Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/005—Compounds of elements of Group 5 of the Periodic Table without metal-carbon linkages
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

• Vanadium complexes with hydrazide-hydrazones, process for their preparation Vanadium complexes with hydrazide-hydrazones, process for their preparation
• the present invention relates to new complexes of vanadium, the method for preparation of new vanadium complexes, pharmaceutical formulations with these complexes, and the use of the complexes and their combinations with other substances for the prevention and treatment of various disorders and diseases from the group of metabolic disorders and/or associated with the activity of various phosphatases.
• diabetes is the most important from the epidemiological viewpoint. It may be caused by the dysfunction of synthesis and secretion of insulin and/or reduction of tissue sensitivity to insulin. This leads to the disorders of metabolism of carbohydrates, lipids and proteins, and consequently to many serious diseases and complications (e.g., obesity, atherosclerosis, retinopathy, nephropathy, diabetic foot) which lower quality of life and are a frequent cause of death.
• diseases and complications e.g., obesity, atherosclerosis, retinopathy, nephropathy, diabetic foot
• Diabetes is treated by lifestyle modification and with appropriate pharmacotherapy.
• Medications are used which act on the two main causes of diabetes: impaired insulin secretion and action on the tissues (e.g., thiazolidinedione derivatives, sulfonylureas).
• medications reducing the absorption of glucose from the gastrointestinal tract are used (e.g., acarbose).
• the pharmacological agents used have many disadvantages, which indicate the need for further exploration of medications with lesser side effects and greater efficiency. This includes the search for medications using the recently discovered mechanisms of antidiabetic action, such as the inhibition of tyrosine phosphatase activity.
• Phosphatases are a large group of enzymes which hydrolyze monoesters of phosphoric acid to a phosphate group and the molecules with a free hydroxyl group. By removing phosphate group, protein phosphatases are capable of modulating the activity of numerous cellular proteins, which is one of the basic regulatory mechanisms of the cells. In an organism, they constitute a group of several hundreds of enzymes affecting the regulation of many signal transduction pathways which determine the course of the basic cell functions, such as metabolism, ability to divide, and death of cells by apoptosis.
• Protein phosphatase activity is essential to the proper functioning of the body, and in light of the numerous studies, regulation of their activity may have important therapeutic implications including, inter alia, glucose metabolism disorders, cancer, inflammatory and autoimmune diseases, diseases of the nervous system (including neurodegenerative disorders, memoryimpairment, various mental disorders), bone diseases, infectious diseases.
• the participation of protein phosphatase in the regulation of numerous functions and systems of the body does not limit their potential use in therapy for mentioned diseases (Xu Y., Fisher G.J., J. Cell. Commun. Signal., 6 (2012) 125; Braithwaite S.P., Voronkov M., Stock J.B., Mouradian M.M., Neurochem.
• vanadium compounds may affect the metabolism of the cells, which may be the basis for the use of these compounds in the treatment of metabolic diseases (Adachi Y., Sakurai H., Chem. Pharm. Bull.,52 (2004) 428; Crans D.C., Yang L., Alfano J.
• vanadium compounds with vanadium in oxidation state were used, including both simple inorganic salts (such as VOS0 4 , VOCl 2 , NaV0 3 ), as well as complexes with organic ligands, and peroxo complexes of vanadium(V).
• simple inorganic salts such as VOS0 4 , VOCl 2 , NaV0 3
• complexes with organic ligands and peroxo complexes of vanadium(V).
• Bidentate ligands of OO, ON, OS, NS, NN types (capital letter represents an atom by which ligand is bound to vanadium, e.g, O represents an oxygen atom), tridentate ligands of ONO type, and tetradentate ligands of ONNO and NONN type were used.
• vanadium complexes with organic ligands are less toxic, and exhibit better properties in the regulation of metabolic processes, compared to inorganic compounds such as vanadyl sulfate and vanadates.
• vanadium compounds being effective inhibitors of protein phosphatase have been described in the literature: Han H., Lu L., Wang Q., Zhu ., Yuan C, Xing S., Fu X., Dalton Trans., 41 (2012) 11 1 16; Mehdi M.Z., Srivastava A.K., Arch. Biochem. Biophys., 440 (2005) 158; Schmid A.C., Byrne R.D., Vilar R., Woscholski R., FEBS Lett., 566 (2004) 35; Tracey A.S., J. Inorg. Biochem., 80 (2000) 11; and disclosed in patents: US 5,583,242.
• vanadium complexes are potent inhibitors of phosphatases, especially protein tyrosine phosphatases such as PTP1B, TCPTP, PTP-MEG2, SHP-1, SHP-2, LAR. It has also been shown that vanadium compounds may also be inhibitors of other phosphatases such as ATP-ase, glucose-6-phosphatase, or fructose-2,6- bisphosphatase.
• the complexes were obtained as a result of reaction of compounds of vanadium(IV) and (V) (mainly VOSO 4 , VO(acac) 2 as well as MVO3 and M 3 VO 4 ) with the respective ligands, in water or an organic solvent (e.g. DMF, EtOH, MeOH), in air or an inert atmosphere (Ar, N 2 ).
• V vanadium(IV) and (V)
• the present invention relates to complexes of vanadium(III), (IV) or (V) with hydrazide hydrazones which have not yet been described in literature or disclosed in patent claims, the method of synthesis and use in the prevention and treatment of metabolic diseases and disorders and/or associated with the activity of phosphatases.
• vanadium complexes are defined by the general formula M[VX(ONO)y(L) consult]-mS, where:
• X is oxygen atom or is absent
• L is Li or L 2 , wherein:
• - Li represents a halogen anion or a neutral or a deprotonated solvent molecule selected from the group consisting of C C ⁇ alcohols, preferably ethyl alcohol or methyl alcohol and/or water;
• L 2 is a neutral or anionic N, NO or OO-donor ligand selected from the group consisting of: polypyridine, preferably 2,2'-bipyridine (bpy), 1 ,10-phenanthroline (phen), pyrones, preferably deprotonated 3-hydroxy-2-methyl-4-pyrone (mal ), quinoline, preferably deprotonated 8-hydroxyquinoline (quin ) or pyridine carboxylic acids, preferably deprotonated 2-picolinic acid;
• S is a neutral solvent molecule selected from the group consisting of C 1 -C4 alcohols, water or sulfuric acid;
• M may be absent, and when present it means monocharged alkali metal cation, preferably Na + , K + , or an ammonium cation or alkylammonium cation, preferably Me 4 N + , ⁇ ⁇ 1" ;
• ONO where the letters O and N represent atom, through which the ligand is bound to the vanadium, means a tridentate ligand of the general formula 1
• R l5 R 2 , R 3 , R independently represent hydrogen atom, fluorine atom, chlorine atom, bromine atom, iodine atom or a S0 3 , hydroxo, nitro, alkoxy, aryloxy, dialkylamino, alkyl or aryl group, wherein at least one of the Ri, R 2 , R 3 , R4 substituents is different from hydrogen atom, R 5 independently stands for hydrogen atom, alkyl or aryl group, R$ is a group selected from:
• R7 substituent independently stands for a hydroxo, nitro, alkoxy, aryloxy, alkyl group or hydrogen atom, fluorine atom, chlorine atom, bromine, iodine atom.
• the complexes exhibit geometry of a deformed tetragonal pyramid or tetragonal bipyramid (A 1?
• a 2 are donor atoms of L 2 ligand, which independently represent a nitrogen atom or an oxygen atom).
• vanadium complexes The process for the preparation of vanadium complexes depends on the type of ligand, the oxidation state of vanadium, and the reaction medium. Complexes prepared by two-step reaction with isolation and / or without isolation of hydrazide-hydrazone under anaerobic conditions or in air.
• the first step of the synthesis involves the condensation reaction of aldehyde or ketone of formula 2
• R6 is as defined above.
• the condensation reaction of aldehyde or ketone with a hydrazide is carried out at a 1 : 1 molar ratio or with a stoichiometric excess of one of the reactants.
• the reaction is carried out in a solvent which is a Cj-C 12 alcohol or water or aqueous alcoholic solution, formed by mixing C 1 -C 12 alcohol with water in any ratio.
