Title of Invention	"BINUCLEAR NIRTOSYL-IRON COMPLEXES WITH BENZO-TRANS-HETEROCYCLIC DERIVATIVES AND A MEHOD FOR THE PRODUCTION THEREOF"
Abstract	The instant invention relates to binuclear nitrosyl iron complexes with benzo-azaheterocycloic derivatives of general formula [Fe2(SR)2(NO)4], wherein R is (I), wherein X is NH, S or O, R1 is lower alkyl, to a method for prreparation thereof, to the use thereof as an anticancer agent, to a nitrogen monoxide donor based thereon, to a pharmaceutical composition containing said complexes in an effective amount, and to a kit used for treating oncological diseases.

Title of Invention

"BINUCLEAR NIRTOSYL-IRON COMPLEXES WITH BENZO-TRANS-HETEROCYCLIC DERIVATIVES AND A MEHOD FOR THE PRODUCTION THEREOF"

Abstract

The instant invention relates to binuclear nitrosyl iron complexes with benzo-azaheterocycloic derivatives of general formula [Fe2(SR)2(NO)4], wherein R is (I), wherein X is NH, S or O, R1 is lower alkyl, to a method for prreparation thereof, to the use thereof as an anticancer agent, to a nitrogen monoxide donor based thereon, to a pharmaceutical composition containing said complexes in an effective amount, and to a kit used for treating oncological diseases.

