Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7135—Compounds containing heavy metals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

• the present invention relates to uses or methods for the treatment of proliferative pathologies, in particular cancers, using ruthenium compounds and compositions containing them.
• Platinum complexes are known to have significant antitumor activity. The best known of these is cisplatin, which is commonly used for the clinical treatment of many cancers. The resistance of certain cancer cells and the intrinsic toxicity of platinum are some of the problems encountered when using this compound. Since the 1970s, research has intensified to find molecules that can replace cisplatin and in recent years ruthenium-containing compounds have emerged as an interesting alternative to those containing platinum. Some ruthenium complexes have already been described as being an alternative for cancer treatments.
• the present invention thus describes ruthenium compounds which have particularly advantageous anti-tumor properties.
• These compounds are organometallic compounds, ie they contain at least one covalent carbon-metal bond (CM, M being ruthenium).
• CM covalent carbon-metal bond
• N-Ru intramolecular nitrogen-ruthenium
• ruthenium is therefore part of a cyclic entity and this class of compounds is commonly called by the chemists of the discipline the class of cyclometallic compounds.
• the cyclic entity containing ruthenium is called a metallocycle.
• the ruthenium is therefore bound to an organic ligand both by a covalent carbon-ruthenium bond and by two donor-acceptor bonds (acid-Lewis base, or coordination link) nitrogen-ruthenium.
• the existence of such a metallocycle in an organometallic molecule gives it particular properties in terms of reactivity and thermodynamic stability.
• Various types of carbon atoms aromatic, benzylic or aliphatic can be metallated and the nature of the bond between the donor atom (nitrogen) and the carbon atom can be varied in many ways.
• the subject of the present invention is therefore a pharmaceutical composition
• a pharmaceutical composition comprising, in a pharmaceutically acceptable carrier, at least one ruthenium complex compound, of the following general formula:
• Li, L 2 , L 3 which may be identical or different, each represent a donor ligand of 2 electrons by a nitrogen, oxygen, phosphorus or sulfur atom, or a halogen atom
• Xi and X 2 different from each other, represent one, a nitrogen atom and the other, a carbon atom, between Xi and X 2 , represented by a curved line, there is a succession of atoms forming, with X 1 , X 2 and R 1 represented on the formula, a ring which consists of 5 to 8 atoms, and between N and X 1, represented by the other curved line, there is a succession of atoms forming with the nitrogen atom, Xi and Ru represented in the formula, a ring which consists of 5 to 8 atoms.
• the term "pharmaceutically acceptable carrier” means substances such as excipients, vehicles, adjuvants, buffers which are conventionally used, in combination with the method (s) active (s), for the preparation of a medicament.
• the choice of such supports depends essentially on the intended route of administration.
• the compounds of the invention may be in the form of pharmaceutically acceptable salts, solvates and / or prodrugs.
• the pro-drugs are variants of the compounds of the invention which can be converted in vivo into compounds of the general formula according to the invention.
• halogen atom is meant a fluorine, chlorine, bromine or iodine atom.
• the halogen atom is a chlorine atom.
• ligands for Li, L 2 and / or L 3 include, in particular, nitrile ligands, for example ligands of formula (d) alkylCN (in particular
• CH 3 CN CH 3 CN
• pyridine ligands optionally substituted on one or more carbon atoms of the pyridine rings, with a halogen atom, an alkyl, aryl, hydroxyl, alkoxyl (O-alkyl) or aryloxyl (O-aryl) radical.
• primary amines C 1-6 alkyl NH 2 , such as methylamine or ethylamine.
• Binding ligands of two electrons by a phosphorus atom include phosphine ligands.
• they are of formula P (Ph) 3-x (alkyl) x , where x represents 0, 1 or 2, preferably x represents 2, and Ph represents the phenyl group.
• P (Ph) (CH 3 ) 2 mention may especially be made of P (Ph) (CH 3 ) 2 .
• alkyl denotes a linear or branched hydrocarbon radical, saturated or unsaturated, advantageously having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and the like.
• alkyl groups may be substituted with an aryl group, in which case it is referred to as an arylalkyl group.
• arylalkyl groups include benzyl and phenethyl.
• the alkyl or arylalkyl groups may optionally have one or more substituents, chosen in particular from a halogen atom, an alkyl, aryl, hydroxyl, alkoxyl (O-alkyl), aryloxyl (O-aryl), carboxylic acid, ester (CO) radical.