• the condensation product is subjected to complexation reaction using the original vanadium complex: VOSO 4 , VO(acac) 2 , VOCl 2 or V(acac) 3 , with or without the addition of deprotonation agent which is ammonia, alkylamine or arylamine.
• the complexation reaction is carried out at a 1 : 1 : 1 molar ratio of vanadium to aldehyde (ketone) and hydrazide in the case of vanadium(IV) or (V) complexes, and at 1 :2:2 molar ratio of vanadium to aldehyde (ketone) and hydrazide in the case of vanadium(III) complexes.
• the complexation reaction can also be carried out with a stoichiometric excess or insufficiency of vanadium.
• reaction products precipitate spontaneously or after the introduction of an additional complexing agent: neutral or anionic double-bond ligand L 2 (so called coligand) NN, NO, or OO-donor.
• Coligand L 2 is introduced at a 1 : 1 molar ratio of vanadium to coligand.
• Coligand may also be introduced in a stoichiometric excess or deficiency relative to vanadium. All of the steps in the synthesis are carried out in the temperature range of -130 to 260°C and in the pressure range of 0.01 to 1 MPa, that is, at the temperature and pressure conditions in which the reaction mixture is in the form of a solution or suspension. Most preferably, these reactions are to be carried out at the boiling point of the solvent under atmospheric pressure. Modification of the described method is the isolation of ligand in the solid state from the reaction medium obtained in the first step of the synthesis, and then using it as a substrate in the second step while retaining the second step reaction conditions described above.
• compositions for use in human and veterinary medicine containing complexes according to the invention and pharmaceutically acceptable excipients.
• the preparations may come in solid or liquid form and can be administered orally, parenterally, transdermally or by inhalation.
• the formulations according to the invention may contain additional substances having proper therapeutic effects, as well as vitamins, mineral salts, plant extracts and dietary supplements.
• the invention also includes the use of the vanadium(III), (IV) or (V) complexes defined above for the manufacture of pharmaceutical products for the prevention and treatment of metabolic diseases and disorders and/or associated with the activity of phosphatases.
• the vanadium complexes are used for the manufacture of preparations for the prevention and treatment of diabetes, impaired glucose tolerance and obesity.
• the vanadium complexes are used for the preparation of inhibiting the activity of protein phosphatases, and most preferably of tyrosine phosphatases.
• the complexes inhibiting the activity of phosphatases, including tyrosine phosphatases can be used in the prevention and treatment of disorders of glucose metabolism, prevention and treatment of cancer, inflammatory and autoimmune diseases, neurological diseases, including neurodegenerative disorders, memory impairment, psychiatric disorders, bone diseases, infectious diseases and other disease entities, in which inhibition of phosphatase activity can have the beneficial prophylactic and/or therapeutic effect.
• the complexes are used alone or in combination with pharmaceutically acceptable carriers, such as water, alcohol, propylene glycol, cellulose derivatives, starch.
• pharmaceutically acceptable carriers such as water, alcohol, propylene glycol, cellulose derivatives, starch.
• the invention includes compounds which act to sensitize target organs for insulin action - enhancing the activity, mimicking or replacing insulin. These compounds also cause an increase of synthesis and/or secretion of insulin by the beta cells of the pancreas. According to the invention, the compounds also show the inhibition activity of the various phosphatases. Proper modulation of the compounds according to the invention can prove to be an effective strategy for prevention and therapy.
• the vanadium complexes obtained according to the invention affect metabolism, as demonstrated in studies on cells of major organs associated with pathogenetic mechanisms of type 2 diabetes, and affect the inhibition of the activity of the PTP1B tyrosine phosphatase, as demonstrated in studies using human recombinant enzyme.
• Example 1 Complex of type [V lu (ONO) 2 ]-mS
• Example 7 Studies of the effect of vanadium complexes on glucose utilization in myocytes, hepatocytes, adipocytes.
• C2C12 cells were maintained in culture medium: DMEM, 10% fetal calf serum, lOO U/ml penicillin and 100 ⁇ g/ml streptomycin. Cultures and experiments were performed at 37 ° C in 5% C0 2 .
• 3T3-L1 fibroblasts were maintained in culture medium: DMEM, 10% calf serum and 100 U/ml penicillin and 100 ⁇ / ⁇ streptomycin. Cultures and experiments were performed at 37 ° C in 5% C0 2 .
• HepG2 hepatocytes were maintained in the culture medium: EMEM, 10% fetal calf serum and 100 U/ml penicillin and 100 ⁇ g/ml streptomycin. Cultures and experiments were performed at 37 0 C in 5% C0 2 .
• 20,000 C2C12 cells were placed in wells of 96-well microplate in culture medium. After 3 days, the cell differentiation was started by the addition of the differentiation medium (DMEM, 2% horse serum, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin). After 24 hours medium was replaced with a differentiation medium. In the eighth day after application of the cells experiments were carried out on the effect of vanadium compounds on glucose utilization. After discarding the differentiation medium, the cells were incubated for 2 hours in the experimental medium (DMEM with 16 mMglucose, 1% bovine albumin and 100 U/ml penicillin and 100 ⁇ g/ml streptomycin).
• the differentiation medium DMEM, 2% horse serum, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
• the medium was replaced with a new portion of the experimental medium, to which solutions of the testedcompounds were added in phosphate buffered saline (PBS) with 1% BSA.
• PBS phosphate buffered saline
• Experiments were performed with or without the addition of human recombinant insulin at a final concentration of 34.5 nM. After stirring the contents, incubation was carried out for 24 hours. After this time the contents of the microplates were mixed and medium were collected for determination of glucose concentration.
• 20,000 HepG2 cells were placed in wells of 96-well microplate in culture medium. After 24 hours of incubation, solutions of the tested compounds were added to the wells of a microplate in phosphate buffered saline (PBS). Experiments were carried out with human recombinant insulin at a final concentration of 34.5nM. After stirring the contents, incubation was carried out for 24 hours. After this time, the contents of the microplate were mixed and medium were collected for determination of glucose concentration.
• PBS phosphate buffered saline
• Glucose concentration in the medium after incubation of the cells with the studied compounds was determined by the oxidase method with fluorimetric detection in 384-well plates. To the tested sample were added an equal volume of the reagent 0.4 U/ml peroxidase, 4 U/ml glucose oxidase and 200 ⁇ 10-Acetyl-3,7-dihydroxyphenoxazine in 50 mM potassium phosphate buffer pH 7.4. After 30 minutes of incubation at 37°C was followed by a measurement of the fluorescence intensity at an excitation wavelength of 530 nm and emission wavelength 580 nm. The glucose concentration was calculated using the standard curve. Glucose utilization by cells was calculated as the difference in glucose concentration in the experimental medium and the medium after incubation of the compounds with the cells.
• Muscle cells are the major organ responsible for glucose utilization and regulating blood glucose levels, and are an important effector organ in the action of various antidiabetic agents.
• the effect of compounds, being the subject of the invention on the glucose utilization was tested using muscle cells (C2C12) and additionally adipocytes (3T3-L1), and hepatocytes (HepG2), and compared to the effects of rosiglitazone, which is used in the treatment of type 2 diabetes and the effect of oxovanadium(IV) maltolate VO(mal) 2 , with proven potent action (Tables 2, 3 and 4).
• Glucose utilization is expressed in relation to the control not containing vanadium compounds.