Full Text	BINUCLEAR NITROSYL IRON COMPLEXES WITH BENZO-AZAHETEROCYCLIC DERIVATIVES AND A METHOD FOR THE PREPARATION THEREOF Field of the invention The invention relates to binuclear nitrosyl iron complexes and may be used in medicinal practice to produce new generation medicinal agents for treating oncological diseases. Background of the invention In recent years in order to create medicinal preparations of a new generation for use in the therapy of oncological diseases, an intensive search is being carried out for anticancer agents based on complexes of transition metals with an improved activity spectrum and reduced side effects as compared with already used clinical preparations, for example, cisplatin, sodium nitroprusside, et al. Interest in nitrosyl metal complexes has increased due to the discovered possibilities for their use as effective NO donors in medicine, in particular, for the therapy of cancer diseases. The use of NO donors as a new class of anticancer agents is related to the important role played by NO in the process of growth of malignant tumors [Wink D., Vodovoz J., Cook J., Biochemistry, 1998, 63, 7, pp. 948-957]. It is shown that nitrogen monoxide changes the level of apoptosis of the tumor cells, the activity of the p53 gene and neoangiogenesis [Brune B., Scheneiderhan N., Nitric oxide evoked p53-accumulation and apoptosis, Toxicol Letters, 2003, 193, 2, pp. 19-23], inhibits the activity of a key reparation protein O6-methyl-guanine-DNA-methyl-transferase of mammals [L Liu, M. Xu-Welliver, A. Kanagula, H.E. Pegg, Inactivation and degradation of 06-alkylguanine-DNA alkyltransferase after reaction with nitric oxide., Cancer Res., 2002, 62, pp. 3037-3043]. Binding NO to active sites of metal enzymes, in particular to non-heme iron-containing proteins, is studied especially intensively [Ford P.C., Lorkovic I.M., Chem. Rev., 2002, 102, 993; Hoshino M., Laverman I.E., Ford P.C., Coord. Chem. Rev., 1999, 187, p. 75]. It has been established that nitrosyl iron complexes with thiol-containing ligands are one of the forms of natural reservoirs of NO [Butler A.R., Megson I.I., Chem. Rev. 2002, 102, pp. 1155-1165], Of the synthetic models, Roussin's "red salt" esters have the formula [Fe2(SR)2(NO)4], where R = Et, t-Bu, (CH2)4-CH3, C6H5F, Ph [T. Thomas, J.H. Robertson, E.G. Cox, Acta. Crystalogr., 1958, 11, p. 599; C. Glidewell, M.E. Harman, M.B. Hursthouse, I.L. Johnson, M. Motevalli, J. Chem. Res., 1998, 212, p. 1676; R.E. Marsh, A.L. Spek, Acta. Crystalogr., Sect. B. Struct. Sci., 2001, 57, p. 800; C. Jinhua, M. Shaoping, H. Jinling, I. Jiaxi, Chinese J. Struct. Sci., 2001, 57, p. 800; C. Jinhua, M. Shaoping, H. Jinling, I. Jiaxi, Chinese J. Struct. Chem., 1983, 2, p. 263; T.B. Rauchfuss, T.D. Weatherill, Inorg. Chem., 1982, 21, pp. 827-830]. These binuclear diamagnetic sulfur-nitrosyl complexes with R = Alk generate NO upon thermo- or photoactivation [J.L. Bourassa, P.C. Ford, Coord. Chem. Rev., 2000, 200-202, pp. 887-900] and may serve as new promising anticancer NO donor agents. Also known are binuclear paramagnetic sulfur-nitrosyl iron complexes with ligands of the µ-N-C-S type, which are synthetic analogues of natural donors of NO - dinitrosyl iron complexes (DNIC) with cysteine, glutathione and other thiol-containing ligands of low molecular weight [O.A. Rakova, N.A. Sanina, S.M. Aldoshin, N.A. Goncharova, G.V. Shilov, Yu.M. Shulga, N.S. Ovanesyan, Inorganic Chemistry Communications 2003, 6, 145-148; A.F. Vanin, N.A. Sanina, V.A. Serezhenkov, D.Sh. Burbaev, V.I. Lozinsky and S.M. Aldoshin, Nitric oxide: biology & chemistry 2007, 16,82-93]. However, the synthetic NO donors of different classes that are known at the present time are not used as therapeutic agents for the treatment of oncological diseases. They are only used to enhance (to a different degree depending on the chemical nature) the action of existing chemotherapeutic agents or of radiotherapy [Wink D., Vodovoz J., Cook J., Biochemistry, 1998, 63, 7, pp. 948-957; N.P. Konovalova, S.A. Goncharova, L.M. Volkova, T.A. Raevskaya, L.T. Eremenko, A.M. Korolev, Nitric Oxide: Biology and Chemistry, 2003, 8, pp. 59-64; Yang W., Rogers P.A., Ding H., J. Biol. Chem., 2002, 277, pp. 12868-12873; O. Siri, A. Tabard, P. Pullumbi, R. Guilard, Inorg. Chim. Acta 2003, 350, p. 633; J.L. Burgaud, E. Jingini, Del Soldata P. Ann., N.Y. Acad. Sci. 2002, 962, p. 360; T.I. Karu, L.V. Pyatibrat, G.S. Kalendo, Toxicology Letters, 2001, 121, p. 57]. In the article by A. Janczyk et al., Nitric Oxide, 2004, 10, 1, pp. 42-50, a study is made of the direct cytotoxic action of a nitrosyl iron complex Na[Fe4S3(NO)7] on the human and mouse melanoma cell lines. However, this nitrosyl iron complex generates NO upon photoactivation (even in the dark) and cannot be used as an anticancer agent either because of the high toxicity in respect to normal cells. So, there is a demand for other anticancer agents with higher effectiveness and reduced toxicity. The object of the instant invention is to expand the arsenal of anticancer agents and create anticancer agents based on complexes of transition metals with an improved activity spectrum and reduced side effects, in particular, an agent based on a tetra-nitrosyl binuclear iron complex, acting as a NO donor, with enhanced activity and reduced toxicity. Summary of the invention In one aspect the invention relates to new binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of formula (Formula Removed) wherein R is (Formula Removed) wherein X is NH, S or 0, R1 is lower alkyl. In another aspect, the invention relates to a method for preparing binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of formula (Formula Removed) by treatment of a thiosulfate nitrosyl iron complex with a corresponding benzo-azaheterocyclic thiol in a stoichiometric ratio in the presence of a reducing agent, in an alkaline medium. This method makes it possible for the first time to obtain the claimed complexes in crystalline form. In an additional aspect, the instant invention relates to a donor of nitrogen monoxide, which is a binuclear nitrosyl iron complex with benzo-azaheterocyclic derivatives that is characterized above. In the next aspect, the instant invention relates to the use of binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of formula (Formula Removed) as an anticancer agent. In an additional aspect, the instant invention relates to the use of binuclear nitrosyl iron complexes benzo-azaheterocyclic derivatives of formula (Formula Removed) for preparation of an anticancer pharmaceutical. In the next aspect, the instant invention relates to a pharmaceutical composition comprising an effective amount of a binuclear nitrosyl iron complex with benzo-azaheterocyclic derivatives of formula (Formula Removed) and a pharmaceutically acceptable carrier. In the next aspect, the instant invention relates to a kit used for treating oncological diseases and comprising (1) a pharmaceutical composition comprising a binuclear nitrosyl iron complex with benzo-azaheterocyclic derivatives of formula (Formula Removed) wherein R has the aforesaid meanings, in a sealed container; and (2) auxiliary agents. Description of the Figures In Fig. 1 - the molecular structure of the complex (Formula Removed) [complex I] is presented. The complex has a centrosymmetrical dimeric binuclear structure (Fig. 1). In the dimer two tetrahedrally coordinated by two NO groups and two benzo-azaheterocyclic thiolyls of the iron atom are linked by a bridge: Fe-S-C-N-Fe'. The complex comprises two molecules of the acetone solvent. In Fig. 2 - the crystalline structure of the complex (Structure Removed) [complex I] is presented. In Fig. 3 - a change of the difference spectra of absorption in time upon interaction of complex I (Formula Removed) with hemoglobin (Hb) is shown. The solvent - 0.05% phosphate buffer, pH = 7.0, comprising 3.3% dimethylsulfoxide, 25°C. Detailed disclosure of the invention The new binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives in accordance with the instant invention have the general formula (Formula Removed) wherein R is wherein X is NH, S or O, R1 is lower alkyl. The term "lower alkyl" that is used here means an alkyl radical with a straight or branched chain comprising from 1 to 6 carbon atoms. Examples of such alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl. Information on the claimed compounds and the claimed method for the preparation thereof is not present in the state of the art. The method for preparing new binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of formula (Formula Removed) wherein R has the aforesaid meanings, consists in that a thiosulfate nitrosyl iron complex bis(µ-thiosulfato-S)-bis(dinitrosylferrate)(2-) sodium Na2 (Formula Removed) (TNIC) is treated with a corresponding benzo-azaheterocyclic thiol in a stoichiometric ratio in the presence of a reducing agent, and the process is carried out in an alkaline medium with the subsequent isolation of the desired product by known techniques. It is preferable that the process be carried out at room temperature, for the most part at 18-25°C. It is preferable that the process be carried out in an oxygen-free atmosphere. It is preferable that benzimidazole-2-thiol, 5-methylbenzimidazole-2-fhiol or benzthiazole-2-thiol be used as the benzo-azaheterocyclic thiol. Hydrogen, thiosulfates of metals, for example, Na2S2O3 5H2O, hydrogen sulfide and aliphatic thiols are used as the reducing agent in the method according to the instant invention. The authors of the invention have established that binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of the instant invention are effective No donors, releasing nitrogen monoxide spontaneously upon decomposition in proton mediums (such as water, blood and components thereof, physiological solutions, etc.) in the absence of hemo-, photo- or enzymatic activation. So, the complexes according to the instant invention are a new, promising class of nitrogen monoxide donors - synthetic analogues of active sites of nitrosyl nonhemo iron-sulfur proteins - natural NO depot. Therefore, the instant invention in the additional aspect relates to nitrogen monoxide donors, which are binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives that are presented above. The binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of natural aromatic thiols of the instant invention have anticancer activity in respect to cancer cells of a mammal, in particular, a human. The instant invention is further directed to the use of binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of formula (Formula Removed) where R has the aforesaid meanings, as an anticancer agent. In particular, the compound of the instant invention may be used for treatment of the following oncological diseases: ovarian carcinoma, breast adenocarcinoma, melanoma B-16, epidermoid Lewis carcinoma. The iron complexes in accordance with the instant invention are suitable for inhibiting the growth of cancer cells in mammals, and it is preferable that they be administered in the form of a pharmaceutical composition comprising an effective antitumor amount of the compound in accordance with the instant invention in combination with at least one pharmaceutically or pharmacologically acceptable carrier and/or excipient. The carrier, also known from the state of the art as an excipient, filler, ancillary substance, additive or diluent, is any substance that is pharmaceutically inert, provides a corresponding consistence or form to the composition and does not weaken the therapeutic efficacy of the anticancer compounds. The carrier is "pharmaceutically or pharmacologically acceptable," if it does not cause an adverse, allergic or other unfavorable reaction upon the administration to a mammal or human, respectively. The instant invention also relates to the use of binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of formula (Formula Removed) wherein R has the aforesaid meanings, for the preparation of an anticancer pharmaceutical. The instant invention additionally proposes pharmaceutical compositions comprising an effective amount of binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of formula (Formula Removed) wherein R has the aforesaid meanings, and a pharmaceutically acceptable carrier. It is preferable that a proton-containing medium be used in the composition according to the invention as the pharmaceutically acceptable carrier. It is also preferable that a mixture of the proton-containing medium and dimethylsulfoxide be used as the pharmaceutically acceptable carrier in the composition according to the invention. It is preferable that water, a physiological solution, water-soluble biopolymers be used as the proton-containing medium. It is preferable that the binuclear nitrosyl iron complex with a benzo-azaheterocyclic derivative be present in the composition in an amount of 50-100 µM. The pharmaceutical compositions containing the anticancer compound in accordance with the instant invention may be produced by any conventional method. The necessary preparative form is selected depending on the selected method of administration. Compositions in accordance with the invention may be produced for any method of administration as long as the target tissue is available via that route of administration. Suitable routes of administration include, but are not limited to, oral, parenteral (e.g., intravenous, intraarterial, subcutaneous, rectal, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal), topical (nasal, transdermal, intraocular), intravesical, intrathecal, into the small intestine, pulmonary, intralymphatic, intracavital, vaginal, transurethral, intradermal, aural, intramammary, buccal, orthotopic, intratracheal, intralesional, percutaneous, endoscopical, transmucosal, sublingual and intestinal administration. Pharmaceutically acceptable carriers for use in the compositions of the present invention are well known to one of ordinary skill in the art and are selected based upon a number of factors: the particular anticancer compound used and its concentration, stability and expected biological availability; the disease, disorder or condition of the human being treated with the composition; the subject, its age, weight and general condition; and the method of administration. Suitable carriers are readily determined by one of ordinary skill in the art [J.G. Nairn in: Remington's Pharmaceutical Science (A. Gennaro, ed.), Mack Publishing Co., Easton, Pa. (1985), pp. 1492-1517]. The compositions are preferably produced as tablets, dispersible powders, pills, capsules, gelcapsules, coated caplets, gels, liposomes, granules, solutions, suspensions, emulsions, syrups, elixirs, troches, dragees, lozenges, or any other dosage form, which can be administered orally. The compositions of the instant invention for oral administration comprise an effective amount of a compound of the invention in a pharmaceutically acceptable carrier. Suitable carriers for solid dosage forms include sugars, starches and other conventional substances including lactose, talc, sucrose, gelatin, carboxymethyl cellulose, agar, mannitol, sorbitol, calcium phosphate, calcium carbonate, sodium carbonate, kaolin, alginic acid, acacia, corn starch, potato starch, sodium saccharin, magnesium carbonate, tragacanth, microcrystalline cellulose, colloidal silicon dioxide, crosscarmellose sodium, talc, magnesium stearate, and stearic acid. Further, such solid