• the "aryl" groups are mono-, bi- or tri-cyclic aromatic hydrocarbon systems, optionally interrupted by at least one heteroatom (in particular O, S or N).
• the aryl groups include monocyclic or bicyclic aromatic hydrocarbon systems having from 6 to 18 carbon atoms, more preferably 6 carbon atoms. Mention may be made, for example, of phenyl, naphthyl and biphenyl groups.
• the aryl groups include pyridyl, imidazoyl, pyrrolyl and furanyl rings.
• the aryl groups may optionally have one or more substituents, chosen in particular from a halogen atom, an alkyl, aryl, hydroxyl, alkoxyl (O-alkyl), aryloxyl (O-aryl), carboxylic acid, ester (C0 2 -alkyl) radical.
• thiol, thioether (S-alkyl), sulfinic acid, sulphonic acid, nitro, nitroxyl, amine (N (alkyl or aryl) x H 2- x where x is an integer which may be 0, 1 or 2), trialkylammonium ( N (alkyl or aryl) y H 3 -y + where y is an integer which may be 0, 1, 2 or 3), hydroxylamine (N (OH) z (alkyl or aryl) 2-z where z is an integer which may be 1 or 2), hydrazine, azo (N N- (alkyl or aryl)), diazonium, amide (CO-NH w (alkyl or aryl) 2-w where w is an integer which may be 0, 1 or 2) or cyano.
• two or three of the groups Li, L 2 and L 3 may be connected to one another by at least one covalent bond.
• the compounds of the invention have one, two or three Li, L 2 and L 3 groups representing a donor ligand of two electrons per nitrogen atom.
• Li, L 2 , and / or L 3 can represent, taken alone, in pairs, or in threes, a pyridine, bipyridine, phenanthroline or terpyridine group, said groups being optionally substituted, in particular by at least one a halogen atom, an alkyl, aryl, hydroxyl, alkoxyl (O-alkyl), aryloxyl (O-aryl), carboxylic acid, ester (CO 2 -alkyl), thiol, thioether (S-alkyl), sulfinic acid , sulfonic acid, nitro, nitroxyl, amine (N (alkyl or aryl) x H 2-x where x is an integer which may be 0, 1 or 2), trialkylammonium (N (alkyl or aryl)
• the compounds according to the invention are such that the groups Li, L 2 and L 3 together form a donor ligand of two electrons per nitrogen atom, for example the groups Li, L 2 , and L 3 may together form terpyridine or 2- (2-pyridyl) -1,10-phenanthroline, optionally substituted, on one or more carbon atoms of its pyridine rings, with at least one halogen atom, an alkyl radical , aryl, hydroxyl, alkoxyl (O-alkyl), aryloxyl (O-aryl), carboxylic acid, ester (CO 2 -alkyl), thiol, thioether (S-alkyl), sulfinic acid, sulfonic acid, nitro, nitroxyl, amine (N (alkyl or aryl) x H 2-x where x is an integer which may be 0, 1 or 2), trialkylammonium (N (alkyl or aryl) y H 3 -
• the substituent is an aryl radical, preferably phenyl, advantageously itself substituted by a methyl or methoxyl radical.
• aryl radical preferably phenyl, advantageously itself substituted by a methyl or methoxyl radical.
• phenanthroline preferably phenyl
• terpyridine advantageously itself substituted by a methyl or methoxyl radical.
• 2- (2-pyhdyl) -1,10-phenanthroline are as defined below, corresponding respectively to the ligands 5, 6, 7 and 8:
• Y " in the compounds of the invention is a counterion and is only present in the compound when the ruthenium complex bears a positive charge.
• Y " is preferably a weakly nucleophilic anion, such as for example BF 4 " , B (C 6 Hs) 4 “, PF 6", CF 3 SO 3 “, tosylate (p-tolylSO 3"), mesylate (MeSO 3 "), SO 4 2", CF 3 CO 2 “, CH 3 CO 2” , bicarbonate (HCO 3 " ), CIO 4 “ , or NO 3 “ , in particular PF 6 " .
• the ligands Li, L 2 and L 3 are so-called ancillary ligands, the presence of which is only necessary to complete the coordination sphere of ruthenium. It seems that their nature does not affect in a significant way the biological activity of the claimed compounds, this being mainly related to the nature of the metallocycle (s).
