthenium (III) complex of formula II can take place in the presence of inorganic reducing agents such as for example Sn 2+ or Ce 3+ or by electrochemistry, or else in the presence of H 2 . In one of its further embodiments, the invention provides a kit for the preparation of the Ruthenium (II) complex of formula I with two components, wherein the first component is the corresponding Ruthenium (III) complex of formula II and the second is a reducing agent, as described above. Both kit components are in the form of solutions present in separate containers to be mixed at the moment of use or immediately before use, or in a solid form as single compounds to be solubilized with suitable solvents when used or immediately before use; as an alternative, the kit is composed of two containers the content of which is to be mixed at the moment of use or immediately before use and wherein the former contains the formula II compound that has been mixed in a dry form with the reducing agent and the latter contains the solvent. The solvents used for the preparation of both the solution of the Ruthenium (III) complex and the reducing agent are aqueous solvents, such as isotonic solutions of 0.9% NaCI or buffered solutions such as phosphate buffer, citrate buffer etc. and are preferably the same in both solutions. Preferably, the Ruthenium (III) complex of formula II is dissolved in a water solution at a concentration ranging from 0.1 to 20 g/l and with a pH ranging from 3 to 8. In a further embodiment the invention refers to the use of the formula I Ruthenium (II) complex of for the preparation of medicaments to be used in the prevention and treatment of tumours and metastases. The use of these compounds according to the invention has proved to be very effective on solid tumours, specifically on those characterized by high metastasising ability, especially those selected in the group consisting of: colon carcinomas or tumours of the gastrointestinal tract, mammary carcinomas, lung tumours or lung metastases from highly invasive tumours. Parenteral, oral, topical or transdermal administration are preferred. Preferably, the Ruthenium (II) complex is prepared at the moment of use or immediately before use, by mixing the corresponding Ruthenium (III) complex with a reducing agent, as previously described. Preferably, the reducing agent is selected in the group consisting of: ascorbic acid, cysteine, glutathione. The mixing can be carried out by mild mechanical stirring or by another method known to the skilled artisan. For the illustrative but not limitative purposes of the invention, the following examples are reported. EXPERIMENTAL PART: Preparation of the formula I Ruthenium (II) complexes. EXAMPLE 1 : Preparation of the Ruthenium (II) complex by reduction of {trans-RuCI 4 (Me 2 SO)(lm)}lmH (lm=lmidazole) with ascorbic acid. To a pH=7.2 buffered 0.15 M NaCI solution of {trans-RuCI 4 (Me 2 SO)(lm)}lmH (lm= imidazole) (10g/L) an equivalent amount of ascorbic acid is added. Soon after the mixing, the solution obtained has been analyzed by means of 1 H-NMR. The recorded spectrum shows the total reduction of the Ruthenium (III) nucleus to Ruthenium (II) with an extremely fast reaction kinetics. The fact that the signal corresponding to Me 2 SO coordinated with ruthenium is read at 3.60 ppm, (whereas the Me 2 SO signal coordinated with the Ruthenium (III) complex must be at -15 ppm) and that its spectral width is equal to 3Hz (while the corresponding value in the spectrum of the Ruthenium (III) complex must be 370 Hz) demonstrated the conversion of the Ruthenium nucleus from paramagnetic to diamagnetic. Moreover, in the spectrum further coordinated imidazolium signals (8.53 ppm, H2; 7.83 and 7.54 ppm H4.H5) and free imidazolium (8.46 ppm, H2; and 7.39 ppm H4, H5) were observed. The signals at 4.83, 4.67, 4,35 ppm corresponded to the signals of the oxidized ascorbic acid. According to the above information, it turned out that the reduction of the ruthenium (III) complex has produced the dianionic species: {trans- RuCI 4 (Me 2 SO)}(lm)} 2" , corresponding to Ruthenium (II). EXAMPLE 2: Preparation of the Ruthenium complex (II) by reduction of the {trans-RuCI 4 (Me 2 SO)(Pyz)}PyzH (Pyz=pyrazine) with ascorbic acid. To an aqueous solution of the Ruthenium (III) complex {trans- RuCI 4 (Me 2 SO)(Pyz)}PyzH}(Pyz=pyrazine) in 0.1 M phosphate buffer pH=7.4, an equivalent amount (1 :1 ) of ascorbic acid