dosage forms may be uncoated or may be coated by known techniques, e.g., to delay disintegration and absorption. The anticancer compounds of the present invention are also preferably used for the production of a dosage form for parenteral administration, e.g., formulated in a dosage form for injection via intravenous, intraarterial, subcutaneous, rectal, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal routes. The compositions of the invention for parenteral administration comprise an effective amount of the anticancer compound in a pharmaceutically acceptable carrier. Dosage forms suitable for parenteral administration include solutions, suspensions, dispersions, emulsions or any other dosage form which can be administered parenterally. Techniques and compositions for making parenteral dosage forms are known from prior art. An insignificant amount of additional components, which are well-known in the pharmaceutical industry, can be included in the composition of the invention. These components to a high degree provide properties which increase the time of retention of the anticancer compound at the site of administration, promote stability of the composition, provide control of the pH, facilitate the introduction of the anticancer compound into the pharmaceutical compositions, and the like. Each of these components is individually present in an amount that is preferably less than about 15 weight %, more preferably less than about 5 weight %, and most preferably less than about 0.5 weight % of the total weight of the composition. Some components, such as fillers or diluents, can constitute up to 90 weight % of the total weight of the composition, as is well-known in the technology of producing medicinal agents. Such additives include cryoprotective components for preventing precipitation of the iron complex with thiophenol, surfactants, wetting or emulsifying agents (e.g., lecithin, polysorbate-80, Tween® 80, pluronic 60, polyoxyethylene stearate), preservatives (e.g., ethyl-n-hydroxybenzoate), agents protecting against the action of microbes (e.g., benzyl alcohol, phenol, m-cresol, chlorobutanol, sorbic acid, thimerosal and paraben), agents for adjusting pH or buffering agents (e.g., acids, bases, sodium acetate, sorbitan monolaurate), components for adjusting osmolarity (e.g., glycerin), thickeners (e.g., aluminum monostearate, stearic acid, cetyl alcohol, stearyl alcohol, guar gum, methyl cellulose, hydroxypropyl cellulose, tristearin, cetyl wax esters, polyethylene glycol), colorants, dyes, flow aids, non-volatile silicones (e.g., cyclomethicone), clays (e.g., bentonites), adhesives, bulking agents, flavorings, sweeteners, adsorbents, fillers (e.g., water, saline, electrolyte solutions), binders (e.g., starches such as maize starch, wheat starch, rice starch or potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, sugars, polymers, acacia), disintegrating agents (e.g., starches such as maize starch, wheat starch, rice starch, potato starch or carboxymethyl starch, structurated polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate, croscarmellose sodium or crospovidone), lubricants (e.g., silica, talc, stearic acid or salts thereof such as magnesium stearate, or polyethylene glycol), coating agents (e.g., concentrated sugar solutions including gum Arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol or titanium dioxide) and antioxidants (e.g., sodium metabisulfite, sodium bisulfite, sodium sulfite, dextrose, phenols and thiophenols). In accordance with a preferred variant of embodiment, the pharmaceutical composition of the invention comprises at least one nonaqueous pharmaceutically acceptable solvent and an anticancer compound having a solubility therein of at least about 10-60 mg/ml. While not being bound to any particular theory, it is believed that the solubility in the dimethylsulfoxide of the anticancer compound may be directly related to its efficacy. It is also preferable that the anticancer compound has an ID100 value (i.e., the concentration of the agent causing 100% inhibition of colony formation) that is at least 4 times less than that of cisplatin when measured according to the method described in the book "Experimental assessment of antitumor preparations in the USSR and the USA," edited by E.P. Sofina, A.B. Sirkin (USSR), A. Goldin, A. Cline (USA), Moscow,: "Meditsina," 1979, pp. 71-105. Dosage form administration by these routes may be continuous or intermittent, depending, for example, upon the patient's physiological condition, on whether the purpose of the administration is therapeutic or prophylactic, and other factors known to or assessable by a skilled practitioner. The dosage and regimens for the administration of the pharmaceutical compositions of the invention can be readily determined by an oncologist. It is understood that the dosage of the anticancer compounds depends on the age, sex, health and weight of the recipient, the kind of concurrent treatment, if any, frequency of treatment and the nature of the desired effect. For any mode of administration, the exact amount of the used anticancer compound and also the prescribed dose that is necessary for achievement of the advantageous effects herewith described also depend in particular on such factors as the bioavailability of the anticancer compound, the human disorder being treated, the desired therapeutic dose and other factors that are obvious to one skilled in the art. The concentration of the anticancer compound of the instant invention in a liquid pharmaceutical composition is, most preferably, 50-100 µM As a rule, relatively low concentrations are preferable, since the anticancer compound is most soluble at low concentrations. Emulsions for parenteral administration can be produced by dissolving an anticancer compound in any pharmaceutically acceptable solvent capable of dissolving the compound (e.g., DMSO, dichloromethane) to form a solution. An appropriate volume of a carrier which is an emulsion, such as a Liposyn II or Liposyn III emulsion, is added to the solution while stirring to form a pharmaceutically acceptable emulsion for parenteral administration to a patient. If desired, such emulsions can be produced with a content of a minimal amount of, or without, a Cremophor® solution. Solutions for parenteral administration can be produced by dissolving an anticancer compound in any pharmaceutically acceptable solvent capable of dissolving the compound (e.g., DMSO) to form a solution. An appropriate volume of a carrier which is a proton-containing medium (saline solutions, sugar solutions, water-soluble polymers, proteins, electrolyte solutions) is added to the solution for parenteral administration to a patient. So, for example, for liquid preparative forms, nonaqueous pharmaceutically acceptable polar solvents, such as oils, alcohols, amides, esters, ethers, ketones, hydrocarbons and mixtures thereof, and also water, saline solutions, dextrose solutions (e.g., DW5), electrolyte solutions or any other pharmaceutically acceptable proton medium, are usually used as the carrier. Suitable nonaqueous pharmaceutically acceptable polar solvents include, but are not limited to, amides (e.g., dimethylacetamide (DMA), dimethylacetamidebenzylbenzoate, dimethylformamide, N-(P-hydroxyethyl) lactamide, N,N-dimethylacetamide, 2-pyrrolidinone, 1 - methyl-2-pyrrolidinone or polyvinylpyrrolidone); esters (e.g., acetic acid esters, such as monoacetin, diacetin and triacetin; aliphatic or aromatic esters, such as ethyl caprylate or ethyl octanoate, alkyl oleate, benzyl benzoate, benzyl acetate, dimethylsulfoxide (DMSO), esters of glycerin such as mono-, di- or triglyceral citrates or tartrates, ethyl benzoate, ethyl acetate, ethyl carbonate, ethyl lactate, ethyl oleate, fatty acid esters of sorbitan, fatty acid derived polyethyleneglycol (PEG) esters, glyceryl monostearate, glyceride esters such as mono-, di- or triglycerides, fatty acid esters such as isopropyl myristrate, fatty acid derived PEG esters such as PEG-hydroxyoleate and PEG-hydroxystearate, N-methyl pyrrolidinone, pluronic-60, polyoxyethylene sorbitol oleic acid polyesters such as poly(ethoxylated)3o-6osorbitol poly(oleate)2-4, poly(oxyethylene) 15-20monooleate, poly(oxyethylene) 15-2omono-12- hydroxystearate and poly(oxyethylene)15-20mono ricinoleate; polyoxyethylene sorbitan esters, such as polyoxyethylene-sorbitan monooleate, polyoxyethylene-sorbitan monopalmitate, polyoxyethylene-sorbitan monolaurate, polyoxyethylene-sorbitan monostearate, and Polysorbate® 20, 40, 60 or 80 from the ICI Americas firm, Wilmington, DE; polyvinylpyrrolidone, alkylene oxides modified fatty acid esters, such as polyoxyl 40 hydrogenated castor oil and polyoxyethylated castor oils (e.g., Cremophor® EL solution or Cremophor® RH 40 solution); fatty acid and saccharide esters (i.e., the condensation product of a monosaccharide (e.g., pentoses, such as ribose, ribulose, arabinose, xylose, lyxose and xylulose; hexoses, such as glucose, fructose, galactose, mannose and sorbose; trioses, tetroses, heptoses, and octoses), disaccharides (e.g., sucrose, maltose, lactose and trehalose) or oligosaccharide or mixtures thereof with a fatty acid (fatty acids) with 4-22 carbon atoms (e.g., saturated fatty acids, such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid and stearic acid, and unsaturated fatty acids, such as palmitoleic acid, oleic acid, elaidic acid, erucic acid and linoleic acid)), or steroidal esters); alkyl, aryl or cyclic ethers having 2-30 carbon atoms (e.g., diethyl ether, tetrahydrofuran, dimethyl isosorbite, diethylene glycol monoethyl ether); glycofurol (tetrahydrofurfuryl alcohol polyethylene glycol ether); ketones having 3-30 carbon atoms (e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone); aliphatic, cycloaliphatic or aromatic hydrocarbons having 4-30 carbon atoms (e.g., benzene, cyclohexane, dichloromethane, dioxolanes, hexane, n-decane, n-dodecane, n-hexane, sulfolan, tetramethylenesulfon, tetramethylenesulfoxide, toluene, dimethylsulfoxide (DMSO), or tetramethylenesulfoxide); alkyl- or arylhalogenides comprising 1-30 carbon atoms and optionally more than one halogen atom as a substituent; dichloromethane; monoethanolamine; petroleum ether; trolamine; omega-3 polyunsaturated fatty acids (e.g., alpha-linolenic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid); polyglycol ester of 12-hydroxystearic acid and polyethylene glycol (Solutol® HS-15 from the BASF firm, Ludwigshafen, Germany); polyoxyethylene glycerol; sodium laurate; sodium oleate; or sorbitan monooleate. In the instant invention it is preferable to use proton-comprising mediums, such as water, saline solutions, water-soluble polymers, proteins, dextrose solutions (e.g., DW5), electrolyte solutions or alcohols from the "PAA Laboratory's" catalogue, (2006), p. 26, as the pharmaceutically acceptable carrier. In the next aspect of the instant invention, a kit is proposed for treatment of oncological diseases, which kit comprises: (1) a pharmaceutical composition comprising a binuclear nitrosyl iron complex with the benzo-azaheterocyclic derivative of the instant invention, in a sealed package; and (2) ancillary agents. The kit may comprise the composition in the form of a single dosage unit or in the form of multiple doses. The kit may contain forms for oral or parenteral administration. The pharmaceutical composition in the kit may be placed in glass or polymeric vials, ampoules, flasks, dosage cartridges for injectors, blisters, capsules, containers with the composition, that are used respectively for the oral or parenteral form. The auxiliary agents comprise liquids for restoration of a composition administered parenterally, if it is presented in the kit in a concentrated form, for example, in the form of a dry substance, dried preparation, etc.; agents for the preparation of oral liquid forms and forms for injection ex tempore. Water for injection, a physiological solution, lidocaine solution, etc. may be used as the liquid for restoration. A solution of glucose, sugars, syrup, etc. may be used for restoration of a composition that is used orally in a liquid form. Optional auxiliary agents of the kit include agents for opening lids, agents for sealing opened reusable lids, enclosed instructions. A pharmaceutical composition which is a solid dosage form for oral administration may be presented in the kit in the form of tablets, capsules in blisters, ampoules, vials, bubbles, packets, etc. A pharmaceutical composition, which is a liquid dosage form for parenteral or oral administration, may be presented in the kit in vials, capsules, ampoules, cartridges, etc. An example of a kit for parenteral administration includes a container in which instructions for use, ampoules or vials with the dry composition and ampoules with a physiological solution for injection are arranged. A device for opening the ampoules is arranged in the container. The ampoules are packed in 10 ampoule blisters. Other variants of the kit are obvious to one skilled in the art in this field from the specification presented above. The following examples are provided only as an additional illustration of the invention and they should not be given consideration as limitation of the invention. Examples of synthesis of the complexes Example 1 Synthesis of the complex (I), where R = benzimidazol-2-yl (Formula Removed) The initial reagents Na2S2O35H2O (Aldrich), NaOH (pro analyze), 2-mercaptobenzimidazole (Aldrich) were used for synthesis. All of the operations as regards the preparation of and mixing the solutions and isolating the complexes were carried out in a technical argon atmosphere at room temperature. An organic solvent was used for recrystallization. A mixture of 0.992 g (4 mmol) of Na2S2O35H2O and 1.074 g (2 mmol) of a thiosulfate nitrosyl iron complex bis(µ-thiosulfato-S)-bis(dinitrosylferrate)(2-) sodium (Formula Removed) (TNIC) was dissolved in distilled water. A mixture consisting of 1.500 g (10 mmol) of 2-mercapto-5-methylbenzimidazole and 0.44 g (11 mmol) of NaOH was also dissolved in water. Both solutions were mixed. The precipitate was filtered off and dried in air. The yield of the product was 0.719 g. After 24 hours the precipitate was recrystallized. The complex dissolves well in ethanol, methanol, acetone, DMSO and DMFA. It does not dissolve in dichloromethane, water, heptane and ether. Yield: 63.50%. Determined for Fe2S2N8C14H10O4, %: Fe, 19.27; S, 10.69; N, 18.54; C, 43.40; H, 4.78; calculated, %: Fe, 18.99; S, 10.90; N, 19.05; C, 44.93; O, 16.32; H, 4.45. IR-spectrum (cm"1): 3207, 1787, 1738, 1515, 1467, 1428, 1384, 1272, 1218, 1183, 1002, 742,713,603. Example 2 Synthesis of the complex (II), where R = 5-methylbenzimidazol-2-yl (Fe2S2N8C16H14CO4 The initial reagents Na2S2O35H2O (Aldrich), NaOH (pro analyze), 2-mercapto-5-methylbenzimidazole (Aldrich) were used for synthesis. All of the operations as regards the preparation of and mixing the solutions and isolating the complexes were carried out in a technical argon atmosphere at room temperature. An organic solvent was used for recrystallization. Distilled water was prepared for the synthesis, passing a flow of technical argon through it for 30 minutes. A mixture of 0.4960 g (2 mmol) of Na2S2O3 5H2O and 0.5740 g (1 mmol) of a thiosulfate nitrosyl iron complex bis(µ-thiosulfato-S)-bis(dinitrosylferrate)(2-) sodium Na2[Fe2(S2O3)2(NO)4] (TNIC) was dissolved in distilled water. A mixture consisting of 0.8250 