• m is equal to 1.
• the curved line, with Xi, X 2 and Ru in the general formula, represents a cycle.
• This ring generally consists of 5 to 8 atoms (including the atoms represented by X 1 , X 2 and Ru in formula (I)), preferably 5 to 6 atoms, and even more preferably 5 atoms.
• the atoms of said ring (other than those represented in the general formula) are chosen from the atoms of carbon, nitrogen, oxygen and sulfur.
• these atoms are only carbon atoms.
• Each of these atoms can independently of said cycle form linear or cyclic structures, saturated or not, for which there are no particular limitations.
• the curved line, with Xi, N and Ru in the general formula, represents another cycle whose Ru-Xi bond is common to the first cycle.
• This ring generally consists of 5 to 8 atoms (including the atoms represented by X-1, N and Ru in formula (I)), preferably from 5 to 6 atoms, and even more preferably 5 atoms.
• the atoms of said ring (other than those represented in the general formula) are chosen from carbon, nitrogen, oxygen and sulfur atoms.
• these atoms are only carbon atoms.
• Each of these atoms can independently of said cycle form linear or cyclic structures, saturated or not, for which there are no particular limitations.
• the compounds of the invention also include optical and geometric isomers, taken individually or as a mixture (especially racemates), of compounds of formula (I).
• the composition according to the invention comprises at least one of the compounds (1) to (12).
• the compounds (8) and (9) are synthesized by metallation of the ligands 2,6-bis (2'pyridyl) -4-carbomethoxybenzene and 2,6-bis (2'pyridyl) -4-methylbenzene respectively.
• the invention also relates to a ruthenium compound, characterized in that it is chosen from compounds (8) and (9):
• compositions according to the invention are particularly advantageous for treating diseases related to cellular hyperproliferation, in particular cancers.
• Cancers include those with solid or liquid tumors.
• the cancers correspond in particular to glioblastomas, neuroblastomas, promyelocytic leukemias, cancers of the prostate, ovaries, lungs, breasts, digestive tract, in particular of the liver, pancreas, head and neck, colon, non-Hodgkin's lymphoma and melanoma.
• the subject of the present invention is also a compound of formula (I), as defined above, for use in the treatment of diseases related to cellular hyperproliferation, in particular cancers.
• the subject of the present invention is also the use of at least one compound of formula (I), as defined above, in the context of the preparation of a pharmaceutical composition intended to treat diseases linked to cellular hyperproliferation. , especially cancers.
• the compounds of the invention exhibit an antiproliferative effect against tumor cells. They are particularly useful for treating cancers by accumulation of tumor cells in G0 / G1 or G2 / M phase and possibly by inducing apoptosis or another type of cell death of tumor cells.
• the compounds of the invention appear in particular capable of accumulating tumor cells in G0 / G1 or G2 / M phase and thus by blocking their cell cycle, but also seem capable of generating their death rapidly, especially when their concentration is increased, sign of a dose-dependent toxicity.
• compositions according to the invention are particularly advantageous for treating tumors resistant to cisplatin or other anticancer drugs.
• compositions according to the invention can be administered in different ways and in different forms.
• they can be administered systemically, orally, by inhalation or by injection, for example intravenously, intramuscularly, subcutaneously, trans-dermally, intra-artery etc., the intravenous routes, intramuscular, subcutaneous, oral and inhalation being preferred.
• the compositions are generally in the form of liquid suspensions, which can be injected by means of syringes or infusions, for example.
• the compounds are generally dissolved in saline, physiological, isotonic, buffered, etc. solutions compatible with pharmaceutical use and known to those skilled in the art.
• compositions according to the invention may contain one or more agents or vehicles chosen from dispersants, solubilizers, stabilizers, preservatives, etc.
• Agents or vehicles that can be used in liquid and / or injectable formulations include methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, etc.
• compositions may also be administered in the form of gels, oils, tablets, suppositories, powders, capsules, aerosols, etc., possibly by means of dosage forms or devices providing sustained and / or delayed release.
• an agent such as cellulose, carbonates or starches is advantageously used.
• the injection rate and / or the injected dose may be adapted by those skilled in the art depending on the patient, the pathology concerned, the mode of administration, etc.