has been added. Immediately after mixing, the solution obtained is analyzed by means of 1 H-NMR. The spectrum recorded highlighted the total reduction of the Ru(lll) to Ru(ll) nucleus, accordingly to what observed in the previous example. The multiplet signals at 9.66 and 8.70 ppm of the spectrum highlighted the presence of pyrazine coordinated to the Ruthenium of the {trans-RuCI 4 (Me 2 SO)(Pyz)} 2 -dianonic species. The signals of the free PyzH + free are read at 8.69 ppm. The protons of Me 2 SO coordinated to Ruthenium (II) have been detected at about 3.65 ppm. EXAMPLE 3: Preparation of the Ruthenium (II) complex by reduction of the {trans-RuCI 4 (Me 2 SO)(lm)}lmH with ascorbic acid (equivalent ratio = 2.5:1). An aqueous solution of the Ruthenium {transRuCI 4 (Me 2 SO)(lm)}(lmH) (1 ,832 g/L) containing ascorbic acid (1.41 10 2 g/L) has been prepared. Immediately after the stirring, the solution was transferred in a quartz cuvette (path length: 1 cm) and the UV7VIS spectra was recorded at defined lenghts of time. The decrease in absorbance (Abs) at 390 nm, the main absorption band demonstrated the Ruthenium (III) complex reduction to Ruthenium (II). Biological Activity Assays EXAMPLE 4. In vivo activity assays of the compositions of the ruthenium (II) complexes according to the present invention on mice affected by MCa mammary carcinoma: prevention activity on the formation of lung metastases. Six groups composed of 6 CBA\Lac kin female mice, weighing 23±3 g were inoculated on day 0 with 10 6 viable cells (as determined with Trypan blue- exclusion test) of MCa mammary carcinoma, in 0.05 ml volume of Dulbecco's buffered at pH 7.4 calcium and magnesium-free saline physiological solution (D- PBS), by intramuscular injection with a sterile insulin syringe. The tumour cells derive from donors of the same strain, were transplanted with an insulin sterile syringe accordingly to the same procedure two weeks before. The tumour cells in PBS suspension were prepared by mechanical mincing of the tumour mass obtained from donor mice and by further removing tissue and cell debris by filtration through a double layer of sterile gauze and further centrifugation at 250xg for 10 minutes. The kin animals were derived from a colony obtained from Chester Beatthy, London (UK) and grown in the University of Trieste pound according to the procedures for the rearing of kin animals. The MCa mammary carcinoma tumour line derives from a stock, kept in liquid nitrogen, of tumour cells obtained originally from Rudjer Boskovic Institute, Zagreb (HR). From day 12 to day 17 after tumour implantation, the six groups of mice were treated intraperitoneally using an insulin sterile syringe, respectively with: Group 1. Control: 10 mg/kg body weight/die of sterile pyrogen-free physiological solution; Group 2. ASC: 6.69 mg/kg body weight/die of ascorbic acid in sterile and pyrogen- free physiological solution; Group 3. CYST: 12 mg/kg body weight/die of cysteine in sterile and pyrogen-free physiological solution; Group 4. RUT: 35 mg/kg body weight/die of Ruthenium (III) complex, (lmH)[trans- RuCI 4 (Me 2 SO)(lm)] in pyrogen-free and sterile physiological solution; Group 5. RUT-ASC: 35 mg/kg body weight/die of the Ruthenium (III) complex, (lmH)[trans-RuCI 4 (Me 2 SO)(lm)] and 12 mg/kg body weight/die of cysteine in sterile and pyrogen-free physiological saline solution; Group 6. RUT-CYST: 35 mg/kg body weight/die of the Ruthenium (III) complex, (lmH)[trans-RuCI 4 (Me 2 SO)(lm)] and 12 mg/kg body weight/die of cysteine in sterile and pyrogen-free physiological saline solution. The reducing agents ascorbic acid and cysteine were used in a equivalent ratio corresponding to 1 with the Ruthenium (III) complex. The redox reaction was immediate. The administration took place after analytical control of the complete reduction of the Ruthenium (III) complex to Ruthenium (II). On day 17 from tumour implantation, the growth of the primary tumour was evaluated and the tumor surgically removed after general anaesthesia of the animals with ketamine. The tumor size was determined by measuring both orthogonal axes of the tumour by means of a caliber; the weight in grams of the rotation solid developed around these axes was measured, considering the tumour density equal to 1 and using the formula: (π/6).a 2 .b, wherein a was the minor and b the major axis. On day 21 from tumour implantation, the mice were sacrificed by cervical