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Abstract

Description

Vanadium complexes with hydrazide-hydrazones, process for their preparation,

pharmaceutical formulations and the use of thereof.

The present invention relates to new complexes of vanadium, the method for preparation of new vanadium complexes, pharmaceutical formulations with these complexes, and the use of the complexes and their combinations with other substances for the prevention and treatment of various disorders and diseases from the group of metabolic disorders and/or associated with the activity of various phosphatases.

There are different kinds of causes for metabolic disorders, of which diabetes is the most important from the epidemiological viewpoint. It may be caused by the dysfunction of synthesis and secretion of insulin and/or reduction of tissue sensitivity to insulin. This leads to the disorders of metabolism of carbohydrates, lipids and proteins, and consequently to many serious diseases and complications (e.g., obesity, atherosclerosis, retinopathy, nephropathy, diabetic foot) which lower quality of life and are a frequent cause of death.

Diabetes is treated by lifestyle modification and with appropriate pharmacotherapy. Medications are used which act on the two main causes of diabetes: impaired insulin secretion and action on the tissues (e.g., thiazolidinedione derivatives, sulfonylureas). In addition, medications reducing the absorption of glucose from the gastrointestinal tract are used (e.g., acarbose).

The pharmacological agents used have many disadvantages, which indicate the need for further exploration of medications with lesser side effects and greater efficiency. This includes the search for medications using the recently discovered mechanisms of antidiabetic action, such as the inhibition of tyrosine phosphatase activity.

Phosphatases are a large group of enzymes which hydrolyze monoesters of phosphoric acid to a phosphate group and the molecules with a free hydroxyl group. By removing phosphate group, protein phosphatases are capable of modulating the activity of numerous cellular proteins, which is one of the basic regulatory mechanisms of the cells. In an organism, they constitute a group of several hundreds of enzymes affecting the regulation of many signal transduction pathways which determine the course of the basic cell functions, such as metabolism, ability to divide, and death of cells by apoptosis.

Protein phosphatase activity is essential to the proper functioning of the body, and in light of the numerous studies, regulation of their activity may have important therapeutic implications including, inter alia, glucose metabolism disorders, cancer, inflammatory and autoimmune diseases, diseases of the nervous system (including neurodegenerative disorders, memoryimpairment, various mental disorders), bone diseases, infectious diseases. The participation of protein phosphatase in the regulation of numerous functions and systems of the body does not limit their potential use in therapy for mentioned diseases (Xu Y., Fisher G.J., J. Cell. Commun. Signal., 6 (2012) 125; Braithwaite S.P., Voronkov M., Stock J.B., Mouradian M.M., Neurochem. Int., 61 (2012) 899; Braithwaite S.P., Stock J.B., Lombroso P. J., Nairn A.C., Prog. Mol. Biol. Transl. Sci., 106 (212) 343; Sun H., Wang Y., Physiology (Bethesda), 27 (2012) 43; Pagano M.A., Tibaldi E., Gringeri E., Brunati A.M., IUBMB Life, 64 (2012) 27; Blunt M.D., Ward S.G., Curr. Opin. Pharmacol., 12 (2012) 444).

Intensive study on the biological activity of vanadium compounds has been carried out for many years. Research is conducted using cellular and animal models. Some of the compounds were also tested clinically. These studies showed a clear effect of vanadium compounds on the various cell regulatory mechanisms, including mechanisms relating to insulin action and inhibition of protein phosphatases. The studies on cells showed that vanadium regulates lipid and glycogen synthesis, inhibits lipolysis and gluconeogenesis and increases glucose transport into cells. These studies proved that vanadium compounds may affect the metabolism of the cells, which may be the basis for the use of these compounds in the treatment of metabolic diseases (Adachi Y., Sakurai H., Chem. Pharm. Bull.,52 (2004) 428; Crans D.C., Yang L., Alfano J. A., Chi L.H., Jin W., Mahroof-Tahir M., Robbins K., Toloue M.M., Chan L.K., Plante A. J., Grayson R.Z., Willsky G.R., Coord. Chem. Rev., 237 (2003) 13; Hamrin K., Henriksson J., Life Sci., 76 (2005) 2329; Katoh A., Matsumura Y., Yoshikawa Y., Yasui H., Sakurai H., J. Inorg. Biochem., 103 (2009) 567; Sakurai H., Watanabe H., Tamura H., Yasui H., Matsushita R., Takada J., Inorg. Chim. Acta, 283 (1998) 175; Thompson K.H., Orvig C, J. Inorg. Biochem., 100 (2006) 1925; US4882171, US5266565, US5278154, US5338759, US6414029).

Studies conducted on animals with diabetes have confirmed the therapeutic effectiveness of vanadium compounds primarily by lowering the concentration of glucose in blood. It was further found that these effects are related to, inter alia, inhibition of the activity of tyrosine phosphatases (Crans D.C., Yang L., Alfano J. A., Chi L.H., Jin W., Mahroof-Tahir M,

Robbins K., Toloue M.M., Chan L.K., Plante A.J., Grayson R.Z., Willsky G.R., Coord.

Chem. Rev., 237 (2003) 13.; Gao L., Niu Y., Liu W., Xie M., Liu X., Chen Z., Li L., Clin.

Chim. Acta 388 (2008) 89; Hamrin K., Henriksson J., Life Sci., 76 (2005) 2329; Islam M.N., Kumbhar A.A., Kumbhar A.S., Zeller M., Butcher R.J., Dusane M.B., Joshi B.N., Inorg

Chem. 49 (2010) 8237; Nilsson J., Degerman E., Haukka M., Lisensky G.C., Garribba E.,

Yoshikawa Y., Sakurai H., Enyedy E.A., Kiss T., Esbak H., Rehder D., Nordlander E., J.

Chem. Soc, Dalton Trans. (38) (2009) 7902; Sakurai H., Katoh A., Yoshikawa Y., Bull.

Chem. Soc. Jpn., 79 (2006) 1645; Thompson K.H., Orvig C, Coord. Chem. Rev., 219-221 (2001) 1033; US4882171, US5266565, US5278154, US6414029).