• the compounds of the invention are administered at doses ranging between 0.1 ⁇ g and 100 mg / kg of body weight, more generally between 0.01 and 10 mg / kg, typically between 0.1 and 10 mg / kg. kg.
• repeated injections can be performed.
• sustained release and / or delayed release systems may be advantageous.
• the invention also relates to a method for treating a pathology related to cellular hyperproliferation, in particular cancer, by administering to a subject afflicted with such a pathology a composition according to the invention.
• benign tumors Among the other types of cell hyperproliferation whose treatment may be envisaged according to the invention may be mentioned benign tumors.
• the term "treatment” refers to the preventive, curative and palliative treatment, as well as the management of the patients (reduction of the suffering, improvement of the lifespan, slowing of the progression of the disease , reduction of tumor growth, etc.).
• the treatment may also be carried out in combination with other chemical or physical agents or treatments (chemotherapy, radiotherapy, gene therapy, etc.).
• the treatments and medicaments of the invention are particularly intended for humans.
• the compounds according to the invention can be advantageously used in combination with an anti-cancer treatment involving radiation, such as radiotherapy.
• compositions according to the invention can be used with other chemical agents or therapeutic anticancer treatments, such as the following therapeutic chemical agents: cisplatin, carboplatin, taxotere or taxol, advantageously taxol.
• therapeutic chemical agents such as the following therapeutic chemical agents: cisplatin, carboplatin, taxotere or taxol, advantageously taxol.
• the compounds of the invention are preferably packaged and administered in a combined, separate or sequential manner with respect to other therapeutic agents or treatments.
• the present invention also relates to a method for inhibiting the proliferation of tumor cells in vivo, in vitro or ex vivo, comprising contacting said tumor cells with a composition according to the invention.
• the tumor cells can be in particular those of the pathologies specified above.
• RDC means "compound derived from ruthenium (Ruthenium Derived Compound)”.
• FIG. 1 Examples of compounds of formula (I)
• FIGURE 2 MTT test results for A172 cells for compounds 1, 2 and 3.
• the graphs are an average of 8 points with standard deviations over one out of four representative experiments performed.
• the horizontal and thick line indicates the IC-50 for each chart.
• FIG. 3 MTT test results for A172 cells for compounds 4 to 12: percentage of cell viability as a function of the concentration ( ⁇ M) of the compound in question. The horizontal and thick line indicates I 1 IC 5 O for each graph.
• FIGURE 4 Cell cycle profile of HCT118 cells treated with compounds 8 and 9 at different concentrations.
• FIG. 5 Expression of the CHOP Protein and Phosphorylation of the H2AX Histone at the Serine 137 Site for A172 Tumor Cells Treated with Compound 8 or Compound 9, and Comparison with Cells Treated with the Reference Compound
• FIGURE 6 Graph showing tumor volume (in mm 3 ) as a function of the number of days of treatment with compound 9 of F10B16 tumor-bearing mice.
• FIG. 7 Graph showing tumor volume (in mm 3 ) as a function of the number of days of treatment with compound 8 of F10B16 tumor-bearing mice.
• the cyclometallic compounds are sensitive to oxygen in the air and acids, especially during the phase of metallation of the ligand by ruthenium. It is therefore advisable to carry out the manipulations in a controlled atmosphere (nitrogen or argon) using the schlenk tube technique.
• the solvents are dried and distilled under nitrogen (or argon) before use.
• Example 2 Analysis of the Cytostatic and Cytotoxic Effects of Derivatives of Ruthenium on Crops of Human Glioblastoma and Colon Carcinoma Cells
• the first step in the characterization of the anticancer effects of ruthenium-derived compounds is to test their activity on lines maintained in culture and compare these effects on lines that have different characteristics of resistance to anticancer treatments.
• a human glioblastoma line (A172) and a colon cancer line (HCT116) were used to test the cytostatic effects of the compounds according to the invention.
• Cisplatin was chosen as a cytotoxic comparator.
• an MTT assay was used to measure the activity of a mitochondhal enzyme, which gives an estimate of the number of cells.
• MTT test The experiments are carried out under a vertical laminar flow hood.
• Trypsin-EDTA 0.25% trypsin in 1 mM Na 4 (EDTA)
• FCS are stored at -15 ° C and thawed before use.