dislocation and lung metastases were analyzed. The lungs removed from the animal immediately after sacrifice, were divided in single lobes, which were then examined immediately with a low-magnification microscope equipped with an ocular-grid for the measurement of the metastases sizes from which the two orthogonal a and b axes (with a <b) are identified. The metastases were then grouped on the basis of their dimensions and the metastatic tumour weight was calculated as the sum of the weight of each single metastasis, each of them regarded as a rotation solid developed around the above mentioned axes, and with a volume calculated by the same formula used for the primary tumour. The experimental data obtained were therefore processed by appropriate statistical tests. The results are reported in Table 1 wherein the number and weight of lung metastases obtained in each group of animal are shown. Table 1 weight in the controls; a.p./a.t.: number of animals devoid of metastases/ total number of animals. The data reported in Table 1 show that cysteine (group 3) and ascorbic acid (group 2) when used alone do not produce any relevant effect on lung metastases, while the Ruthenium (III) complex (group 4) and the compositions of the Ruthenium (II) complexes according to the invention (groups 5 and 6) caused a marked reduction in the number and an even more marked decrease in the weight of lung metastases with respect to the controls. It is evident that the compositions of Ruthenium (II) according to the inventions (groups 5 and 6) have a more pronounced effect than the corresponding Ruthenium (III) (group 4) on the number of metastases. With regard tothe metastases weight, the stronger effect was seen in the compositions containing cysteine as a reducing agent (group 6). It is moreover relevant that, with respect to the treatment with the Ruthenium (III) complexes according to the prior art (group 4), the use of Ruthenium (II) complexes according to the invention (groups 5 and 6) prevented the formation of metastases as shown by the ap/at ratio between animals devoid of metastases and number of total animals with respect to the control treatments and as confirmed by statistical analysis (Fisher test) . EXAMPLE 5. In vivo activity assay of the compositions according to the present invention on mice affected by MCa mammary carcinoma: prevention activity in the formation of lung metastases. In a further experiment similar to the previous one, six groups of animals, each of them composed of 7 CBA Lac kin female mice, weighing 23±3 g were inoculated on day 0 with cells of MCa mammary carcinoma, according to the protocol described in the previous example. From day 10 to day 15 after tumour implantation, the 6 groups of mice were treated intraperitoneally using a sterile insulin syringe, respectively with: Group 1 : Control: 10 ml/kg body weight/die of sterile and pyrogen-free physiological solution; Group 2: GLU: 23.5 mg/kg body weight die of glutathione in sterile and pyrogen- free physiological solution; Group 3: CYST: 12 mg/kg body weight/die of cysteine in sterile and pyrogen-free saline physiological solution; Group 4: RUT: 35 mg/kg body weight/die of Ruthenium (III) complex, (lmH)[trans- RuCI 4 (Me 2 SO)(lm)] in sterile and pyrogen-free physiological saline solution; Group 5: RUT-GLU: 35 mg/kg body weight/die of Ruthenium (III) [trans- RuCI 4 (Me 2 SO)(lm)] and 23.5 mg/kg body weight/die of glutathione in sterile and pyrogen-free saline physiological solution; Group 6: RUT-CYST: 35 mg/kg of body weight/die of Ruthenium (III) complex (lmH)[trans-RuCI 4 (Me 2 SO)(lm)] and 12 mg/kg body weight/die of cysteine in sterile and pyrogen-free saline physiological solution. The reducing agents glutathione and cysteine were used in an equivalent ratio of 1 with the Ruthenium (III) complex. The redox reaction was immediate. The administration took place after the analytical control of total reduction of the Ruthenium (III) to Ruthenium (II) complexes. On day 16 from tumour implantation, the growth of the primary tumour as described in the previous example was evaluated. On day 23 from tumour implantation, the mice were sacrificed and the metastases were evaluated as in previous example. The experimental data obtained were processed by appropriate statistical tests and reported in Table 2, wherein the number and weight of lung metastases obtained in the treated group of animals are also