The effect of the antidiabetic action of vanadium complexes, maltol and oxovanadium (IV) ethyl maltolate, has been confirmed in humans in clinical trials. (Hamrin K., Henriksson J., Life Sci., 76 (2005) 2329.; Sakurai H., Katoh A., Yoshikawa Y., Bull. Chem. Soc. Jpn., 79 (2006) 1645; Scior T., Guevara-GarciaA., BernardP., DoQ., Domeyer D., Laufer S., Mini- Rev.Med. Chem., 5 (2005) 995; Vardatsikos G., Mehdi M.Z., Srivastava A.K., Int. J. Mol. Med., 24 (2009) 303.

In the trials vanadium compounds with vanadium in oxidation state (III to V) were used, including both simple inorganic salts (such as VOS0₄, VOCl₂, NaV0₃), as well as complexes with organic ligands, and peroxo complexes of vanadium(V). Bidentate ligands of OO, ON, OS, NS, NN types (capital letter represents an atom by which ligand is bound to vanadium, e.g, O represents an oxygen atom), tridentate ligands of ONO type, and tetradentate ligands of ONNO and NONN type were used. (Sakurai H., Katoh A., Yoshikawa Y.,Bull. Chem. Soc. Jpn., 79 (2006) 1645; Thompson K. H., McNeill J. H., Orvig C, Chem. Rev., 99 (1999) 2561. In general, vanadium complexes with organic ligands are less toxic, and exhibit better properties in the regulation of metabolic processes, compared to inorganic compounds such as vanadyl sulfate and vanadates.

Examples of vanadium compounds being effective inhibitors of protein phosphatase have been described in the literature: Han H., Lu L., Wang Q., Zhu ., Yuan C, Xing S., Fu X., Dalton Trans., 41 (2012) 11 1 16; Mehdi M.Z., Srivastava A.K., Arch. Biochem. Biophys., 440 (2005) 158; Schmid A.C., Byrne R.D., Vilar R., Woscholski R., FEBS Lett., 566 (2004) 35; Tracey A.S., J. Inorg. Biochem., 80 (2000) 11; and disclosed in patents: US 5,583,242. US 7,692,012 B2; GB0328157; US6432941; US8242092; US8008035; US5877210; US 5155031; US 6579540. It has been shown that vanadium complexes are potent inhibitors of phosphatases, especially protein tyrosine phosphatases such as PTP1B, TCPTP, PTP-MEG2, SHP-1, SHP-2, LAR. It has also been shown that vanadium compounds may also be inhibitors of other phosphatases such as ATP-ase, glucose-6-phosphatase, or fructose-2,6- bisphosphatase.

The ability to inhibit the phosphatase activity has been shown for this group of compounds in studies using isolated enzymes, cell-based and animal models. The studies conducted on cells and animals have shown that these compounds may affect the overall level of phosphorylation of proteins and the various receptors and regulatory proteins, as well as transcription factors such as INS-R, Akt, GSK-3, NF-kappaB, FKHR, FKHR-1 , FOXO, ERK 1/2. This leads to the modulation of various cellular functions and may be the basis for the therapeutic use of vanadium complexes in, inter alia, neoplastic, metabolic, inflammatory, neurodegenerative and other diseases. The beneficial effects of vanadium complexes have been observed in studies using cells (including cancer) and in animal studies demonstrating the potential therapeutic efficacy of this group of phosphatase inhibitors in, inter alia, diabetes, cancer, neurodegenerative diseases and cardiovascular diseases.

A number of vanadium complexes with N-salicylidene hydrazide and hydrazides of l-(2- hydroxyphenyl)-alkyl ketones as tridentate ONO donor ligands have beenobtained, and review articles about them have been published. (Plass, W., Coord. Chem. Rev., 255 (2011) 2378; Maurya, M. R., Coord. Chem. Rev., 237 (2003) 163; Seena E. B., Mathew N., Kuriakose M., Kurup M. R. P., Polyhedron, 27 (2008) 1455; Sreeja P. B., Kurup M. R. P., Spectrochim. Acta A, 61 (2005) 331). These compounds include core types [V^IVO]²⁺, [V^vO]³⁺ and [V^v0₂]⁺, wherein vanadium(III) complexes are unknown, and vanadium complexes (IV) are rare. The vast majority of the described connections contain ONO donor ligands in which the fragment derived from o- hydroxybenzaldehyde or o-hydroxyphenylketone possesses the unsubstituted aromatic ring. The complexes were obtained as a result of reaction of compounds of vanadium(IV) and (V) (mainly VOSO₄, VO(acac)₂ as well as MVO3 and M₃VO₄) with the respective ligands, in water or an organic solvent (e.g. DMF, EtOH, MeOH), in air or an inert atmosphere (Ar, N₂).

The literature describes the potential use of such compounds in catalysis and oxidation of organic compounds (Monafared H.H., Bikas R., Mayer P., Inorg. Chim. Acta, 363 (2010) 2574; Maurya M. R., Agarwal S., Bader C, Ebel M., Rehder D., Dalton Trans., (3) (2005) 537).

The present invention relates to complexes of vanadium(III), (IV) or (V) with hydrazide hydrazones which have not yet been described in literature or disclosed in patent claims, the method of synthesis and use in the prevention and treatment of metabolic diseases and disorders and/or associated with the activity of phosphatases.

According to the present invention vanadium complexes are defined by the general formula M[VX(ONO)y(L)„]-mS, where:

X is oxygen atom or is absent

L is Li or L₂, wherein:

- Li represents a halogen anion or a neutral or a deprotonated solvent molecule selected from the group consisting of C C^ alcohols, preferably ethyl alcohol or methyl alcohol and/or water;

L₂ is a neutral or anionic N, NO or OO-donor ligand selected from the group consisting of: polypyridine, preferably 2,2'-bipyridine (bpy), 1 ,10-phenanthroline (phen), pyrones, preferably deprotonated 3-hydroxy-2-methyl-4-pyrone (mal ), quinoline, preferably deprotonated 8-hydroxyquinoline (quin ) or pyridine carboxylic acids, preferably deprotonated 2-picolinic acid;

wherein, when X is absent, then y = 2 and n = 0, and when X is present then y = 1, wherein, when then n is 1 or 2, and when n is 2 then Li are the same or different, and when L=L₂, then n is 1,

S is a neutral solvent molecule selected from the group consisting of C₁-C4 alcohols, water or sulfuric acid;

- m varies from 0 to 4,

M may be absent, and when present it means monocharged alkali metal cation, preferably Na⁺, K⁺, or an ammonium cation or alkylammonium cation, preferably Me₄N⁺, Εί ΝΓ^1"; ONO, where the letters O and N represent atom, through which the ligand is bound to the vanadium, means a tridentate ligand of the general formula 1

Formula 1 in the keto or enol form, neutral or deprotonated, wherein R_l5 R₂, R₃, R» independently represent hydrogen atom, fluorine atom, chlorine atom, bromine atom, iodine atom or a S0₃, hydroxo, nitro, alkoxy, aryloxy, dialkylamino, alkyl or aryl group, wherein at least one of the Ri, R₂, R₃, R4 substituents is different from hydrogen atom, R₅ independently stands for hydrogen atom, alkyl or aryl group, R$ is a group selected from:

(A) (B) (C) (D) (E)

(F) (G) (H) (I) wherein R7 substituent independently stands for a hydroxo, nitro, alkoxy, aryloxy, alkyl group or hydrogen atom, fluorine atom, chlorine atom, bromine, iodine atom.