g (5 mmol) of 2-mercapto-5-methylbenzimidazole and 0.2890 g (7 mmol) of NaOH was also dissolved in water. Both solutions were mixed. The precipitate was filtered off and dried in air. The yield of the product was 2.2017 g. After 24 hours the precipitate was recrystallized. The complex dissolves well in acetone, DMSO and DMFA. It does not dissolve in dichloromethane, water, heptane and ether. Yield: 45.53%. Determined for Fe2S2N8C16H14O4, %: Fe, 19.27; S, 10.69; N, 19.54; C, 34.26; H, 2.48; calculated, %: Fe, 20.01; S, 11.49; N, 20.08; C, 34.43; O, 11.47; H, 2.53. IR-spectrum (cm'1): 3133, 1790, 1724, 1619, 1490, 1443, 1411, 1373, 1276, 1223, 1189, 1011,854,800,720,596,543. Example 3 Synthesis of complex (III), wherein R = benzothiazol-2-yl (Fe2S4N6C14H8O4) The initial reagents Na2S2O3 5H2O (Aldrich), NaOH (Pro analyze), 2-mercaptobenzothiazole (Aldrich) were used for synthesis. All of the operations as regards the preparation of and mixing the solutions and isolating the complexes were carried out in a technical argon atmosphere at room temperature. An organic solvent was used for recrystallization. Distilled water was prepared for the synthesis, passing a flow of technical argon through it for 30 minutes. A mixture of 0.4960 g (2 mmol) of Na2S2O3 5H2O and 0.5740 g (1 mmol) of a thiosulfate nitrosyl iron complex bis(µ-thiosulfato-S)-bis(dinitrosylferrate)(2-) sodium Na2[Fe2(S2O3)2(NO)4] (TNIC) was dissolved in distilled water. A mixture consisting of 0.8402 g (5 mmol) of 2-mercaptobenzothiazole and 0.2890 g (7 mmol) of NaOH was also dissolved in water. Both solutions were mixed. The precipitate was filtered off and dried in air. The yield of the product was 1.1182 g. After 24 hours the precipitate was recrystallized. The complex dissolves well in ethanol, methanol, acetone, methylene chloride, acetone nitrile, ether, THF, DMSO and DMFA. It does not dissolve in water or heptane. Determined for Fe2S2N6C14H8O4, %: Fe, 18.96; S, 22.01; N, 14.54; C, 29.39; H, 1.57; calculated, %: Fe, 19.78; S, 22.72; N, 14.89; C, 29.79; O, 11.34; H, 1.43. IR-spectrum (cm"1): 3448, 2923, 2852, 2379, 1787, 1721, 1468, 1429, 1384, 1313, 1270, 1006,852,754,724,706,611. The IR-spectra of all of the samples were registered on the Fourier SPECTRUM BX-II spectrometer. The sample was prepared in the form of tablets with KBr (1 mg of the studied substance per 300 mg of KBr). An X-ray diffraction analysis of the complex Fe2S4N8C14H10O4 was carried out on an automatic four-circuitous difractometer P-4 of the BRUKER firm [graphitic monochromator, (Mo-Kα)=0.71073 A, temperature 200K, 9/20-scanning]. Black monocrystals in the form of a parallelepiped having dimensions 0.35x0.20x0.15 mm were used in the experiment. The crystals are not stable at room temperature and break down at the rate of 10% in 1 hour. The structure is deciphered by the direct method. The positions and thermal parameters of non-hydrogen atoms are further defined in an isotrope and then anisotrope approach by the full matrix method of least squares (MLS). Hydrogen atoms are detected from mutually spaced Fourier synthesis and further defined in an isotrope approach. The molecular structure of complex I is presented in Fig. 1. The complex has a centrosymmetrical dimeric binuclear structure. In the dimer two, tetrahedrally coordinated by two groups of NO and two benzo-azaheterocyclic thiolyls, iron atoms are linked by a bridge: Fe-S-C-N-Fe'. The complex contains two molecules of a solvent - acetone. Crystallographic data and the main parameters of the further definition are presented in Table 1. The interatomic distance and the angles are presented in Table 2. All of the calculations are made with use of the complex of SHELXTL programs (G.M. Sheldrick, SHELXTL v.6.14, Structure Determination Software Suite, Bruker AXS, Madison, Wisconsin, USA, 2000). Distance Fe(l) Fe(la) -4.057 A (seeTable 2). Table 1Crystallographic data and characteristics of analysis for complex I Table 2 Interatomic distance and valence angles in the structure of complex I (Table Removed) The matrixes of generation of equivalent positions of the atoms: #1 -x, -y, -z The crystalline structure of complex I is presented in Fig. 2. The Moessbauer spectra of absorption were taken on the WissEl installation operating in the constant acceleration regimen. Co57 in the Rh matrix served as the source. Measurements of the spectra at low temperatures were carried out with the aid of a flow helium cryostat CF-506 (Oxford Instruments) with an adjustable temperature. Treating the Moessbauer spectra was carried out by the least square method with the presumption of the Lorentz shape of individual spectral components. The results are presented in Table 3. Table 3 (Table Removed) Parameters of Moessbauer spectra of Fe of complexes I-III at T=290K Study of NO-donor activity of binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives NO-donor activity of complexes I-III was first established upon a study of the reaction thereof with hemoglobin (Hb). At the base of the method of study is the reaction of the formation of Hb-NO upon the interaction of hemoglobin with free NO in the solution [R. Cassoly, Q.H. Gibson, Conformation, co-operativity and ligand binding human hemoglobin, J. Mol. Biol. 91 (1975) 301-313; T.J. McMahon, J.S. Stamler, Concerted nitric oxide/oxygen delivery by hemoglobin, in: L. Packer (Ed.), Methods In Enzymology, Academic Press, 301 (1999) Part C, 99-114]. The Hb to NO binding constant is 31010 M-1, which is three times more than a similar constant for CO and six times more than the binding constant for O2 [E. antonini, M. Brunori, Hemoglobin and myoglobin in the reactions with ligands, in: A. Neuberger, E.L. Tatum (Eds.) North-Holland research monographs. Fronties of biology. Vol. 21, North-Holland Publishing Company, Amsterdam-London, 21 (1971) 276]. The bimolecular constant of interaction of Hb with NO is close to diffusion constant: 1.02 108 M-1s-1 in a 0.05 M phosphate buffer, pH=7.0 at 20°C. Therefore, Hb is a very convenient trap for NO and is actively used to study NO-donor activity of compounds generating nitrogen monoxide. Wherein the spectra of optical absorption of free Hb and Hb-NO significantly differ, which makes it easy to detect the formation of a nitrosyl adduct. The results of a study are presented for clarity in Fig. 3 for complex I. A homogeneous bovine Hb preparation was prepared from bovine hemoglobin of the "MP Biomedicals" firm, which is a mixture of metHb and HbO2. 0.05 M phosphate buffer, pH 7.0 was used in all the steps of preparation of Hb and in all of the experiments with Hb. In order to transform the mixture of metHb and HbO2 to Hb, a 2x15 cm column was preliminarily prepared with Sephadex G-25 and transferred to an anaerobic condition. At the output from the column, 5 ml of Hb with a concentration of 6 10-4 moll-1 were collected. Hb was stored in a frozen state in the form of balls in liquid nitrogen. Before use, Hb was unfrozen in 5 ml volume vessels in a nitrogen flow. 2.8 ml of a buffer, 0.1 ml of Hb 6 10-4 moll-1 were put in an anaerobic 4 ml test cuvet with a 1 cm length of an optical path and the spectrum of absorption was recorded. An anaerobic absolute DMSO was added to the charge of nitrosyl complex in a vessel filled with nitrogen so as to obtain a solution of the complex with a concentration of 6 10-3 moll-1 Then the solution was stirred for 3-5 minutes to complete dissolution and the obtained solution was administered by 0.1 ml into a test cuvet with Hb and into a comparison cuvet comprising 2.9 ml of the anaerobic buffer. The final concentration of the nitrosyl complex was equal to 2 10 moll-1, DMSO - 3.3%. Then the difference spectra of absorption were recorded, the first - 1 minute after the beginning of the reaction (shown in Fig. 3), then at intervals of 3 minutes for the first 30 minutes of the reaction, and then at intervals of 15, 30, 60 minutes (in Fig. 3 only a portion of these spectra is shown for clarity). Registration of the spectra was carried out to the complete conversion of Hb to HbNO, when the spectrum stopped changing. The amount of the formed NO was evaluated spectrophotometrically according to the amount of the formed HbNO. In order to determine the concentration of the HbNO, the spectrum of absorption of the reaction system comprising Hb and HbNO was broken down with the aid of computer processing with the MathCad program to components - Hb and HbNO spectrum. The calculation was carried out in a wavelength range of 450-650 nm in accordance with 200 experimental points. The spectrophotometer Specord M-40, provided with an interface for computer registration of the spectra and with a thermostatically controlled cuvet separation, was used for registration of the absorption spectra. The spectra were registered at 25°C. As a result of these studies, it was established for the first time that the complexes generate NO in aqueous solutions of dimethylsulfoxide spontaneously (i.e., in the absence of hemo-, photo- or enzymatic activation). Study of cytotoxicity of tetranitrosyl iron complexes with benzo-azaheterocyclic thiolyls on human cancer cells in vitro Experimental technique Before the beginning of the experiment, all of the compounds were dissolved in 200 µl of DMSO and then brought to the necessary concentration by the nutrient medium RPMI1640. The final concentration of DMSO in the samples did not exceed 0.2% and did not affect the growth of cells. Cell lines - human ovary cancer SKOV3. The cells were grown in a monolayer in an RPMI1640 medium comprising 10% fetal bovine serum at 37°C and a 5% content of CO2. For the experiments, the cells were seeded in 96-well plates and grown under the same conditions. A test of the cytotoxic effect was carried with the aid of an MTT-test, based on the capability of the dehydrogenase of live cells to reduce the uncolored salt of tetrazolium to the blue crystals of formazan that are dissolved in dimethylsulfoxide (DMSO). All of the compounds were put in wells in a volume of 20 µl in 4 final concentrations of 100, 50, 25 and 10 (µM). The total volume of incubation was 200 µl. Cells with preparations were incubated in the aforesaid conditions for 72 hours. At the end of incubation, an MTT reagent was added to the cells and incubated under the same conditions for 2 hours. Then the formed formazan crystals were dissolved in 100 ul of DMSO at 37°C for 20 minutes. The optical absorption of the DMSO solutions was measured on an optical counter for multi-well plates at a wavelength of 540 nm. The results were expressed in the form of average meanings for four parallel measurements. The cytotoxic effect was assessed according to the survival of the cells in experimental samples in respect to the control in %. The compound was regarded as active if at a concentration of 100 µM the number of live cells was 50% or less (IC50 ≤ 100 µM). The measurement error did not exceed 5%. The results of the study are presented in Table 4. Two of the studied nitrosyl iron complexes - I and III, comprising thiobenzimidazole and thiobenzthiazole ligands, exhibited cytotoxic activity on cell line SCOV3. The IC50 was 57 µM and 25 µM, respectively, for the I and III complexes. The studied nitrosyl iron complexes I and III demonstrated a different cytotoxic effect against human cancer cells in vitro. Complex III exhibited maximum activity (IC50 = 25 µM). Cisplatin (cis-DDP), an anticancer preparation that is used in Russian and foreign clinics (IC50 = 16 µM), was used as the comparison preparation. It was established that upon a reduction of the concentration by two times, the complex III retains the same activity as at a concentration of 100 µM (the survival of the cancer cells is 12.5%) (Table 4). The fact that the toxicity of the complex III is significantly less (the general toxicity index thereof LD100 is 50 µg/kg) than that of cisplatin (LD100 - 16 mg/kg) may be of significance in high-dose chemotherapy, accompanied by severe side effects, as a method for increasing the sensitivity of cancer cells to therapeutic action and makes it possible to reduce the concentration of the used cytostatics. Table 4 Cytotoxic activity of nitrosyl iron complexes I-III against SKOV3 cell line (Table Removed) In accordance with the methodical indications in respect to a study of the anticancer activity of pharmacological substances, studies were carried out on grafted mice tumors Ca-755, lymphatic leukemia P-388 and melanoma B-16 [on first generation hybrid mice BDF1 (C57B1/6 x DBA/2) and DBA/2 with a weight of 18-25 g, obtained from the department of laboratory animals GU RONTs in the name of N.N. Blokhin RAMS]. A statistically significant anticancer effect of the claimed preparations has been detected. CLAIMS 1. A binuclear nitrosyl iron complex with benzo-azaheterocyclic derivatives of general formula wherein R is (Formula Removed) wherein X is NH, S or O, R1 is lower alkyl. 2. The binuclear nitrosyl iron complex according to claim 1, wherein R is benzimidazol-2-yl, 5-methylbenzimidazol-2-yl or benzthiazol-2-yl. 3. A method for preparing binuclear nitrosyl iron complexes according to claim 1, consisting in that a thiosulfate nitrosyl iron complex is treated with a corresponding benzo-azaheterocyclic thiol in a stoichiometric ratio in the presence of a reducing agent, and the process is carried out in an alkaline medium with the subsequent isolation of the desired product by known techniques. 4. The method according to claim 3, characterized in that the process is carried out at room temperature, mainly at 18-25°C. 5. The method according to claim 3, characterized in that the process is carried out in an oxygen-free atmosphere. 6. The method according to claim 3, characterized in that benzimidazole-2-thiol, 5-methylbenzimidazole-2-thiol or benzthiazole-2-thiol is used as the benzo-azaheterocyclic thiol. 7. The method according to claim 3, characterized in that hydrogen, metal thiosulfates, hydrogen sulfide and aliphatic thiols are used as the reducing agent. 8. A donor of nitrogen monoxide that is the binuclear nitrosyl iron complex according to claim 1. 9. Use of the binuclear nitrosyl iron complex according to claim 1 as an anticancer agent. 10. Use of the binuclear nitrosyl iron complex according to claim 1 for the preparation of an anticancer pharmaceutical. 11. A pharmaceutical composition comprising an effective amount of the binuclear nitrosyl iron complex with benzo-azaheterocyclic derivatives according to claim 1, and a pharmaceutically acceptable carrier. 12. The pharmaceutical composition according to claim 11, wherein a proton-containing medium is used as the pharmaceutically acceptable carrier. 13. The pharmaceutical composition according to claim 11, wherein a mixture of a proton-containing medium and dimethylsulfoxide is used as the pharmaceutically acceptable carrier. 14. The pharmaceutical composition according to any one of claim 12 or claim 13, wherein water, a physiological solution, water-soluble biopolymers are used as the proton-containing medium. 15. The pharmaceutical composition according to claim 11, wherein the binuclear nitrosyl iron complex with benzo-azaheterocyclic derivatives is present in an amount of 50-100 µM. 16. A kit used for treating oncological diseases, comprising (1) a pharmaceutical composition comprising a binuclear nitrosyl iron complex with benzo-azaheterocyclic derivatives according to claim 1, in a sealed container; and (2) auxiliary components.