reported. Table 2 %T/C= percentage of the metastases weight in the treated animals/metastases weight in the controls. From the data shown in Table 2, the antimetastatic effect of the Ruthenium (II) complexes according to the invention (groups 5 and 6) is clearly seen in respect to the effect of the corresponding compound of Ruthenium (III) (group 4); this effect refers both to the reduction of the number of metastases and, even more significantly, to the reduction of their weight. EXAMPLE 6: In vivo activity assays of the Ruthenium (III) and Ruthenium (II) complexes of the state of the art, in BD2F1 mice affected by Lewis lung carcinoma (LLC). Groups of 7 BD2F1 female mice were inoculated by means of intramuscular injection on day 0 with cells of Lewis lung carcinoma (LLC). From day 8 to day 13 the animals were treated with the Ruthenium (III) complex [trans- RuCI 4 (DMSO)(lm)](lmH), (compound of formula II) and with the Ruthenium (II) complex, trans-RuCI 2 (DMSO) 4 , both belonging to the state of the art. On day 20 from tumour implantation, the mice were sacrificed and the metastases evaluated. All the procedures of the experiments were carried out as reported in the previous examples. The experimental data obtained, processed by appropriate statistical tests are shown in Table 3. Table 3 animals/metastases weight or number in the controls. The data reported in Table 6, show that the Ruthenium (III) complex of formula II ([trans-RuCI 4 (DMSO)(lm)] (ImH)) belonging to the state of the art is more effective in the reduction of lung metastases than the Ruthenium (II) complex (trans- RuCI 2 (DMSO) 4 ) of the state of the art. Toxicity assays. EXAMPLE 7: Effects of the in vivo treatment of the compositions according to the present invention on mice affected by MCa mammary carcinoma. Four groups, each of them composed of 7 CBA Lac-kin female mice weighing 23±3 g on day 0 were inoculated with MCa mammary carcinoma cells according to the protocol described in the previous examples. From day 11 to day 16 after tumour implantation, the four groups of mice were treated intraperitoneally using a sterile insulin syringe, respectively with: Group 1 : Control: 10 ml/kg body weight/die of sterile and pyrogen-free physiological solution; Group 2: CYST: 2 mg/kg body weight/die of cysteine in sterile and pyrogen-free physiological solution; Group 3: RUT: 35 mg/kg body weight/die of Ruthenium (III) complex (lmH)[trans- RuCI 4 (Me 2 SO)(lm)] in sterile and pyrogen-free physiological solution; Group 4: RUT-CYST: 35 mg/kg body weight/die of Ruthenium (III) complex , (lmH)[trans-RuCI 4 (Me 2 SO)(lm)] and 12 mg/kg body weight/die of cysteine in sterile and pyrogen-free physiological solution. The reducing agent (cysteine) was used in a equivalent ratio of 1with the Ruthenium (III) complex. The redox reaction was immmediate. The administration took place after analytical control of the complete reduction of the Ruthenium (III) to Ruthenium (II) complexes. On day 17 from tumour implantation, the mice were sacrificed, the primary tumour and the spleen were removed and analysed as in the previous example. The results are reported in Table 4 which shows: the weight variation of the primary tumour (% variation with respect to the controls) from day 11 to day 17, the animal weight on day 11 and 17, the body weight variation (expressed in percentage with respect to the controls) and the spleen weight on day 17. Table 4 Treatment % Variation Body Body % Spleen of the primary weight Weight variation weight tumour (day 11) (day 17) (day 17) 1 Controls - 31.4±0.5 31.0±0.7 - 273± 13.3 2 CYST - 6.4 30.5±0.6 28.8±0.6 -2.5 297± 3.3 3 RUT -30.3 30.0±1.0 26.5±0.8 -8.8 197± 8.8 4 RUT+CYST -13.6 30.8±0.6 29.0±0.4 -2.3 282± 20.3 5 The data reported in the Table 4 indicate how the body weight and the spleen weight, which are indicators of the systemic toxicity of the treatment, turned out to be more similar to the control (group 1) when the mice were treated with the Ruthenium (II) complexes according to the invention (group 4) than when treated with Ruthenium (III) (group 3) only. These data are in agreement with a lower 0 systemic toxicity of the Ruthenium (II) complexes according to the invention with respect to the Ruthenium (III) complexes of corresponding formula. EXAMPLE 8. Effects of the in vivo treatment of the compositions according to the present invention in mice affected by MCa mammary carcinoma. Four