According to the invention, the complexes exhibit geometry of a deformed tetragonal pyramid or tetragonal bipyramid (A_1? A₂ are donor atoms of L₂ ligand, which independently represent a nitrogen atom or an oxygen atom).

The process for the preparation of vanadium complexes depends on the type of ligand, the oxidation state of vanadium, and the reaction medium. Complexes prepared by two-step reaction with isolation and / or without isolation of hydrazide-hydrazone under anaerobic conditions or in air.

According to the invention, the first step of the synthesis involves the condensation reaction of aldehyde or ketone of formula 2

Formula 2 wherein Ri, R₂, R₃, R4 and R₅ are as defined above, with a hydrazide of formula 3

Formula 3 wherein R6 is as defined above. The condensation reaction of aldehyde or ketone with a hydrazide is carried out at a 1 : 1 molar ratio or with a stoichiometric excess of one of the reactants. The reaction is carried out in a solvent which is a Cj-C₁₂ alcohol or water or aqueous alcoholic solution, formed by mixing C₁-C₁₂ alcohol with water in any ratio.

In the second step the condensation product is subjected to complexation reaction using the original vanadium complex: VOSO₄, VO(acac)₂, VOCl₂ or V(acac)₃, with or without the addition of deprotonation agent which is ammonia, alkylamine or arylamine. The complexation reaction is carried out at a 1 : 1 : 1 molar ratio of vanadium to aldehyde (ketone) and hydrazide in the case of vanadium(IV) or (V) complexes, and at 1 :2:2 molar ratio of vanadium to aldehyde (ketone) and hydrazide in the case of vanadium(III) complexes. The complexation reaction can also be carried out with a stoichiometric excess or insufficiency of vanadium.

The reaction products precipitate spontaneously or after the introduction of an additional complexing agent: neutral or anionic double-bond ligand L₂ (so called coligand) NN, NO, or OO-donor. Coligand L₂ is introduced at a 1 : 1 molar ratio of vanadium to coligand. Coligand may also be introduced in a stoichiometric excess or deficiency relative to vanadium. All of the steps in the synthesis are carried out in the temperature range of -130 to 260°C and in the pressure range of 0.01 to 1 MPa, that is, at the temperature and pressure conditions in which the reaction mixture is in the form of a solution or suspension. Most preferably, these reactions are to be carried out at the boiling point of the solvent under atmospheric pressure. Modification of the described method is the isolation of ligand in the solid state from the reaction medium obtained in the first step of the synthesis, and then using it as a substrate in the second step while retaining the second step reaction conditions described above.

Proper combinations of the mentioned parameters and the use of coligand allow for obtaining complexes being the subject of the invention with a good yield.

Selected examples of obtained complexes are summarized in Table 1. The substituents R correspond to numbers shown in formula 1, and R is a group selected from groups A-I. For all the complexes R4 = R5 = H.

Table 1. i R₂ R3 R* (A-I) R7 L_1? L₂ mS

Complexes of the types [VO(ONO)(L₁)(L₁)] ^•mS

CI H CI C - = EtOH - L_t = EtO^"

H H OCH3 C - = EtOH - = EtO^"

Br OCH3 Br C - Li = EtOH - = EtO^"

Complexes oft lie types [VO(ONO)(Li)]-mS

OCH₃ H Br E - = H₂0 -

Br H CI E - = H₂0 -

H H N0₂ E - Li = H₂0 - lBu H ¾u E - Li = H₂0 -

H H OH E - = H₂0 -

OH H H E - Li = H₂0 -

H H OCH₃ E - Li = H₂0 0,5H₂O

H H N0₂ B - L = EtO^" -

OH H H C - L = EtO^" -

H H CI B - L = EtO^" -

H H N0₂ C - L = EtO^" 1H₂0 OH H H D - L = OH^" 1H₂0

Complexes of the types [VO(ONO)(L₂)]-mS

H H Br A OH L₂ = phen 2H₂0

H H CI A OH L₂ = phen 2H₂0

OCH₃ H Br A OH L₂ = phen 2H₂0

Br H CI A OH L₂ = phen 1H₂0

CI H CI A OH L₂ = phen 1H₂0

H H Br A OCH3 L₂ = phen -

H Et₂N H A CI L₂ = phen 1H₂0

H H Br A 'Bu L₂ = phen 0,5H₂O lBu H ¾u A H L₂ = bpy lMeOH lBu H ¾u A H L₂ = mal^" lEtOH

Complexes of the types M[VO(ONO)(L₂)]-mS

H H SO3 A OH L₂ = phen 4H₂0

H H SO3 A OCH₃ L₂ = phen 4H₂0

Complexes of the types M[VO(ONO)(L)]-mS

H H S0₃ C - L = OH^" -

H H SO3 D - L = EtO^" 1H₂S0₄

Complexes of the types [V(ONO)₂]-mS

H H Br A N0₂ - -

H H Br I - - -

H H Br G - - -

H H Br H - - -

OCH3 H Br C - - -

H Et₂N H C - - -

H H Br A CI - 1H₂0

Another aspect of the invention are the pharmaceutical preparations for use in human and veterinary medicine, containing complexes according to the invention and pharmaceutically acceptable excipients. The preparations may come in solid or liquid form and can be administered orally, parenterally, transdermally or by inhalation.

The formulations according to the invention, in addition to vanadium complexes, may contain additional substances having proper therapeutic effects, as well as vitamins, mineral salts, plant extracts and dietary supplements. The invention also includes the use of the vanadium(III), (IV) or (V) complexes defined above for the manufacture of pharmaceutical products for the prevention and treatment of metabolic diseases and disorders and/or associated with the activity of phosphatases. Preferably, according to the invention, the vanadium complexes are used for the manufacture of preparations for the prevention and treatment of diabetes, impaired glucose tolerance and obesity.

Preferably, the vanadium complexes are used for the preparation of inhibiting the activity of protein phosphatases, and most preferably of tyrosine phosphatases. The complexes inhibiting the activity of phosphatases, including tyrosine phosphatases can be used in the prevention and treatment of disorders of glucose metabolism, prevention and treatment of cancer, inflammatory and autoimmune diseases, neurological diseases, including neurodegenerative disorders, memory impairment, psychiatric disorders, bone diseases, infectious diseases and other disease entities, in which inhibition of phosphatase activity can have the beneficial prophylactic and/or therapeutic effect.

The complexes are used alone or in combination with pharmaceutically acceptable carriers, such as water, alcohol, propylene glycol, cellulose derivatives, starch.

The invention includes compounds which act to sensitize target organs for insulin action - enhancing the activity, mimicking or replacing insulin. These compounds also cause an increase of synthesis and/or secretion of insulin by the beta cells of the pancreas. According to the invention, the compounds also show the inhibition activity of the various phosphatases. Proper modulation of the compounds according to the invention can prove to be an effective strategy for prevention and therapy.