Full Text

BINUCLEAR NITROSYL IRON COMPLEXES WITH BENZO-AZAHETEROCYCLIC DERIVATIVES AND A METHOD FOR THE PREPARATION THEREOF
Field of the invention
The invention relates to binuclear nitrosyl iron complexes and may be used in medicinal practice to produce new generation medicinal agents for treating oncological diseases.
Background of the invention
In recent years in order to create medicinal preparations of a new generation for use in the therapy of oncological diseases, an intensive search is being carried out for anticancer agents based on complexes of transition metals with an improved activity spectrum and reduced side effects as compared with already used clinical preparations, for example, cisplatin, sodium nitroprusside, et al.
Interest in nitrosyl metal complexes has increased due to the discovered possibilities for their use as effective NO donors in medicine, in particular, for the therapy of cancer diseases. The use of NO donors as a new class of anticancer agents is related to the important role played by NO in the process of growth of malignant tumors [Wink D., Vodovoz J., Cook J., Biochemistry, 1998, 63, 7, pp. 948-957]. It is shown that nitrogen monoxide changes the level of apoptosis of the tumor cells, the activity of the p53 gene and neoangiogenesis [Brune B., Scheneiderhan N., Nitric oxide evoked p53-accumulation and apoptosis, Toxicol Letters, 2003, 193, 2, pp. 19-23], inhibits the activity of a key reparation protein O6-methyl-guanine-DNA-methyl-transferase of mammals [L Liu, M. Xu-Welliver, A. Kanagula, H.E. Pegg, Inactivation and degradation of 06-alkylguanine-DNA alkyltransferase after reaction with nitric oxide., Cancer Res., 2002, 62, pp. 3037-3043].
Binding NO to active sites of metal enzymes, in particular to non-heme iron-containing proteins, is studied especially intensively [Ford P.C., Lorkovic I.M., Chem. Rev., 2002, 102, 993; Hoshino M., Laverman I.E., Ford P.C., Coord. Chem. Rev., 1999, 187, p. 75]. It has been established that nitrosyl iron complexes with thiol-containing ligands are one of the forms of natural reservoirs of NO [Butler A.R., Megson I.I., Chem. Rev. 2002, 102, pp. 1155-1165],
Of the synthetic models, Roussin's "red salt" esters have the formula [Fe2(SR)2(NO)4], where R = Et, t-Bu, (CH2)4-CH3, C6H5F, Ph [T. Thomas, J.H. Robertson, E.G. Cox, Acta. Crystalogr., 1958, 11, p. 599; C. Glidewell, M.E. Harman, M.B. Hursthouse, I.L. Johnson, M. Motevalli, J. Chem. Res., 1998, 212, p. 1676; R.E. Marsh, A.L. Spek, Acta. Crystalogr., Sect. B. Struct. Sci., 2001, 57, p. 800; C. Jinhua, M. Shaoping, H. Jinling, I. Jiaxi, Chinese J. Struct. Sci., 2001, 57, p. 800; C. Jinhua, M. Shaoping, H. Jinling, I. Jiaxi, Chinese J. Struct. Chem., 1983, 2, p. 263; T.B. Rauchfuss, T.D. Weatherill, Inorg. Chem., 1982, 21, pp. 827-830].