groups each composed of 6 CBA Lac kin female rats weighing 23+3 g. were 5 inoculated on day 0 with tumour cells according to the protocol described in the previous examples. From day 12 to day 17, after tumour implantation, the four groups of mice were treated intraperitoneally using a sterile insulin syringe, respectively with: Group 1 : Control: 10 ml/kg body weight/die of sterile and pyrogen-free 0 physiological solution; Group 2: ASC: 6.69 mg/kg body weight/die of ascorbic acid in sterile and pyrogen- free physiological solution; Group 3: RUT: 35 mg/kg body weight/die of Ruthenium (III) complex, (lmH)[trans- RuCI 4 (Me 2 SO)(lm)] in sterile and pyrogen-free physiological solution; Group 4: RUT+ASC: 35 mg/kg body weight/die of Ruthenium (III) complex (lmH)[trans-RuCI 4 (Me 2 SO)(lm)] and 6.69 mg/kg body weight/ die of ascorbic acid in sterile and pyrogen-free physiological saline solution. The reducing agent (ascorbic acid) was used in a equivalent ratio of 1 with the Ruthenium (III) complex. The redox reaction was immediate. The administration took place after analytical control of the complete reduction of the Ruthenium (III) complex to Ruthenium (II). On day 18 from tumour implantation, the primary tumour and the spleen were removed and weighed, as described previously. The results are reported in Table 5 which shows: the weight variation of the primary tumour (variation in % with respect to the controls) from day 12 to day 18, the animal weight on days 12 and 18, and the body weight variation (variation in % with respect to the controls). Table 5 The experiment confirms the lower toxicity of the Ruthenium (II) complexes of the invention (group 4) with respect to the prior art Ruthenium (III) complexes (group 3). It is evident from the data reported in Tables 4 and 5 that the compositions according to the invention are less toxic than the corresponding Ruthenium (III) complexes of formula II. As a matter of fact, not only the decrease in body weight is lower, but also the spleen weight is substantially unaltered, contrary to what occurs in group 3 with the Ruthenium (III) compound. As the result of all the activity and toxicity tests performed, it is therefore possible to conclude that the Ruthenium (II) compounds of the invention, are markedly more active than the corresponding Ruthenium (III) complexes and than the Ruthenium (II) complexes belonging to the state of the art. Moreover, unexpectedly, the complexes according to the invention are endowed with a lower systemic toxicity contrarily to what expected from the state of the art. Altogether the results show that a new therapeutical alternative for the treatment of tumours with a higher and more selective efficacy and with lower systemic toxicity with respect to all known treatments based on ruthenium complexes is herein disclosed.

Claims: CLAIMS 1. A Ruthenium (II) complex of formula I: wherein, R 1( R 2 , R 3 , either the same or different from one another, are chosen in the groups consisting of: H, C C 6 alkyl either linear or branched, saturated or unsaturated, C 3 -C 7 cycloalkyl, aryl; or is a 5-7 membered nitrogen heterocycle, saturated or unsaturated, containing possibly 1 or more atoms selected in the group consisting O, S and N or N substituted with a C r C 4 alkylthio residue, aryl or benzyl; said nitrogen heterocycle being possibly benzocondensed and/or substituted with C C 4 alkyl, C,-C 4 alkoxy, C,-C 4 alkylthio, aryl or benzyl; and wherein: Q + = NH + R 1 R 2 R 3 , wherein R R 2 and R 3 maintain the above mentioned meanings; and wherein: R 4 and R 5 , either the same or different from one another, are selected in the group consisting of: H, C C 6 alkyl, C 3 -C 7 cycloalkyl, aryl; or R 4 and R 5 form together with the S atom to which they are bound, a 4-7-membered heterocycle, for use in therapy. 2. The complex according to claim 1 , wherein R 1f R 2 and R 3 are selected in the group consisting of: H, ethyl. 3. The complex according to claim 1 , wherein NR 1 R 2 R 3 is a 5-membered nitrogen heterocycle selected in the group consisting of: imidazole, N-methylimidazole, pyrazole and oxazole. 4. The complex according to claim 1 , wherein NR^Ra is a 6-membered heterocycle selected in the group consisting of: pyridine, 3,5-lutidine and 4- methylpyridine. 5. The complex according to claim 1 , wherein NR^Ra is a 7-membered heterocycle selected in the group consisting of: azepine, diazepine and oxazepine. 