The vanadium complexes obtained according to the invention affect metabolism, as demonstrated in studies on cells of major organs associated with pathogenetic mechanisms of type 2 diabetes, and affect the inhibition of the activity of the PTP1B tyrosine phosphatase, as demonstrated in studies using human recombinant enzyme.

Studies have been conducted on the effects of vanadium complexes on glucose uptake by myocytes (C2C12 cells), hepatocytes (HepG2 cells) and adipocytes (3T3-L1 cells). The effectiveness of the complexes was compared with the action of oxovanadium(IV) ethyl maltolate [VO(mal)₂], whose strong antidiabetic activity has been widely described in the literature.

Demonstration of the inhibitory effects of the studied compounds on the activity of PTPIB allows for an assumption of the same effect of these compounds against other phosphatases. This results from the structural similarities of different phosphatases and mechanisms of action of vanadium compounds described in literature.

The invention is illustrated by following examples. Example 1. Complex of type [V^lu(ONO)₂]-mS

Ri = MeO, R₂, R4, R₅ = H, R₃ = Br, R<; = substituent C, m = 0

5-bromo-3-methoxy salicylaldehyde (695 mg, 3.00 mmol), phenylacetic acid hydrazide (451 mg, 3.00 mmol) and EtOH (28 mL) were heated to reflux under Ar atmosphere for about 10 minutes to give a yellow solution with a white precipitate. Then, solid [V(acac)₃] (522 mg, 1.50 mmol) was added and further heated to reflux for approximately 25 minutes. Initially, the mixture became brown, whereupon the product precipitated. The precipitate was filtered, washed twice with EtOH and dried in air. Yield: 487 mg, 41.9 %. Elemental analysis: calculated for C₃₂H₂₇Br₂N₄0₆V: C, 49.64; H, 3.51 ; N, 7.24%. Found: C, 49.24; H, 3.64; N, 7.09%. IR (KBr, cm^"1): ^vcN(imina) 1592s. Magnetic moment: fx_ef ~ 2.7 μ_Β Example 2. Complex of type [V^vO(ONO) Li] mS

Ri, R₂, R4, R₅ = H, R₃ = N0₂, Re = substituent B, Li

5-nitrosalicylic aldehyde (500 mg, 3.00 mmol), salicylic acid hydrazide (457 mg, 3.00 mmol) and 40 ml of EtOH was heated to reflux for approximately 10 minutes. Then VOS0₄-xH₂0 (665 mg; 3.00 mmol) was added and refluxing was continued for approximately 90 minutes. The clear solution was left to crystallize for approximately 2 days. The product was filtered and washed twice with a small amount of cold EtOH. Yield: 843 mg, 68.0 %. Elemental analysis: calculated for C₁₆H₁₄N₃0₇V: C, 46.73; H, 3.43; N, 10.22%. Found: C, 46.58; H, 3.66; N, 10.08%. IR (KBr, cm^"1): v_CN(imina) 1608s, 1623s; v_vo 968s. Magnetic moment: diamagneticcompound.

Crystallographic data: triclinic system, space group P-l, a = 7.4393(4), b = 8.6931(5), c = 13.2880(8) A, a = 88.914(5), β = 80.414(5), y = 82.214(5)°, V= 839.53(8) A³, T= 293(2) K, Z = 2, D_c = 1.627 Mg m^"3, μ = 0.638 mm^"1, 8897 measured reflections, 2556 independent ( ?int = 0.0457), 2187 observed, [/ > 2σ(Ι)]. R_{ = 0.1389; wR₂ = 0.4652 [for 2187 observed reflections].

Example 3. Complex of type [V^lvO(ONO)L_!]-mS

R₂, R4, R₅ = H, R_b R₃ = CI, R₆ = substituent E, Lj = H₂0 m= 0

3,5-dichlorosalicylaldehyde (573 mg, 3.00 mmol), nicotinic acid hydrazide (411 mg, 3.00 mmol) and 50 ml of EtOH were heated to reflux for approximately 15 minutes under anaerobice conditions (under Ar). Formation of a yellow solution was observed. Then VO(acac)₂ (795 mg, 3.00 mmol) was added, and the system was still heated to reflux for approximately 60 minutes under anaerobicconditions. During this time an orange compound precipitated, which was then filtered, washed twice with EtOH and dried in air. Yield: 938 mg, 80.0 %. Elemental analysis: calculated for Ci₃H₉Cl₂N₃0₄V: C, 39.72; H, 2.31; N, 10.69%. Found: C, 40.12; H, 2.18; N, 10.67%. IR (KBr, cm^"1): v_CN(i_mi„a) 1613s; v_v0 882s. Magnetic moment: μ^ = 1.3 μ_Β.

Example 4. Complex of type [V^vO(ONO)(Li)₂] mS

R₂, R4, R₅ = H, Ri, R₃ = CI, R₆ = substituent C, = EtOH, i EtO^", m = 0

3,5-dichlorosalicylaldehyde (717 mg, 3.75 mmol), phenylacetic acid hydrazide (564 mg, 3.75 mmol) and 125 mL of EtOH were heated to reflux for approximately 15 minutes under Ar atmosphere to give a yellow solution. Then, solid [VO(acac)₂] (994 mg, 3.75 mmol) was added and further heated to reflux for approximately 60 minutes. Yellow-brown solution was then concentrated to approximately 50 ml and allowed to cool. The brown precipitate was filtered, washed twice with EtOH and dried in air. Yield: 1.04 g, 72.3 %.. Elemental analysis: calculated for C₁₉H₂₁C1₂N₂0₅V: C, 47.62; H, 4.42; N, 5.85%. Found: C, 47.27; H, 4.27; N, 5.82%. IR (KBr, cm^"1): _CN(i_mj_na) 1609s; v_v0 973s, 954m. Magnetic moment: diamagneticcompound.

Crystallographic data: orthorhombic system, space group na2i, a = 38.045(5), b = 7.608(5), c = 15.221(5) A, V = 4406(3) A³, T = 293(2) K, Z = 4, _c = 1.445 Mg m^"3, μ = 0.724 mm^"1, 14347 measured reflections, 7820 independent (Rm = 0.0243), 5327 observed [I > 2σ(Ι)]. R_x = 0.0366; wR₂ = 0.072 [for 5327 observed reflections].

Example 5. Complex of type [V^lvO(ONO)(L₂)]-mS

R₂, R4, R5 = H, Ri, R₃ = t-Bu, R6 = substituted C, L₂ = phen, m = 0

3,5-Di-tert-butylsalicylaldehyde (254 mg; 1.50 mmol), phenylacetic acid hydrazide (227 mg, 1.5 mmol) and 25 ml of EtOH were heated to reflux under Ar for approximately 15 minutes. To the resulting clear solution [VO(acac)₂] (398 mg; 1.50 mmol) was added, then the mixture was heated to reflux for approximately 40 minutes. To the dark yellow solution 1 ,10- phenanthroline (271 mg, 1.50 mmol) was added and refluxing was continued for approximately 10 minutes. The solvent was evaporated to approximately 12 ml and cooled in ice. The crystals which formed on the next day were filtered, washed twice with EtOH and dried in air. Yield: 346 mg, 37.4 %. Elemental analysis: calculated for C₃5H3₈N₄03V: C, 68.28; H, 6.55; N, 9.10%. Found: C, 67.34; H, 5.62; N, 9.42%. IR (KBr, cm^-1): ν_{0Ν(ίπώΐ3)} 1625s; v_v0953s. Magnetic moment: ⁼ 1.7 μ_Β.