These binuclear diamagnetic sulfur-nitrosyl complexes with R = Alk generate NO upon thermo- or photoactivation [J.L. Bourassa, P.C. Ford, Coord. Chem. Rev., 2000, 200-202, pp. 887-900] and may serve as new promising anticancer NO donor agents.
Also known are binuclear paramagnetic sulfur-nitrosyl iron complexes with ligands of the µ-N-C-S type, which are synthetic analogues of natural donors of NO - dinitrosyl iron complexes (DNIC) with cysteine, glutathione and other thiol-containing ligands of low molecular weight [O.A. Rakova, N.A. Sanina, S.M. Aldoshin, N.A. Goncharova, G.V. Shilov, Yu.M. Shulga, N.S. Ovanesyan, Inorganic Chemistry Communications 2003, 6, 145-148; A.F. Vanin, N.A. Sanina, V.A. Serezhenkov, D.Sh. Burbaev, V.I. Lozinsky and S.M. Aldoshin, Nitric oxide: biology & chemistry 2007, 16,82-93].
However, the synthetic NO donors of different classes that are known at the present time are not used as therapeutic agents for the treatment of oncological diseases. They are only used to enhance (to a different degree depending on the chemical nature) the action of existing chemotherapeutic agents or of radiotherapy [Wink D., Vodovoz J., Cook J., Biochemistry, 1998, 63, 7, pp. 948-957; N.P. Konovalova, S.A. Goncharova, L.M. Volkova, T.A. Raevskaya, L.T. Eremenko, A.M. Korolev, Nitric Oxide: Biology and Chemistry, 2003, 8, pp. 59-64; Yang W., Rogers P.A., Ding H., J. Biol. Chem., 2002, 277, pp. 12868-12873; O. Siri, A. Tabard, P. Pullumbi, R. Guilard, Inorg. Chim. Acta 2003, 350, p. 633; J.L. Burgaud, E. Jingini, Del Soldata P. Ann., N.Y. Acad. Sci. 2002, 962, p. 360; T.I. Karu, L.V. Pyatibrat, G.S. Kalendo, Toxicology Letters, 2001, 121, p. 57].
In the article by A. Janczyk et al., Nitric Oxide, 2004, 10, 1, pp. 42-50, a study is made of the direct cytotoxic action of a nitrosyl iron complex Na[Fe4S3(NO)7] on the human and mouse melanoma cell lines. However, this nitrosyl iron complex generates NO upon photoactivation (even in the dark) and cannot be used as an anticancer agent either because of the high toxicity in respect to normal cells.
So, there is a demand for other anticancer agents with higher effectiveness and reduced toxicity.
The object of the instant invention is to expand the arsenal of anticancer agents and create anticancer agents based on complexes of transition metals with an improved activity spectrum and reduced side effects, in particular, an agent based on a tetra-nitrosyl binuclear iron complex, acting as a NO donor, with enhanced activity and reduced toxicity.
Summary of the invention
In one aspect the invention relates to new binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of formula
(Formula Removed)
wherein R is
(Formula Removed)
wherein X is NH, S or 0, R1 is lower alkyl.
In another aspect, the invention relates to a method for preparing binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of formula
(Formula Removed)
by treatment of a thiosulfate nitrosyl iron complex with a corresponding benzo-azaheterocyclic thiol in a stoichiometric ratio in the presence of a reducing agent, in an alkaline medium. This method makes it possible for the first time to obtain the claimed complexes in crystalline form.
In an additional aspect, the instant invention relates to a donor of nitrogen monoxide, which is a binuclear nitrosyl iron complex with benzo-azaheterocyclic derivatives that is characterized above.
In the next aspect, the instant invention relates to the use of binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of formula
(Formula Removed)
as an anticancer agent.
In an additional aspect, the instant invention relates to the use of binuclear nitrosyl iron complexes benzo-azaheterocyclic derivatives of formula
(Formula Removed)
for preparation of an anticancer pharmaceutical.
In the next aspect, the instant invention relates to a pharmaceutical composition comprising an effective amount of a binuclear nitrosyl iron complex with benzo-azaheterocyclic derivatives of formula
(Formula Removed)
and a pharmaceutically acceptable carrier.
In the next aspect, the instant invention relates to a kit used for treating oncological diseases and comprising (1) a pharmaceutical composition comprising a binuclear nitrosyl iron complex with benzo-azaheterocyclic derivatives of formula
(Formula Removed)
wherein R has the aforesaid meanings, in a sealed container; and (2) auxiliary agents.
Description of the Figures
In Fig. 1 - the molecular structure of the complex
(Formula Removed)
[complex I] is presented.
The complex has a centrosymmetrical dimeric binuclear structure (Fig. 1). In the dimer two tetrahedrally coordinated by two NO groups and two benzo-azaheterocyclic thiolyls of the iron atom are linked by a bridge: Fe-S-C-N-Fe'. The complex comprises two molecules of the acetone solvent.
In Fig. 2 - the crystalline structure of the complex
(Structure Removed)
[complex I] is presented.
In Fig. 3 - a change of the difference spectra of absorption in time upon interaction of
complex I
(Formula Removed)
with hemoglobin (Hb) is shown.
The solvent - 0.05% phosphate buffer, pH = 7.0, comprising 3.3% dimethylsulfoxide, 25°C.
Detailed disclosure of the invention
The new binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives in accordance with the instant invention have the general formula
(Formula Removed)
wherein R is
wherein X is NH, S or O, R1 is lower alkyl.
The term "lower alkyl" that is used here means an alkyl radical with a straight or branched chain comprising from 1 to 6 carbon atoms. Examples of such alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl.
Information on the claimed compounds and the claimed method for the preparation thereof is not present in the state of the art.
The method for preparing new binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of formula
(Formula Removed)
wherein R has the aforesaid meanings, consists in that a thiosulfate nitrosyl iron complex bis(µ-thiosulfato-S)-bis(dinitrosylferrate)(2-) sodium Na2
(Formula Removed)
(TNIC) is treated with a corresponding benzo-azaheterocyclic thiol in a stoichiometric ratio in the presence of a reducing agent, and the process is carried out in an alkaline medium with the subsequent isolation of the desired product by known techniques.
It is preferable that the process be carried out at room temperature, for the most part at 18-25°C.
It is preferable that the process be carried out in an oxygen-free atmosphere.
It is preferable that benzimidazole-2-thiol, 5-methylbenzimidazole-2-fhiol or benzthiazole-2-thiol be used as the benzo-azaheterocyclic thiol.
Hydrogen, thiosulfates of metals, for example, Na2S2O3 5H2O, hydrogen sulfide and aliphatic thiols are used as the reducing agent in the method according to the instant invention.
The authors of the invention have established that binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of the instant invention are effective No donors, releasing nitrogen monoxide spontaneously upon decomposition in proton mediums (such as water, blood and components thereof, physiological solutions, etc.) in the absence of hemo-, photo- or enzymatic activation. So, the complexes according to the instant invention are a new, promising class of nitrogen monoxide donors - synthetic analogues of active sites of nitrosyl nonhemo
iron-sulfur proteins - natural NO depot. Therefore, the instant invention in the additional aspect relates to nitrogen monoxide donors, which are binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives that are presented above.
The binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of natural aromatic thiols of the instant invention have anticancer activity in respect to cancer cells of a mammal, in particular, a human.
The instant invention is further directed to the use of binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of formula
(Formula Removed)
where R has the aforesaid meanings, as an anticancer agent.
In particular, the compound of the instant invention may be used for treatment of the following oncological diseases: ovarian carcinoma, breast adenocarcinoma, melanoma B-16, epidermoid Lewis carcinoma.
The iron complexes in accordance with the instant invention are suitable for inhibiting the growth of cancer cells in mammals, and it is preferable that they be administered in the form of a pharmaceutical composition comprising an effective antitumor amount of the compound in accordance with the instant invention in combination with at least one pharmaceutically or pharmacologically acceptable carrier and/or excipient. The carrier, also known from the state of the art as an excipient, filler, ancillary substance, additive or diluent, is any substance that is pharmaceutically inert, provides a corresponding consistence or form to the composition and does not weaken the therapeutic efficacy of the anticancer compounds. The carrier is "pharmaceutically or pharmacologically acceptable," if it does not cause an adverse, allergic or other unfavorable reaction upon the administration to a mammal or human, respectively.
The instant invention also relates to the use of binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of formula
(Formula Removed)
wherein R has the aforesaid meanings, for the preparation of an anticancer pharmaceutical.
The instant invention additionally proposes pharmaceutical compositions comprising an effective amount of binuclear nitrosyl iron complexes with benzo-azaheterocyclic derivatives of formula
(Formula Removed)
wherein R has the aforesaid meanings, and a pharmaceutically acceptable carrier.
It is preferable that a proton-containing medium be used in the composition according to the invention as the pharmaceutically acceptable carrier.
It is also preferable that a mixture of the proton-containing medium and dimethylsulfoxide be used as the pharmaceutically acceptable carrier in the composition according to the invention.
It is preferable that water, a physiological solution, water-soluble biopolymers be used as
the proton-containing medium.
It is preferable that the binuclear nitrosyl iron complex with a benzo-azaheterocyclic derivative be present in the composition in an amount of 50-100 µM.
The pharmaceutical compositions containing the anticancer compound in accordance with the instant invention may be produced by any conventional method. The necessary preparative form is selected depending on the selected method of administration. Compositions in accordance with the invention may be produced for any method of administration as long as the target tissue is available via that route of administration. Suitable routes of administration include, but are not limited to, oral, parenteral (e.g., intravenous, intraarterial, subcutaneous, rectal, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal), topical (nasal, transdermal, intraocular), intravesical, intrathecal, into the small intestine, pulmonary, intralymphatic, intracavital, vaginal, transurethral, intradermal, aural, intramammary, buccal, orthotopic, intratracheal, intralesional, percutaneous, endoscopical, transmucosal, sublingual and intestinal administration.
Pharmaceutically acceptable carriers for use in the compositions of the present invention are well known to one of ordinary skill in the art and are selected based upon a number of factors: the particular anticancer compound used and its concentration, stability and expected biological availability; the disease, disorder or condition of the human being treated with the composition; the subject, its age, weight and general condition; and the method of administration. Suitable carriers are readily determined by one of ordinary skill in the art [J.G. Nairn in: Remington's Pharmaceutical Science (A. Gennaro, ed.), Mack Publishing Co., Easton, Pa. (1985), pp. 1492-1517].
The compositions are preferably produced as tablets, dispersible powders, pills, capsules, gelcapsules, coated caplets, gels, liposomes, granules, solutions, suspensions, emulsions, syrups, elixirs, troches, dragees, lozenges, or any other dosage form, which can be administered orally.
The compositions of the instant invention for oral administration comprise an effective amount of a compound of the invention in a pharmaceutically acceptable carrier. Suitable carriers for solid dosage forms include sugars, starches and other conventional substances including lactose, talc, sucrose, gelatin, carboxymethyl cellulose, agar, mannitol, sorbitol, calcium phosphate, calcium carbonate, sodium carbonate, kaolin, alginic acid, acacia, corn starch, potato starch, sodium saccharin, magnesium carbonate, tragacanth, microcrystalline cellulose, colloidal silicon dioxide, crosscarmellose sodium, talc, magnesium stearate, and stearic acid. Further, such solid dosage forms may be uncoated or may be coated by known techniques, e.g., to delay disintegration and absorption.
The anticancer compounds of the present invention are also preferably used for the
production of a dosage form for parenteral administration, e.g., formulated in a dosage form for injection via intravenous, intraarterial, subcutaneous, rectal, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal routes. The compositions of the invention for parenteral administration comprise an effective amount of the anticancer compound in a pharmaceutically acceptable carrier. Dosage forms suitable for parenteral administration include solutions, suspensions, dispersions, emulsions or any other dosage form which can be administered parenterally. Techniques and compositions for making parenteral dosage forms are known from prior art.
An insignificant amount of additional components, which are well-known in the pharmaceutical industry, can be included in the composition of the invention. These components to a high degree provide properties which increase the time of retention of the anticancer compound at the site of administration, promote stability of the composition, provide control of the pH, facilitate the introduction of the anticancer compound into the pharmaceutical compositions, and the like. Each of these components is individually present in an amount that is preferably less than about 15 weight %, more preferably less than about 5 weight %, and most preferably less than about 0.5 weight % of the total weight of the composition. Some components, such as fillers or diluents, can constitute up to 90 weight % of the total weight of the composition, as is well-known in the technology of producing medicinal agents. Such additives include cryoprotective components for preventing precipitation of the iron complex with thiophenol, surfactants, wetting or emulsifying agents (e.g., lecithin, polysorbate-80, Tween® 80, pluronic 60, polyoxyethylene stearate), preservatives (e.g., ethyl-n-hydroxybenzoate), agents protecting against the action of microbes (e.g., benzyl alcohol, phenol, m-cresol, chlorobutanol, sorbic acid, thimerosal and paraben), agents for adjusting pH or buffering agents (e.g., acids, bases, sodium acetate, sorbitan monolaurate), components for adjusting osmolarity (e.g., glycerin), thickeners (e.g., aluminum monostearate, stearic acid, cetyl alcohol, stearyl alcohol, guar gum, methyl cellulose, hydroxypropyl cellulose, tristearin, cetyl wax esters, polyethylene glycol), colorants, dyes, flow aids, non-volatile silicones (e.g., cyclomethicone), clays (e.g., bentonites), adhesives, bulking agents, flavorings, sweeteners, adsorbents, fillers (e.g., water, saline, electrolyte solutions), binders (e.g., starches such as maize starch, wheat starch, rice starch or potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, sugars, polymers, acacia), disintegrating agents (e.g., starches such as maize starch, wheat starch, rice starch, potato starch or carboxymethyl starch, structurated polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate, croscarmellose sodium or crospovidone), lubricants (e.g., silica, talc, stearic acid or salts thereof such as magnesium stearate, or