• MTT 4,5-dimethylthiazol-2-diphenyltetrazolium bromide
• MTT 4,5-dimethylthiazol-2-diphenyltetrazolium bromide
• MTT 4,5-dimethylthiazol-2-diphenyltetrazolium bromide
• MTT 4,5-dimethylthiazol-2-diphenylt
• HCT-116 or glial (A-172) cells were purchased from European Type Culture Collection, and placed in a 37 ° C, 5% CO 2 incubator in round Petri dishes (diameter 10 cm) with 10 ml of medium. When they are quite numerous (70% confluence), they are washed with PBS at room temperature and then mixed with 1.5 ml of Trypsin-EDTA to unhook them from the petri dish. This cell suspension is placed in culture medium heated to 37 ° C., then this solution is spread in 96-well cell culture plates (100 ⁇ L / well) which is incubated for 24 to 48 hours, until reach a cell confluence of 50%.
• the medium is renewed by cell medium containing different concentrations of RDCs and cisplatin at 37 ° C, which is incubated. After 48 hours, the medium is replaced by a solution at 37 ° C. of MTT in medium, which is placed in the incubator for at least one hour or until violet crystals resulting from the complexation of the MTT are formed quantitatively at the bottom of each well. Finally, this medium is replaced by 100 ⁇ l / well of a 0.04 M HCl / 1 PrOH solution at room temperature to dissolve the crystals. A reading of the optical density of the solutions obtained is made. The optical densities of the RDC or cisplatin treated wells are compared with those of untreated wells (controls).
• One manipulation is to treat 4 plates (3 X RDC and cisplatin). Each plate contains a single product at different concentrations. 9 columns are treated at 50, 20, 15, 10, 7.5, 5, 2.5, 1 and 0.2 ⁇ M and 3 columns are left as controls. The control column located at each end of the plate is not taken into account. The third column control is taken into account for calculations.
• A172 cells were cultured in 96-well plates in DMEM medium with 10% calf serum. At 50% confluency, the cells were treated for 48 hours with cisplatin (cisp) or the various compounds derived from ruthenium at the indicated concentrations (1, 5, 15, 50 ⁇ M). The quantity of cells present in the wells was evaluated by an MTT test (MTT, Sigma) whose reaction products are quantified with an Elisa plate reader (Metertech, USA) (490-650 nm). The results obtained were related to the values of the control condition (100% viability).
• Example 3 Study of the Inhibition of the Proliferation of Cancer Lines by the Compounds According to the Invention
• the ability of compounds 8 and 9 to inhibit the proliferation of cancer cells was studied using the MTT method described above, which gives an estimate the number of cells.
• These studies were carried out on cancerous lines of various origins (see Table 2) under the culture conditions described above.
• the IC 50 ( ⁇ M) were determined for each of the lines and compared to the values obtained with a ruthenium derivative of reference.
• the two compounds according to the invention are more effective than the reference compound described in WO 2006/016069 and having the following formula:
• the HCT116 cells were treated for 48 hours at the indicated doses (1 ⁇ M and 5 ⁇ M). After 48 hours of treatment, the cells were washed with PBS (phosphate buffered saline) three times and fixed with 90% ethanol. The cells were then labeled with propidium iodide / RNAse solution. The labeled cells (10,000 per sample) were then counted with a FACScan flow cytometer and CelIQuest software (Becton Dickinson) to determine the DNA content and the cell cycle profile.
• PBS phosphate buffered saline
• CHOP protein has pro-apoptotic properties (induction of cell death).
• Phosphorylation of histone H2AX is a marker of DNA damage that leads to the activation of p53, a protein with pro-apoptotic properties or slowing of cell proliferation.
• the A172 tumor cells were treated for 6 hours with the compounds at two concentrations (5 ⁇ M and 10 ⁇ M). After 6 hours of treatment, the cells were lysed and 30 .mu.g of soluble proteins were separated on 12% polyacrylamide gel. After migration, the proteins were transferred to nitrocellulose by the Western blot technique. The CHOP and H2AX proteins were then detected using specific primary antibodies, themselves recognized by corresponding secondary antibodies whose peroxidase activity was revealed using an ECL kit.
• PB16 F1 cells (200,000 cells) were implanted subcutaneously in 8 weeks old black 6 mice. Groups of 6 mice per condition were made. The treatments started when the tumors were palpable with a volume of the order of 100 mm 3 .

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