Crystallographic data: monoclinic system, space group P2_!/c, a = 14.9200(3), b = 16.2740(3), c = 19.9320(3) A, /? = 130.2810(10)°, V = 3692.08(12) A³, T = 293(2) K, Z = 4, D_c = 1.100 Mg m^"3, μ = 0.303 mm^"1, 30452 measured reflections, 8445 independent (R^ = 0.0351), 6015 observed, [/> 2σ(Ι)]. Ri = 0.0937; wR₂ = 0.2649 [for 6015 observed reflections].

Example 6. Complex of type M[V^IVO(ONO)(L₂)]- mS

M = K⁺, Ri, R₂, t, R₅ = H, R₃ =S0₃, Re = substituent A, R₇ = MeO, L₂ = phen, m = 4, S = H₂0

The potassium salt of 5-sulfosalicylic aldehyde (367 mg, 1.50 mmol), 4-methoxy- benzohydrazide (250 mg, 1.50 mmol) and EtOH (45 ml) were heated to reflux under Ar for approximately 20 minutes. Then, [VO(acac)₂] (378 mg; 1.50 mmol) was added to the pale yellow solution and refluxing was continued for approximately 20 minutes. 1 ,10- phenanthroline (271 mg; 1.50 mmol) was added to the brown solution. The red solution was concentrated to approximately 50% of volume and allowed to cool. The resulting orange precipitate was filtered, washed twice with EtOH and dried in air. Yield: 599 mg; 56.6 %. Elemental analysis: calculated for C₂₇H₂₉KN₄OnSV: C, 45.96; H, 3.86; N, 7.94; S, 4.54%. Found: C, 45.65; H, 3.70; N, 7.83; S, 4.40%. ER. (KBr, cm^"1): v_CN(imina) 1608s; v_v0 961s. Magnetic moment: μ_εί = 1 ·3 μ_Β·

Example 7. Studies of the effect of vanadium complexes on glucose utilization in myocytes, hepatocytes, adipocytes.

Cell cultures

C2C12 cells were maintained in culture medium: DMEM, 10% fetal calf serum, lOO U/ml penicillin and 100 μg/ml streptomycin. Cultures and experiments were performed at 37 ° C in 5% C0₂.

3T3-L1 fibroblasts were maintained in culture medium: DMEM, 10% calf serum and 100 U/ml penicillin and 100 μ§/τα\ streptomycin. Cultures and experiments were performed at 37 ° C in 5% C0₂.

HepG2 hepatocytes were maintained in the culture medium: EMEM, 10% fetal calf serum and 100 U/ml penicillin and 100 μg/ml streptomycin. Cultures and experiments were performed at 37 ⁰ C in 5% C0₂.

Glucose utilization.

20,000 C2C12 cells were placed in wells of 96-well microplate in culture medium. After 3 days, the cell differentiation was started by the addition of the differentiation medium (DMEM, 2% horse serum, 100 U/ml penicillin and 100 μg/ml streptomycin). After 24 hours medium was replaced with a differentiation medium. In the eighth day after application of the cells experiments were carried out on the effect of vanadium compounds on glucose utilization. After discarding the differentiation medium, the cells were incubated for 2 hours in the experimental medium (DMEM with 16 mMglucose, 1% bovine albumin and 100 U/ml penicillin and 100 μg/ml streptomycin). Then, the medium was replaced with a new portion of the experimental medium, to which solutions of the testedcompounds were added in phosphate buffered saline (PBS) with 1% BSA. Experiments were performed with or without the addition of human recombinant insulin at a final concentration of 34.5 nM. After stirring the contents, incubation was carried out for 24 hours. After this time the contents of the microplates were mixed and medium were collected for determination of glucose concentration.

10,000 3T3-L1 cells were placed in wells of 96-well microplate coated with polylysine in culture medium. After reaching confluence, cell differentiation into adipocytes was started by replacing medium with the culture medium supplemented with 1 μΝΜ6Χ3ΐη6ΐη38οη6 and 0.5 mM 3-isobutyl-l-methylxanthine. After 48 hours of incubation, the medium was replaced with a culture medium supplemented with 10 μg/ml human recombinant insulin. After one hour, solutions of the tested compounds were added to the wells of a microplate in phosphate buffered saline (PBS). After stirring the contents, incubation was carried out for 24 hours. After this time, the contents of the microplate were mixed and medium were collected for determination of glucose concentration.

20,000 HepG2 cells were placed in wells of 96-well microplate in culture medium. After 24 hours of incubation, solutions of the tested compounds were added to the wells of a microplate in phosphate buffered saline (PBS). Experiments were carried out with human recombinant insulin at a final concentration of 34.5nM. After stirring the contents, incubation was carried out for 24 hours. After this time, the contents of the microplate were mixed and medium were collected for determination of glucose concentration.

Determination of glucose concentration.

Glucose concentration in the medium after incubation of the cells with the studied compounds was determined by the oxidase method with fluorimetric detection in 384-well plates. To the tested sample were added an equal volume of the reagent 0.4 U/ml peroxidase, 4 U/ml glucose oxidase and 200 μΜ 10-Acetyl-3,7-dihydroxyphenoxazine in 50 mM potassium phosphate buffer pH 7.4. After 30 minutes of incubation at 37°C was followed by a measurement of the fluorescence intensity at an excitation wavelength of 530 nm and emission wavelength 580 nm. The glucose concentration was calculated using the standard curve. Glucose utilization by cells was calculated as the difference in glucose concentration in the experimental medium and the medium after incubation of the compounds with the cells.

Muscle cells are the major organ responsible for glucose utilization and regulating blood glucose levels, and are an important effector organ in the action of various antidiabetic agents. The effect of compounds, being the subject of the invention on the glucose utilizationwas tested using muscle cells (C2C12) and additionally adipocytes (3T3-L1), and hepatocytes (HepG2), and compared to the effects of rosiglitazone, which is used in the treatment of type 2 diabetes and the effect of oxovanadium(IV) maltolate VO(mal)₂, with proven potent action (Tables 2, 3 and 4). Glucose utilizationis expressed in relation to the control not containing vanadium compounds.

Table 2. The effect of vanadium complexes (50 μΜ) and rosiglitazone (50 μΜ) on glucose utilizationby C2C12 cells in the presence (34.5 nM) and absence of human recombinant insulin.

Table 3. Effect of vanadium complexes (50 μΜ) and rosiglitazone (50 μΜ) on glucose utilizationby 3T3-L1 cells.