polyethylene glycol), coating agents (e.g., concentrated sugar solutions including gum Arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol or titanium dioxide) and antioxidants (e.g., sodium metabisulfite, sodium bisulfite, sodium sulfite, dextrose, phenols and thiophenols).
In accordance with a preferred variant of embodiment, the pharmaceutical composition of the invention comprises at least one nonaqueous pharmaceutically acceptable solvent and an anticancer compound having a solubility therein of at least about 10-60 mg/ml. While not being bound to any particular theory, it is believed that the solubility in the dimethylsulfoxide of the anticancer compound may be directly related to its efficacy. It is also preferable that the anticancer compound has an ID100 value (i.e., the concentration of the agent causing 100% inhibition of colony formation) that is at least 4 times less than that of cisplatin when measured according to the method described in the book "Experimental assessment of antitumor preparations in the USSR and the USA," edited by E.P. Sofina, A.B. Sirkin (USSR), A. Goldin, A. Cline (USA), Moscow,: "Meditsina," 1979, pp. 71-105.
Dosage form administration by these routes may be continuous or intermittent, depending, for example, upon the patient's physiological condition, on whether the purpose of the administration is therapeutic or prophylactic, and other factors known to or assessable by a skilled practitioner.
The dosage and regimens for the administration of the pharmaceutical compositions of the invention can be readily determined by an oncologist. It is understood that the dosage of the anticancer compounds depends on the age, sex, health and weight of the recipient, the kind of concurrent treatment, if any, frequency of treatment and the nature of the desired effect. For any mode of administration, the exact amount of the used anticancer compound and also the prescribed dose that is necessary for achievement of the advantageous effects herewith described also depend in particular on such factors as the bioavailability of the anticancer compound, the human disorder being treated, the desired therapeutic dose and other factors that are obvious to one skilled in the art.
The concentration of the anticancer compound of the instant invention in a liquid pharmaceutical composition is, most preferably, 50-100 µM As a rule, relatively low concentrations are preferable, since the anticancer compound is most soluble at low concentrations.
Emulsions for parenteral administration can be produced by dissolving an anticancer compound in any pharmaceutically acceptable solvent capable of dissolving the compound (e.g., DMSO, dichloromethane) to form a solution. An appropriate volume of a carrier which is an emulsion, such as a Liposyn II or Liposyn III emulsion, is added to the solution while stirring to
form a pharmaceutically acceptable emulsion for parenteral administration to a patient. If desired, such emulsions can be produced with a content of a minimal amount of, or without, a Cremophor® solution.
Solutions for parenteral administration can be produced by dissolving an anticancer compound in any pharmaceutically acceptable solvent capable of dissolving the compound (e.g., DMSO) to form a solution. An appropriate volume of a carrier which is a proton-containing medium (saline solutions, sugar solutions, water-soluble polymers, proteins, electrolyte solutions) is added to the solution for parenteral administration to a patient.
So, for example, for liquid preparative forms, nonaqueous pharmaceutically acceptable polar solvents, such as oils, alcohols, amides, esters, ethers, ketones, hydrocarbons and mixtures thereof, and also water, saline solutions, dextrose solutions (e.g., DW5), electrolyte solutions or any other pharmaceutically acceptable proton medium, are usually used as the carrier.
Suitable nonaqueous pharmaceutically acceptable polar solvents include, but are not
limited to, amides (e.g., dimethylacetamide (DMA), dimethylacetamidebenzylbenzoate,
dimethylformamide, N-(P-hydroxyethyl) lactamide, N,N-dimethylacetamide, 2-pyrrolidinone, 1 -
methyl-2-pyrrolidinone or polyvinylpyrrolidone); esters (e.g., acetic acid esters, such as
monoacetin, diacetin and triacetin; aliphatic or aromatic esters, such as ethyl caprylate or ethyl
octanoate, alkyl oleate, benzyl benzoate, benzyl acetate, dimethylsulfoxide (DMSO), esters of
glycerin such as mono-, di- or triglyceral citrates or tartrates, ethyl benzoate, ethyl acetate, ethyl
carbonate, ethyl lactate, ethyl oleate, fatty acid esters of sorbitan, fatty acid derived
polyethyleneglycol (PEG) esters, glyceryl monostearate, glyceride esters such as mono-, di- or
triglycerides, fatty acid esters such as isopropyl myristrate, fatty acid derived PEG esters such as
PEG-hydroxyoleate and PEG-hydroxystearate, N-methyl pyrrolidinone, pluronic-60,
polyoxyethylene sorbitol oleic acid polyesters such as poly(ethoxylated)3o-6osorbitol
poly(oleate)2-4, poly(oxyethylene) 15-20monooleate, poly(oxyethylene) 15-2omono-12-
hydroxystearate and poly(oxyethylene)15-20mono ricinoleate; polyoxyethylene sorbitan esters, such as polyoxyethylene-sorbitan monooleate, polyoxyethylene-sorbitan monopalmitate, polyoxyethylene-sorbitan monolaurate, polyoxyethylene-sorbitan monostearate, and Polysorbate® 20, 40, 60 or 80 from the ICI Americas firm, Wilmington, DE; polyvinylpyrrolidone, alkylene oxides modified fatty acid esters, such as polyoxyl 40 hydrogenated castor oil and polyoxyethylated castor oils (e.g., Cremophor® EL solution or Cremophor® RH 40 solution); fatty acid and saccharide esters (i.e., the condensation product of a monosaccharide (e.g., pentoses, such as ribose, ribulose, arabinose, xylose, lyxose and xylulose; hexoses, such as glucose, fructose, galactose, mannose and sorbose; trioses, tetroses, heptoses, and octoses), disaccharides (e.g., sucrose, maltose, lactose and trehalose) or
oligosaccharide or mixtures thereof with a fatty acid (fatty acids) with 4-22 carbon atoms (e.g., saturated fatty acids, such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid and stearic acid, and unsaturated fatty acids, such as palmitoleic acid, oleic acid, elaidic acid, erucic acid and linoleic acid)), or steroidal esters); alkyl, aryl or cyclic ethers having 2-30 carbon atoms (e.g., diethyl ether, tetrahydrofuran, dimethyl isosorbite, diethylene glycol monoethyl ether); glycofurol (tetrahydrofurfuryl alcohol polyethylene glycol ether); ketones having 3-30 carbon atoms (e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone); aliphatic, cycloaliphatic or aromatic hydrocarbons having 4-30 carbon atoms (e.g., benzene, cyclohexane, dichloromethane, dioxolanes, hexane, n-decane, n-dodecane, n-hexane, sulfolan, tetramethylenesulfon, tetramethylenesulfoxide, toluene, dimethylsulfoxide (DMSO), or tetramethylenesulfoxide); alkyl- or arylhalogenides comprising 1-30 carbon atoms and optionally more than one halogen atom as a substituent; dichloromethane; monoethanolamine; petroleum ether; trolamine; omega-3 polyunsaturated fatty acids (e.g., alpha-linolenic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid); polyglycol ester of 12-hydroxystearic acid and polyethylene glycol (Solutol® HS-15 from the BASF firm, Ludwigshafen, Germany); polyoxyethylene glycerol; sodium laurate; sodium oleate; or sorbitan monooleate.
In the instant invention it is preferable to use proton-comprising mediums, such as water, saline solutions, water-soluble polymers, proteins, dextrose solutions (e.g., DW5), electrolyte solutions or alcohols from the "PAA Laboratory's" catalogue, (2006), p. 26, as the pharmaceutically acceptable carrier.
In the next aspect of the instant invention, a kit is proposed for treatment of oncological diseases, which kit comprises: (1) a pharmaceutical composition comprising a binuclear nitrosyl iron complex with the benzo-azaheterocyclic derivative of the instant invention, in a sealed package; and (2) ancillary agents.
The kit may comprise the composition in the form of a single dosage unit or in the form of multiple doses. The kit may contain forms for oral or parenteral administration.
The pharmaceutical composition in the kit may be placed in glass or polymeric vials, ampoules, flasks, dosage cartridges for injectors, blisters, capsules, containers with the composition, that are used respectively for the oral or parenteral form.
The auxiliary agents comprise liquids for restoration of a composition administered parenterally, if it is presented in the kit in a concentrated form, for example, in the form of a dry substance, dried preparation, etc.; agents for the preparation of oral liquid forms and forms for injection ex tempore. Water for injection, a physiological solution, lidocaine solution, etc. may be used as the liquid for restoration. A solution of glucose, sugars, syrup, etc. may be used for
restoration of a composition that is used orally in a liquid form.
Optional auxiliary agents of the kit include agents for opening lids, agents for sealing opened reusable lids, enclosed instructions.
A pharmaceutical composition which is a solid dosage form for oral administration may be presented in the kit in the form of tablets, capsules in blisters, ampoules, vials, bubbles, packets, etc. A pharmaceutical composition, which is a liquid dosage form for parenteral or oral administration, may be presented in the kit in vials, capsules, ampoules, cartridges, etc.
An example of a kit for parenteral administration includes a container in which instructions for use, ampoules or vials with the dry composition and ampoules with a physiological solution for injection are arranged. A device for opening the ampoules is arranged in the container. The ampoules are packed in 10 ampoule blisters.
Other variants of the kit are obvious to one skilled in the art in this field from the specification presented above.
The following examples are provided only as an additional illustration of the invention and they should not be given consideration as limitation of the invention.
Examples of synthesis of the complexes
Example 1
Synthesis of the complex (I), where R = benzimidazol-2-yl
(Formula Removed)
The initial reagents Na2S2O35H2O (Aldrich), NaOH (pro analyze), 2-mercaptobenzimidazole (Aldrich) were used for synthesis. All of the operations as regards the preparation of and mixing the solutions and isolating the complexes were carried out in a technical argon atmosphere at room temperature. An organic solvent was used for recrystallization.
A mixture of 0.992 g (4 mmol) of Na2S2O35H2O and 1.074 g (2 mmol) of a thiosulfate
nitrosyl iron complex bis(µ-thiosulfato-S)-bis(dinitrosylferrate)(2-) sodium
(Formula Removed)
(TNIC) was dissolved in distilled water. A mixture consisting of 1.500 g (10 mmol) of 2-mercapto-5-methylbenzimidazole and 0.44 g (11 mmol) of NaOH was also dissolved in water.
Both solutions were mixed. The precipitate was filtered off and dried in air. The yield of the product was 0.719 g. After 24 hours the precipitate was recrystallized.
The complex dissolves well in ethanol, methanol, acetone, DMSO and DMFA. It does not dissolve in dichloromethane, water, heptane and ether.
Yield: 63.50%.
Determined for Fe2S2N8C14H10O4, %: Fe, 19.27; S, 10.69; N, 18.54; C, 43.40; H, 4.78; calculated, %: Fe, 18.99; S, 10.90; N, 19.05; C, 44.93; O, 16.32; H, 4.45.
IR-spectrum (cm"1): 3207, 1787, 1738, 1515, 1467, 1428, 1384, 1272, 1218, 1183, 1002, 742,713,603.
Example 2 Synthesis of the complex (II), where R = 5-methylbenzimidazol-2-yl (Fe2S2N8C16H14CO4
The initial reagents Na2S2O35H2O (Aldrich), NaOH (pro analyze), 2-mercapto-5-methylbenzimidazole (Aldrich) were used for synthesis. All of the operations as regards the preparation of and mixing the solutions and isolating the complexes were carried out in a technical argon atmosphere at room temperature. An organic solvent was used for recrystallization. Distilled water was prepared for the synthesis, passing a flow of technical argon through it for 30 minutes.
A mixture of 0.4960 g (2 mmol) of Na2S2O3 5H2O and 0.5740 g (1 mmol) of a thiosulfate
nitrosyl iron complex bis(µ-thiosulfato-S)-bis(dinitrosylferrate)(2-) sodium
Na2[Fe2(S2O3)2(NO)4] (TNIC) was dissolved in distilled water. A mixture consisting of 0.8250 g (5 mmol) of 2-mercapto-5-methylbenzimidazole and 0.2890 g (7 mmol) of NaOH was also dissolved in water.
Both solutions were mixed. The precipitate was filtered off and dried in air. The yield of the product was 2.2017 g. After 24 hours the precipitate was recrystallized.
The complex dissolves well in acetone, DMSO and DMFA. It does not dissolve in dichloromethane, water, heptane and ether.
Yield: 45.53%.
Determined for Fe2S2N8C16H14O4, %: Fe, 19.27; S, 10.69; N, 19.54; C, 34.26; H, 2.48; calculated, %: Fe, 20.01; S, 11.49; N, 20.08; C, 34.43; O, 11.47; H, 2.53.
IR-spectrum (cm'1): 3133, 1790, 1724, 1619, 1490, 1443, 1411, 1373, 1276, 1223, 1189, 1011,854,800,720,596,543.
Example 3 Synthesis of complex (III), wherein R = benzothiazol-2-yl (Fe2S4N6C14H8O4)
The initial reagents Na2S2O3 5H2O (Aldrich), NaOH (Pro analyze), 2-mercaptobenzothiazole (Aldrich) were used for synthesis. All of the operations as regards the preparation of and mixing the solutions and isolating the complexes were carried out in a technical argon atmosphere at room temperature. An organic solvent was used for recrystallization. Distilled water was prepared for the synthesis, passing a flow of technical argon through it for 30 minutes.
A mixture of 0.4960 g (2 mmol) of Na2S2O3 5H2O and 0.5740 g (1 mmol) of a thiosulfate
nitrosyl iron complex bis(µ-thiosulfato-S)-bis(dinitrosylferrate)(2-) sodium
Na2[Fe2(S2O3)2(NO)4] (TNIC) was dissolved in distilled water. A mixture consisting of 0.8402 g
(5 mmol) of 2-mercaptobenzothiazole and 0.2890 g (7 mmol) of NaOH was also dissolved in water.
Both solutions were mixed. The precipitate was filtered off and dried in air. The yield of the product was 1.1182 g. After 24 hours the precipitate was recrystallized.
The complex dissolves well in ethanol, methanol, acetone, methylene chloride, acetone nitrile, ether, THF, DMSO and DMFA. It does not dissolve in water or heptane.
Determined for Fe2S2N6C14H8O4, %: Fe, 18.96; S, 22.01; N, 14.54; C, 29.39; H, 1.57; calculated, %: Fe, 19.78; S, 22.72; N, 14.89; C, 29.79; O, 11.34; H, 1.43.
IR-spectrum (cm"1): 3448, 2923, 2852, 2379, 1787, 1721, 1468, 1429, 1384, 1313, 1270, 1006,852,754,724,706,611.
The IR-spectra of all of the samples were registered on the Fourier SPECTRUM BX-II spectrometer. The sample was prepared in the form of tablets with KBr (1 mg of the studied substance per 300 mg of KBr).
An X-ray diffraction analysis of the complex Fe2S4N8C14H10O4 was carried out on an automatic four-circuitous difractometer P-4 of the BRUKER firm [graphitic monochromator, (Mo-Kα)=0.71073 A, temperature 200K, 9/20-scanning]. Black monocrystals in the form of a parallelepiped having dimensions 0.35x0.20x0.15 mm were used in the experiment. The crystals are not stable at room temperature and break down at the rate of 10% in 1 hour. The structure is deciphered by the direct method. The positions and thermal parameters of non-hydrogen atoms are further defined in an isotrope and then anisotrope approach by the full matrix method of least squares (MLS). Hydrogen atoms are detected from mutually spaced Fourier synthesis and further defined in an isotrope approach.
The molecular structure of complex I is presented in Fig. 1. The complex has a centrosymmetrical dimeric binuclear structure. In the dimer two, tetrahedrally coordinated by two groups of NO and two benzo-azaheterocyclic thiolyls, iron atoms are linked by a bridge: Fe-S-C-N-Fe'. The complex contains two molecules of a solvent - acetone.
Crystallographic data and the main parameters of the further definition are presented in Table 1.
The interatomic distance and the angles are presented in Table 2.
All of the calculations are made with use of the complex of SHELXTL programs (G.M. Sheldrick, SHELXTL v.6.14, Structure Determination Software Suite, Bruker AXS, Madison, Wisconsin, USA, 2000).
Distance Fe(l) Fe(la) -4.057 A (seeTable 2).
Table 1Crystallographic data and characteristics of analysis for complex I