6. The complex according to claim 1 , wherein NR^Ra is a benzocondensed nitrogen heterocycle selected in the group consisting of: indazole, isoquinoline, benzimidazole and 1 ,5,6-trimethyl-benzimidazole. 7. The complex according to anyone of claims 1-6, wherein R 4 -SO-R 5 is selected in the group consisting of: dimethylsulfoxide and diethylsulfoxide. 8. The complex according to any of claims 1 , 3 and 7, wherein is imidazole, Q + is imidazolium and R 4 -SO-R 5 is dimethylsulfoxide. 9. The complex according to any of claims 1 , 2 and 7, wherein NR.,R 2 R 3 is imidazole, Q + is ammonium and R 4 -SO-R 5 is dimethylsulfoxide. 10. Pharmaceutical compositions containing the Ruthenium (II) complex as the active compound, as defined in anyone of claims 1-9, in combination with suitable excipients and/or diluents and/or pharmacologically acceptable stabilizers. 1 1. The pharmaceutical composition according to claim 10, characterised in that it is in the form of solution or suspension. 12. The pharmaceutical composition according to claim 10, characterised to be in the form of: gel cream, granular powder, tablet, pill, capsule or insert. 13. The pharmaceutical composition according to claim 10, wherein said Ruthenium (II) complexes are in combination with one or more antitumour drugs. 14. The pharmaceutical composition according to anyone of claims 10-13, preparable immediately before use by mixing the corresponding Ruthenium (III) complex with a physiologically compatible reducing agent. 15. The pharmaceutical composition according to claim 14, wherein the reducing agent is selected in the group consisting of: ascorbic acid, cysteine and glutathione. 16. The pharmaceutical composition according to any of claims 14 and 15, wherein the Ruthenium (III) complex is used in a ratio of equivalents with the reducing agent ranging from 5:1 to 1 :2. 17. The pharmaceutical composition according to Claim 16, wherein the ratio of equivalents between the Ruthenium (III) complex and the reducing agent is 1. 18. A kit for the preparation of the Ruthenium (II) complex as defined in anyone of claims 1-9, wherein the first component is the corresponding Ruthenium (III) complex and the second is a reducing agent. 19. The kit according to claim 18, wherein both components are present in a solid form in separate containers to be mixed and solubilised immediately before use in a suitable solvent. 20. The kit according to claim 18, wherein both components are present in the form of solutions to be mixed immediately before use. 21. The kit according to claim 18, wherein the reducing agent is selected in the group consisting of: ascorbic acid, glutathione and cysteine. 22. The kit according to anyone of claims 18-21 , wherein both components are present in a ratio of equivalents of the Ruthenium (III) complex with the reducing agent ranging from 5:1 to 1 :2. 23. The kit according to claim 22, wherein the ratio of equivalents is 1. 24. Use of a Ruthenium (II) complex as defined in anyone of claims 1-9, for the preparation of medicaments for the prevention and treatment of tumours and metastases. 25. The use according to claim 24, wherein said tumours are solid and characterised by a high metastasising ability. 26. The use according to claim 25, wherein said tumours are selected in the group consisting of: colon carcinomas, mammary carcinomas, lung tumours and lung metastases from metastatic tumours. 27. The use according to anyone of claims 24-26, wherein the administration is parenteral, oral, topical or transdermal. 28. The use according to anyone of claims 24-27, wherein the Ruthenium (II) complex is prepared immediately before use, by mixing the corresponding Ruthenium (III) complex with a reducing agent. 29. The use according to claim 28, wherein the reducing agent is selected in the group consisting of: ascorbic acid, cysteine and glutathione.

Inventors: ALESSIO ENZO, BERGAMO ALBERTA, IENGO ELISABETTA, MESTRONI GIOVANNI, SAVA GIANNI, ZORZET SONIA, ALESSIO, ENZO, BERGAMO, ALBERTA, IENGO, ELISABETTA, MESTRONI, GIOVANNI, SAVA, GIANNI, ZORZET, SONIA
Applicants: ALESSIO ENZO, BERGAMO ALBERTA, IENGO ELISABETTA, MESTRONI GIOVANNI, SAVA GIANNI, SIGEA SRL, ZORZET SONIA, ALESSIO, ENZO, BERGAMO, ALBERTA, IENGO, ELISABETTA, MESTRONI, GIOVANNI, SAVA, GIANNI, SIGEA S.R.L., ZORZET, SONIA
Application year: 2000
Main classification: C07F15
IPC codes: A61K9, A61K31, A61K45, A61K47, A61P35, A61P43, C07C317, C07F15