Complex Valency of Substituents in the formula Glucose

vanadium 1 , R6 of group A-I utilization(%)

[V(ONO)₂] III R₁ ⁼R₂ ⁼R4⁼R5= H 124

R₃=Br

R6=A

R₇= t-Bu

[VO(ONO)(phen)]^«2H₂0 IV R₁=R₂=R₄=R₅= H 136

R₃=Br R6=A

R₇= OH

[VO(ONO)(OC₂H₅)] V 110

[VO(mal)₂] IV 116

Rosiglitazone 112

Table 4. The effect of vanadium complexes (50 μΜ) on glucose utilization by HepG2 cells.

Example 8. Studies on inhibition of tyrosine phosphatase IB (PTP1B)

To the solution of the studied compound in the well of a 96- well microplate were added equal volumes of 0.6 mM6,8-difluoro-4-methylumbelliferyl phosphate and 1.5 U/ml human recombinant PTP1B phosphatase in the reaction buffer pH 7, 0:25 mM of 3 - (N-morpholino) propanesulfonic acid, 50 mM of sodium chloride, 1 mM of dithiothreitol and 0.05% of Tween-20. After 30 minutes of incubation at 37°C a measurement of the fluorescence intensity was measuredat an excitation wavelength of 355 nm and emission wavelength of 560 nm (Welte, S., Baringhaus KH, Schmider W., Muller, G., S. Petry, Tennagels N., Anal. Biochem ., 338 (2005) 32). The results were expressed as percentage of the activity relative to the control sample not containing an inhibitor. The study demonstrated a strong inhibitory effect of complex vanadium compounds phosphatase activity (Table 5).

Table 5. Effect of vanadium complexes (1 μΜ) on the activity of human recombinant PTPIB phosphatase.

The enzymatic activity of PTPIB was given in relation to the control not containing vanadium compounds whose activity was assumed to be 100%. The complex bis(maltolato)oxo- vanadium(IV) [VO(mal)₂], is a reference complex described in the literature.

Claims

Patent Claims 1. Vanadium complexes of the general formula M[VX(ONO)_y(L)_n]-mS, where:

- X is oxygen atom or is absent

- L is Li or L₂, wherein:

- L_t represents a halogen anion or a neutral or a deprotonated solvent molecule selected from the group consisting of C Ci₂ alcohols and/or water;

- L₂ is a neutral or anionic NN, NO or OO-donor ligand selected from the group consisting of: polypyridine, 1,10-phenanthroline, pyrones, quinoline, or pyridine carboxylic acids;

- wherein, when X is absent, then y = 2 and n = 0;

- and when X is present then y = 1, wherein, when L=L_\ then n is 1 or 2, and when n is 2 then Li are the same or different, and when L=L₂, then n is 1 ;

- S is a neutral solvent molecule selected from the group consisting of C 1-C4 alcohols, water or sulfuric acid;

- m varies from 0 to 4;

- M may be absent, and when present it means monocharged alkali metal cation, ammonium cation or alkylammonium cation;

- ONO, where the letters O and N represent atom, through which the ligand is bound to vanadium, it means a tridentate ligand of the general formula 1 ;

in the keto or enol form, neutral or deprotonated, wherein Ri, R₂, R₃, R, independently represent hydrogen atom, fluorine atom, chlorine atom, bromine atom, iodine atom or a hydroxo, nitro, alkoxy, aryloxy, dialkylamino, alkyl or aryl group, wherein at least one of the Ri, R₂, R₃, R4 substituents is different from hydrogen atom, R5 independently stands for hydrogen atom, alkyl or aryl group, R^ is a group selected from: hydrogen atom, alkyl or aryl group, R6 is a group selected from:

(A) (B) (C) (D) (E)

(F) (G) (H) (I) wherein R7 substituent independently stands for a hydroxo, nitro, alkoxy, aryloxy, alkyl group or hydrogen atom, fluorine atom, chlorine atom, bromine atom, iodine atom.

2. Complexes, according to claim 1 , characterized in that LI indicates ethyl alcohol or methyl alcohol.

3. Complexes, according to claim 1, characterized in that L2 indicates 2,2 '-bipyridine, deprotonated 3-hydroxy-2-methyl-4-pyrone, deprotonated 8-hydroxyquinoline, deprotonated 2-picolinic acid.

4. Complexes according to claim 1 , characterized in that M is Na⁺, K⁺, Me₄N⁺, Et₄ N⁺.

5. Complexes according to claim 1, characterized in that they exhibit the geometry of a deformed tetragonal pyramid or deformed tetragonal bipyramid.

6. A process for preparation of vanadium complexes of the general formula defined in claim 1, in a two-step reaction, characterized in that in the first step, the compound of formula 2.

Formula 2

wherein Rl, R2, R3, R4 and R5 are as defined in claim 1, is condensed with a hydrazide of formula 3

Formula 3

in which R6 is as defined in claim 1 wherein the condensation reaction is carried out at a 1 :1 molar ratio or with a stoichiometric excess of one of the reactants, in a solvent which is a Ci- Ci₂ alcohol or water or aqueous alcoholic solution, and in the second step the condensation product is subjected to complexation reaction using the original vanadium complex: VOSO4, VO(acac)₂, VOCl₂ or V(acac)₃, possibly with the addition of deprotonation agent, wherein the complexation reaction is carried out at a 1 :1 : 1 molar ratio of vanadium to compound 2 and compound 3 and in the case of vanadium(IV) or (V) , and at 1 :2:2 molar ratio of vanadium to compound 2 and compound 3 in the case of vanadium(III) complexes, or with a stechiometric excess or deficiency of vanadium, after which ligand L₂ described in claim 1 may be added wherein ligand L₂ is introduced at a 1:1 molar ratio of vanadium to ligand or in a stoichiometric excess or deficiency relative to vanadium.

7. A method according to claim 6, characterized in that the two steps of the synthesis are carried out in the temperature range from -130 to 260 ⁰ C and in the pressure range from 0.01 to 1 MPa.

8. A method according to claim 6 or 7, characterized in that ammonia, alkylamine or arylamine are used as deprotonation agent.

9. A method according to claim 6 or 7, characterized in that the product of the first step is separated from the reaction medium and then introduced into the second reaction step.

10. Pharmaceutical preparations for use in human and veterinary medicine containing vanadium complexes defined in claim 1 and pharmaceutically acceptable excipients.

11. Preparations according to claim 10, characterized in that they are in solid or liquid form.

12. Preparations according to claim 10, characterized in that they contain additional substances having a good therapeutic effect and/or vitamins and/or mineral salts and/ or plant extracts and/or dietary supplements.

13. The use of vanadium complexes as defined in claim 1 for the preparation of pharmaceutical products for the prevention and treatment of metabolic diseases and disorders and/or diseases or disorders associated with the activity of phosphatases.

14. The use according to claim 13, characterized in that the vanadium complexes are used for the making of preparations for the prevention and treatment of diabetes, impaired glucose tolerance and obesity.

15. The use according to claim 13, characterized in that the phosphatase is a protein phosphatase.

16. The use according to claim 15, characterized in that the protein phosphatase is a tyrosine phosphatase.

17. The use according to claim 15, characterized in that the vanadium complexes are used in the prevention and treatment of glucose metabolism disorders, the prevention and treatment of neoplastic diseases, inflammatory diseases, autoimmune diseases and the nervous system disorders (neurodegenerative, memory, psychiatric), bone diseases, infectious diseases.

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