Table 2 Interatomic distance and valence angles in the structure of complex I

(Table Removed)
The matrixes of generation of equivalent positions of the atoms:
#1 -x, -y, -z
The crystalline structure of complex I is presented in Fig. 2.
The Moessbauer spectra of absorption were taken on the WissEl installation operating in the constant acceleration regimen. Co57 in the Rh matrix served as the source. Measurements of the spectra at low temperatures were carried out with the aid of a flow helium cryostat CF-506 (Oxford Instruments) with an adjustable temperature. Treating the Moessbauer spectra was
carried out by the least square method with the presumption of the Lorentz shape of individual spectral components.
The results are presented in Table 3.
Table 3
(Table Removed)
Parameters of Moessbauer spectra of Fe of complexes I-III at T=290K

Study of NO-donor activity of binuclear nitrosyl iron complexes with benzo-azaheterocyclic
derivatives
NO-donor activity of complexes I-III was first established upon a study of the reaction thereof with hemoglobin (Hb). At the base of the method of study is the reaction of the formation of Hb-NO upon the interaction of hemoglobin with free NO in the solution [R. Cassoly, Q.H. Gibson, Conformation, co-operativity and ligand binding human hemoglobin, J. Mol. Biol. 91 (1975) 301-313; T.J. McMahon, J.S. Stamler, Concerted nitric oxide/oxygen delivery by hemoglobin, in: L. Packer (Ed.), Methods In Enzymology, Academic Press, 301 (1999) Part C, 99-114]. The Hb to NO binding constant is 31010 M-1, which is three times more than a similar constant for CO and six times more than the binding constant for O2 [E. antonini, M. Brunori, Hemoglobin and myoglobin in the reactions with ligands, in: A. Neuberger, E.L. Tatum (Eds.) North-Holland research monographs. Fronties of biology. Vol. 21, North-Holland Publishing Company, Amsterdam-London, 21 (1971) 276]. The bimolecular constant of interaction of Hb with NO is close to diffusion constant: 1.02 108 M-1s-1 in a 0.05 M phosphate buffer, pH=7.0 at 20°C. Therefore, Hb is a very convenient trap for NO and is actively used to study NO-donor activity of compounds generating nitrogen monoxide. Wherein the spectra of optical absorption of free Hb and Hb-NO significantly differ, which makes it easy to detect the formation of a nitrosyl adduct.
The results of a study are presented for clarity in Fig. 3 for complex I.
A homogeneous bovine Hb preparation was prepared from bovine hemoglobin of the "MP Biomedicals" firm, which is a mixture of metHb and HbO2. 0.05 M phosphate buffer, pH 7.0 was used in all the steps of preparation of Hb and in all of the experiments with Hb. In order to transform the mixture of metHb and HbO2 to Hb, a 2x15 cm column was preliminarily prepared with Sephadex G-25 and transferred to an anaerobic condition. At the output from the column, 5 ml of Hb with a concentration of 6 10-4 moll-1 were collected. Hb was stored in a frozen state in the form of balls in liquid nitrogen. Before use, Hb was unfrozen in 5 ml volume
vessels in a nitrogen flow. 2.8 ml of a buffer, 0.1 ml of Hb 6 10-4 moll-1 were put in an anaerobic 4 ml test cuvet with a 1 cm length of an optical path and the spectrum of absorption was recorded. An anaerobic absolute DMSO was added to the charge of nitrosyl complex in a vessel filled with nitrogen so as to obtain a solution of the complex with a concentration of 6 10-3 moll-1 Then the solution was stirred for 3-5 minutes to complete dissolution and the obtained solution was administered by 0.1 ml into a test cuvet with Hb and into a comparison cuvet comprising 2.9 ml of the anaerobic buffer. The final concentration of the nitrosyl complex was equal to 2 10 moll-1, DMSO - 3.3%. Then the difference spectra of absorption were recorded, the first - 1 minute after the beginning of the reaction (shown in Fig. 3), then at intervals of 3 minutes for the first 30 minutes of the reaction, and then at intervals of 15, 30, 60 minutes (in Fig. 3 only a portion of these spectra is shown for clarity). Registration of the spectra was carried out to the complete conversion of Hb to HbNO, when the spectrum stopped changing. The amount of the formed NO was evaluated spectrophotometrically according to the amount of the formed HbNO. In order to determine the concentration of the HbNO, the spectrum of absorption of the reaction system comprising Hb and HbNO was broken down with the aid of computer processing with the MathCad program to components - Hb and HbNO spectrum. The calculation was carried out in a wavelength range of 450-650 nm in accordance with 200 experimental points. The spectrophotometer Specord M-40, provided with an interface for computer registration of the spectra and with a thermostatically controlled cuvet separation, was used for registration of the absorption spectra. The spectra were registered at 25°C.
As a result of these studies, it was established for the first time that the complexes generate NO in aqueous solutions of dimethylsulfoxide spontaneously (i.e., in the absence of hemo-, photo- or enzymatic activation).
Study of cytotoxicity of tetranitrosyl iron complexes with benzo-azaheterocyclic thiolyls on
human cancer cells in vitro Experimental technique
Before the beginning of the experiment, all of the compounds were dissolved in 200 µl of DMSO and then brought to the necessary concentration by the nutrient medium RPMI1640. The final concentration of DMSO in the samples did not exceed 0.2% and did not affect the growth of cells.
Cell lines - human ovary cancer SKOV3. The cells were grown in a monolayer in an RPMI1640 medium comprising 10% fetal bovine serum at 37°C and a 5% content of CO2. For the experiments, the cells were seeded in 96-well plates and grown under the same conditions.
A test of the cytotoxic effect was carried with the aid of an MTT-test, based on the capability of the dehydrogenase of live cells to reduce the uncolored salt of tetrazolium to the
blue crystals of formazan that are dissolved in dimethylsulfoxide (DMSO).
All of the compounds were put in wells in a volume of 20 µl in 4 final concentrations of 100, 50, 25 and 10 (µM). The total volume of incubation was 200 µl. Cells with preparations were incubated in the aforesaid conditions for 72 hours. At the end of incubation, an MTT reagent was added to the cells and incubated under the same conditions for 2 hours. Then the formed formazan crystals were dissolved in 100 ul of DMSO at 37°C for 20 minutes. The optical absorption of the DMSO solutions was measured on an optical counter for multi-well plates at a wavelength of 540 nm. The results were expressed in the form of average meanings for four parallel measurements. The cytotoxic effect was assessed according to the survival of the cells in experimental samples in respect to the control in %. The compound was regarded as active if at a concentration of 100 µM the number of live cells was 50% or less (IC50 ≤ 100 µM). The measurement error did not exceed 5%.
The results of the study are presented in Table 4.
Two of the studied nitrosyl iron complexes - I and III, comprising thiobenzimidazole and thiobenzthiazole ligands, exhibited cytotoxic activity on cell line SCOV3. The IC50 was 57 µM and 25 µM, respectively, for the I and III complexes.
The studied nitrosyl iron complexes I and III demonstrated a different cytotoxic effect against human cancer cells in vitro. Complex III exhibited maximum activity (IC50 = 25 µM).
Cisplatin (cis-DDP), an anticancer preparation that is used in Russian and foreign clinics (IC50 = 16 µM), was used as the comparison preparation. It was established that upon a reduction of the concentration by two times, the complex III retains the same activity as at a concentration of 100 µM (the survival of the cancer cells is 12.5%) (Table 4). The fact that the toxicity of the complex III is significantly less (the general toxicity index thereof LD100 is 50 µg/kg) than that of cisplatin (LD100 - 16 mg/kg) may be of significance in high-dose chemotherapy, accompanied by severe side effects, as a method for increasing the sensitivity of cancer cells to therapeutic action and makes it possible to reduce the concentration of the used cytostatics.
Table 4
Cytotoxic activity of nitrosyl iron complexes I-III against SKOV3 cell line

(Table Removed)
In accordance with the methodical indications in respect to a study of the anticancer
activity of pharmacological substances, studies were carried out on grafted mice tumors Ca-755, lymphatic leukemia P-388 and melanoma B-16 [on first generation hybrid mice BDF1 (C57B1/6 x DBA/2) and DBA/2 with a weight of 18-25 g, obtained from the department of laboratory animals GU RONTs in the name of N.N. Blokhin RAMS]. A statistically significant anticancer effect of the claimed preparations has been detected.

CLAIMS
1. A binuclear nitrosyl iron complex with benzo-azaheterocyclic derivatives of general
formula
wherein R is
(Formula Removed)
wherein X is NH, S or O, R1 is lower alkyl.
2. The binuclear nitrosyl iron complex according to claim 1, wherein R is benzimidazol-2-yl, 5-methylbenzimidazol-2-yl or benzthiazol-2-yl.
3. A method for preparing binuclear nitrosyl iron complexes according to claim 1, consisting in that a thiosulfate nitrosyl iron complex is treated with a corresponding benzo-azaheterocyclic thiol in a stoichiometric ratio in the presence of a reducing agent, and the process is carried out in an alkaline medium with the subsequent isolation of the desired product by known techniques.
4. The method according to claim 3, characterized in that the process is carried out at room temperature, mainly at 18-25°C.
5. The method according to claim 3, characterized in that the process is carried out in an oxygen-free atmosphere.
6. The method according to claim 3, characterized in that benzimidazole-2-thiol, 5-methylbenzimidazole-2-thiol or benzthiazole-2-thiol is used as the benzo-azaheterocyclic thiol.
7. The method according to claim 3, characterized in that hydrogen, metal thiosulfates, hydrogen sulfide and aliphatic thiols are used as the reducing agent.
8. A donor of nitrogen monoxide that is the binuclear nitrosyl iron complex according to claim 1.
9. Use of the binuclear nitrosyl iron complex according to claim 1 as an anticancer agent.

10. Use of the binuclear nitrosyl iron complex according to claim 1 for the preparation of an anticancer pharmaceutical.
11. A pharmaceutical composition comprising an effective amount of the binuclear nitrosyl iron complex with benzo-azaheterocyclic derivatives according to claim 1, and a pharmaceutically acceptable carrier.
12. The pharmaceutical composition according to claim 11, wherein a proton-containing medium is used as the pharmaceutically acceptable carrier.
13. The pharmaceutical composition according to claim 11, wherein a mixture of a proton-containing medium and dimethylsulfoxide is used as the pharmaceutically acceptable

carrier.
14. The pharmaceutical composition according to any one of claim 12 or claim 13, wherein water, a physiological solution, water-soluble biopolymers are used as the proton-containing medium.
15. The pharmaceutical composition according to claim 11, wherein the binuclear nitrosyl iron complex with benzo-azaheterocyclic derivatives is present in an amount of 50-100 µM.
16. A kit used for treating oncological diseases, comprising (1) a pharmaceutical
composition comprising a binuclear nitrosyl iron complex with benzo-azaheterocyclic
derivatives according to claim 1, in a sealed container; and (2) auxiliary components.

Documents:

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Patent Number

268939

Indian Patent Application Number

7581/DELNP/2009

PG Journal Number

40/2015

Publication Date

02-Oct-2015

Grant Date

24-Sep-2015

Date of Filing

23-Nov-2009

Name of Patentee

INSTITUTE OF PROBLEMS OF CHEMICAL PHYSICS RAS (IPCP RAS)

Applicant Address

PROSPEKT AKADEMIKA SEMENOVA, 1, IPCP RAS, CHERNOGOLOVKA, NOGINSKY R-ON, MOSKOVSKAYA OBL., 142432, RUSSIAN FEDERATION

Inventors:

#	Inventor's Name	Inventor's Address
1	SANINA, NATALIA ALEXEEVNA	UL. PERVAYA, D. 6-A, KV. 3, CHERNOGOLOVKA, MOSKOVSKYAYA OBL., 142432 RUSSIAN FEDERATION
2	ZHUKOVA, OLGA STEPANOVNA	UL. KIROVOGRADSKAYA, D. 2, KV. 314, MOSCOW 117587, RUSSIAN FEDERATION
3	SMIRNOVA, ZOYA SERGEEVNA	UL. DZHAMILYA, 5-1-210, MOSCOW 115580 RUSSIAN FEDERATION
4	RUDNEVA, TATYANA NIKOLAEVNA	UL. TSENTRALNAYA, D. 24, KV. 131 CHERNOGOLOVKA, MOSKOVSKAYA OBL., 142432 RUSSIAN FEDERATION
5	SHILOV, GENNADY VIKTOROVICH	UL. TSENTRALNAYA, D. 22, KV. 131 CHERNOGOLOVKA, MOSKOVSKAYA OBL., 142432 RUSSIAN FEDERATION
6	ALDOSHIN, SERGEI MIKHAILOVICH	UL. TRETYA, 17, CHERNOGOLOVKA, MOSKOVSKAYA OBL., 142432, RUSSIAN FEDERATION
7	DAVYDOV, MIKHAIL IVANOVICH	UL. AKADEMIKA MILLIONSCHIKOVA, 35-311, MOSCOW, 115446, RUSSIAN FEDERATION

PCT International Classification Number

C07F 15/02

PCT International Application Number

PCT/RU2007/000286

PCT International Filing date

2007-05-30

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA