WO2008147243A1 - Binuclear nirtosyl-iron complexes with benzo-trans-heterocyclic derivatives and a method for the production thereof - Google Patents

Abstract

The invention relates to binuclear nirtosyl-iron complexes with benzo-trans-heterocyclic derivatives of general formula [Fe2(SR)2(NO)4], wherein R is of (I), in which x is NH, S or O and R1 is lower alkyl, to a method for production thereof, to the use thereof in the form of an antitumoral medicinal agent, to a nitrogen monoxide donor based thereon, to a pharmaceutical composition containing said complexes in an effective quantity and to a set used for treating oncological diseases.

Description

 BINADER NITROZYL IRON COMPLEXES WITH BENZAZAHETEROCYCLIC DERIVATIVES, METHOD FOR THEIR

OBTAINING

Technical field

The invention relates to binuclear nitrosyl iron complexes and can be used in medical practice to create new generation drugs for the treatment of cancer. State of the art

In recent years, in order to create new-generation drugs used in the treatment of cancer, an intensive search for antitumor agents based on transition metal complexes with an improved spectrum of activity and reduced side effects compared with already used clinical drugs, for example, cisplatin, sodium nitroprusside and etc.

The interest in nitrosyl metal complexes has increased due to the open possibilities of their use as effective NO donors in medicine, in particular for the treatment of tumor diseases. The use of NO donors as a new class of antitumor agents is associated with the important role of NO in the growth of malignant tumors [Wipk D., Vodovoz J., Soc. J., Biochemistru, 1998, 63, 7, pp. 948-957]. Nitrogen monoxide has been shown to alter the level of tumor cell apoptosis, p53 gene activity, and neoangiogenesis [Vrupe V., Scheneiderhan N., Nitriс okhide evoked p53-accumulation a d aportosis, Toxol Letters, 2003, 193, 2, pp.19-23], inhibits the activity of key repair protein

Mammalian Ob-methylguanine DNA Methyl Transferase [L. Liu, M. Hu-Welliver,

S. Capagula, N.E. Reg, Iptivatiop apd degradatiop оf 06-alkylguapipe-DNA alkultrаspеrаase аftеr reаstiop with pitriс ohide., Sapser. Res., 2002, 62, pp. 3037-3043].

The binding of NO to active sites of metal enzymes, in particular, to non-heme iron-containing proteins, has been studied especially intensively [Ford P.C., Lorkovis I.M., Chem. Rev., 2002, 102, 993; Hoshipo M., Lavermap L.E., Ford P. C, Sord. Chem. Rev., 1999, 187, p. 75]. Found that one of the forms

SUBSTITUTE SHEET (RULE 26) Natural reservoirs of NO are nitrosyl iron complexes with thiol-containing ligands [Butler AR, Megson LL, Chem. Rev., 2002, 102, pp. 155-1165].

Their synthetic models — Russen’s red salt esters — have the composition [Fe ₂ (SR) ₂ (NO) ₄ ], where R = Et, t-Bu, (CH ₂ ) ₄ -CH ₃ , C ₆ H ₅ F, Ph [T. Thomas, J.N. Robbertsop, E.G. Sokh, Asta. Crastalogr., 1958, 11, p. 599; C. Glidewell, M.E. Narmap, M.V. Shrstous, LL. Johnsop, M. Motewalli, J. Chem. Res., 1998, 212, p. 1676; R.E. Marsh, A.L. Srek, Asta. Crastalogr., Sest. B. Struct. Ssi., 2001, 57, p. 800; C. Jiphuah, M. Shaipipg, H. Jiplipg, L. Lachi, Chinese J. Struct. Chem., 1983, 2, p. 263; T. B. Raushfoss, T.D. Wеthеrill, Ipoorg. Chem., 1982, 21, pp. 827-830].

These binuclear diamagnetic seranitrosyl complexes with R = AIk generate NO upon thermal or photoactivation [J.L. Borussa, P. C. Ford, Co. Chem. Rev., 2000, 200-202, pp. 887-900] and can serve as new promising antitumor NO-donating agents. Also known are binuclear paramagnetic seranitrosyl iron complexes with μ-N-С-S ligands, which are synthetic analogues of natural donors of NO dinitrosyl iron complexes (DNIC) with cysteine, glutathione and other thiol-containing ligands of small molecular weight [O.A. Rakova, N.A. Sapipa, S.M. Aldoship, N.A. Gopsharova, G.V.Shilov, Yu.M. Shulga, N.S. Ovapesuap, morgapis Chemistru Commutisopiops 2003, 6, 145-148; A. F.Vapip, N.A. Sapipa, V. A. Serzhepkov, D. Sh. Vurbaev, V.I. Lozipsku and S.M. Aldoship, Nitrikh okhide: biologu & schemistra 2007, 16, 82-93].

However, the currently known synthetic NO donors of various classes are not used as therapeutic agents for the treatment of cancer, but are used only to enhance (to varying degrees, depending on the chemical nature) the effects of existing chemotherapeutic agents or radiotherapy [Wipk D., Vodóvoz J ., Soc J., Biochemistru, 1998, 63, 7, pp. 948-957; N.R. Kopovalova, S.A. Gopsharova L.M. Volkova, T.A. Raevskaua, L.T. Eremepko, A.M. Korolev, Nitris Ohide: Biolog apd Chemistru, 2003, 8, pp. 59-64; Yapg, W., Rogers P.A., Ding H., J. Biol. Chem., 2002, 277, pp. 12868-12873; O. Siri, A. Tabard, P. Bulmbi, R. Guilard, Iporg. Chim. Assa 2003, 350, p. 633; J.L. Burgaud, E. Jpgipi, DeI Soldato P. App, N. Y. Acad. Sсi. 2002,

SUBSTITUTE SHEET (RULE 26) 962, p. 360; TJ. Kata, LV Ruibibrat, GS Kalepdo, Toxiolothers, 2001, 121, P-57].

In the work of A. Japszuk et al. Nitriс Ohide, 2004, 10, 1, pp. 42-50, the direct cytotoxic effect of the nitrosyl iron complex Na [Fe ₄ S ₃ (NO) ₇ ] on human and mouse melanoma cells was studied. However, this nitrosyl iron complex generates NO during photoactivation (even in the dark), and also cannot be used as an antitumor drug due to its high toxicity to normal cells.

Thus, there is a need for other antitumor agents with increased efficacy and reduced toxicity.

The objective of the present invention is to expand the arsenal of antitumor agents and the creation of antitumor drugs based on transition metal complexes with an improved activity spectrum and reduced side effects, in particular, medicines based on binuclear nitrosyl iron complexes with benzazheterocyclic derivatives acting as NO donors with increased activity and reduced toxicity.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to novel binuclear nitrosyl iron complexes with benzazheterocyclic derivatives of the formula [Fe ₂ (SR) ₂ (NO) ₄ ], where R is

где X представляет собой NH, S или О, R¹ представляет собой низший алкил.

where X represents NH, S or O, R ¹ represents lower alkyl.

In another aspect, the invention relates to a method for producing binuclear nitrosyl iron complexes with benzazheterocyclic derivatives of the formula [Fe ₂ (SR) ₂ (NO) ₄ ] by treating the thiosulfate nitrosyl iron complex with an appropriate benzazheterocyclic thiol in a stoichiometric ratio in the presence of a reducing agent in an alkaline medium. This method allows for the first time to obtain the claimed complexes in

SUBSTITUTE SHEET (RULE 26) crystalline form.

In an additional aspect, the present invention relates to a donor of nitric oxide, which is a binuclear nitrosyl complex of iron with benzazheterocyclic derivatives, described above. In a further aspect, the present invention relates to the use of binuclear nitrosyl iron complexes with benzazheterocyclic derivatives of the formula [Fe ₂ (SR) ₂ (NO) ₄ ] as an antitumor drug.

In an additional aspect, the present invention relates to the use of binuclear nitrosyl iron complexes with benzazheterocyclic derivatives of the formula [Fe ₂ (SR) ₂ (NO) ₄ ] for the preparation of antitumor drugs.

In a further aspect, the present invention relates to a pharmaceutical composition comprising an effective amount of a binuclear nitrosyl iron complex with benzazheterocyclic derivatives of the formula [Fe ₂ (SR) ₂ (NO) ₄ ] and a pharmaceutically acceptable carrier.

In a further aspect, the present invention relates to a kit used to treat cancer, comprising: (1) a pharmaceutical composition comprising a binuclear nitrosyl iron complex with benzazheterocyclic derivatives of the formula [Fe ₂ (SR) ₂ (NO) ₄ ], where R has the above meanings in sealed packaging; and (2) auxiliaries.

Description of figures

Figure l shows the molecular structure of the complex (Fe ₂ S ₂ N ₈ C ₁₄ H ₁₀ O ₄ ) [complex I].

The complex has a centrosymmetric dimeric binuclear structure (Fig. 1). In the dimer, two tetrahedrally coordinated two NO groups and two benzazheterocyclic thiol iron atoms are connected by a bridge: Fe-S-C-N-Fe '. The complex contains two solvent molecules - acetone. Figure 2 shows the crystal structure of the complex

(Fe ₂ S ₂ N ₈ C ₁₄ H ₁₀ O ₄ ) [complex I].

Fig. 3 shows the change in the difference absorption spectra over time.

SUBSTITUTE SHEET (RULE 26) in the interaction of complex I (Fe ₂ S ₂ N ₈ C ₁₄ H ₁₀ O ₄ ) with hemoglobin (Hb). The solvent is 0.05% phosphate buffer, pH = 7.0, containing 3.3% dimethyl sulfoxide, 25 ° C.

DETAILED DESCRIPTION OF THE INVENTION

The novel binuclear nitrosyl iron complexes with the benzazheterocyclic derivatives of the present invention have the general formula [Fe ₂ (SR) ₂ (NO) ₄ ], where R is

где X представляет собой NH, S или О, R¹ представляет собой низший алкил.

where X represents NH, S or O, R ¹ represents lower alkyl.

Preferably, R is benzimidazol-2-yl, 5-methyl-benzimidazol-2-yl or benzyl thiazol-2-yl.

The term “lower alkyl” as used herein means a straight or branched chain alkyl radical containing 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.

In the prior art there is no information about the claimed compounds and the method for their preparation. The method for producing new binuclear nitrosyl iron complexes with benzazheterocyclic derivatives of the formula [Fe ₂ (SR) ₂ (NO) ₄ ], where R has the above meanings, is that the thiosulfate nitrosyl iron complex is bis (μ-thiocylphato-S) -bis ( dinitrosylphosphate) (2-) sodium Na ₂ [Fe ₂ (S ₂ O ₃ ) ₂ (NO) ₄ ] (TNLC) is treated with the corresponding benzazheterocyclic thiol in a stoichiometric ratio in the presence of a reducing agent, and the process is carried out in an alkaline medium followed by isolation of the target product by known receptions.

Preferably, the process is carried out at room temperature, preferably at 18-25 ° C. Preferably, the process is conducted in an oxygen free atmosphere.

As a benzazheterocyclic thiol, preferably

SUBSTITUTE SHEET (RULE 26) use benzimidazole-2-thiol, 5-methylbenzimidazole-2-thiol or benzethiazole-2-thiol.

Hydrogen, metal thiosulfate, for example, Na ₂ S ₂ O ₃ -5H ₂ O, hydrogen sulfide and aliphatic thiols are used as reducing agent in the method of the present invention.

The inventors have found that binuclear nitrosyl iron complexes with the benzazheterocyclic derivatives of the present invention are effective NO donors, generating nitrogen monoxide spontaneously when decomposed in proton media (such as water, blood and its components, physiological solutions, etc.), in the absence of chemo, photo, or enzymatic activation. Thus, the complexes of the present invention represent a new promising class of nitrogen monoxide donors — synthetic analogues of active sites of nitrosyl non-heme iron-sulfur proteins — natural NO depots. Therefore, the present invention in an additional aspect relates to nitric oxide donors, which are binuclear nitrosyl iron complexes with benzazheterocyclic derivatives described above.

Binuclear nitrosyl iron complexes with the benzazheterocyclic derivatives of the natural aromatic thiols of the present invention possess antitumor activity against tumor cells of a mammal, in particular a human.

The present invention is further directed to the use of binuclear nitrosyl iron complexes with benzazheterocyclic derivatives of the formula [Fe ₂ (SR) ₂ (NO) ₄ ], where R has the above meanings, as an antitumor drug.

In particular, the compounds of the present invention can be used to treat the following oncological diseases: ovarian cancer, breast adenocarcinoma, B-16 melanoma, Lewis epidermoid carcinoma. The iron complexes of the present invention are suitable for inhibiting the growth of tumors in mammals, and are preferably administered in the form of a pharmaceutical composition comprising an effective

SUBSTITUTE SHEET (RULE 26) an antitumor amount of a compound of the present invention in combination with at least one pharmaceutically or pharmacologically acceptable carrier and / or excipient. A carrier, also known in the art as an excipient, excipient, excipient, additive or diluent, is any substance that is pharmaceutically inert, gives the appropriate consistency or form of the composition and does not impair the therapeutic efficacy of the antitumor compounds. A carrier is “pharmaceutically or pharmacologically acceptable” if it does not cause an adverse, allergic or other adverse reaction when administered to a mammal or human, respectively.

The present invention also relates to the use of binuclear nitrosyl iron complexes with benzazheterocyclic derivatives of the formula [Fe ₂ (SR) ₂ (NO) ₄ ], where R has the above meanings, to obtain an antitumor drug.

The present invention further provides pharmaceutical compositions comprising an effective amount of a binuclear nitrosyl iron complex with benzazheterocyclic derivatives of the formula

[Fe ₂ (SR) ₂ (NO) ₄ ], where R has the above meanings, and a pharmaceutically acceptable carrier.

Preferably, a proton-containing medium is used as a pharmaceutically acceptable carrier in the composition of the invention.

As a pharmaceutically acceptable carrier, a mixture of a proton-containing medium and dimethyl sulfoxide is also preferably used in the composition of the invention.

Preferably, water, physiological saline, and water-soluble biopolymers are used as the proton-containing medium.

Preferably, a binuclear nitrosyl iron complex with a benzazheterocyclic derivative is present in the composition in an amount of 50-100 μM.

Pharmaceutical compositions containing antitumor compounds of the present invention can be prepared by any

SUBSTITUTE SHEET (RULE 26) in the usual way. The required formulation is selected depending on the chosen route of administration. Compositions according to the invention can be prepared for any route of administration so that the target tissue is accessible with this 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), local (nasal, percutaneous, intraocular, intraocular) into the small intestine, pulmonary, intralymphatic, intracavitary, vaginal, trasurethral, intradermal, auricular, intramammary, buccal, orthotopic, intratracheal, intrafocal, percutaneous, endoscopic, transmucosal, sublingual and intestinal injections.

Pharmaceutically acceptable carriers for use in the compositions of the present invention are well known to those skilled in the art and are selected depending on a number of factors: the particular antitumor compound used and its concentration, stability and expected bioavailability; the disease, disorder or condition of the person being treated with the composition; subject, his age, weight and general condition; and method of administration. Suitable carriers are readily identifiable by a person skilled in the art (J.G. Nairp at: Remptop's Pharmaceut Scalse (published by A. Gepparo), Mask Publishing Co., Eastop, Pa, 1985, pp. 1492-1517).

The compositions are preferably prepared in the form of tablets, dispersible powders, pills, capsules, gelatin capsules, coated droplets, gels, liposomes, granules, solutions, suspensions, emulsions, syrups, elixirs, lozenges, dragees, lozenges, or any other dosage form, which can be administered orally.

Compositions of the present invention for oral administration comprise an effective antitumor 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,

SUBSTITUTE SHEET (RULE 26) carboxymethyl cellulose, agar, beckon, sorbitol, calcium phosphate, calcium carbonate, sodium carbonate, kaolin, alginic acid, gum arabic, corn starch, potato starch, sodium saccharinate, magnesium carbonate, tragacanth, microcrystalline cellulose, colloidal sodium silica, colloidal sodium hydroxide, magnesium and stearic acid. Further, such solid dosage forms may be uncoated or may be coated by known methods, for example, to delay disruption and absorption.

The antitumor compounds of the present invention are also preferably used for the preparation of a parenteral dosage form, for example, in the form of a dosage form for injection by intravenous, intraarterial, subcutaneous, rectal, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal or intrasterial route. Compositions according to the invention for parenteral administration include an effective antitumor amount of an antitumor compound in a pharmaceutically acceptable carrier. Formulations suitable for parenteral administration include solutions, suspensions, dispersions, emulsions, or any other dosage form that can be administered parenterally. Techniques and compositions for preparing parenteral dosage forms are known in the art.

A minor amount of additional components well known in the pharmaceutical industry may be included in the compositions of the invention for various purposes. These components for the most part give properties that increase the retention time of the antitumor compound at the injection site, contribute to the stability of the composition, provide pH control, facilitate the introduction of the antitumor compound into pharmaceutical compositions, and the like. Each of these components is individually present in an amount, preferably, less than about 15 mass%, more preferably less than about 5 mass%, and most preferably less than about 0.5 mass%, based on the total weight of the composition. Some components, such as

SUBSTITUTE SHEET (RULE 26) fillers or diluents can be up to 90 mass%, based on the total weight of the composition, as is well known in the technology for the preparation of medicines. Such additives include cryoprotective component to prevent precipitation of the iron complex with thiophenol, surfactants, wetting or emulsifying agents (e.g. lecithin, policopbat-80, Tween ^® 80, plyuponik-60, polyoxyethylene stearate), preservatives (e.g., ethyl-n- hydroxybenzoate), anti-microbial agents (e.g. benzyl alcohol, phenol, m-cresol, chlorobutanol, sorbic acid, thimerosal and paraben), pH adjusting agents or buffering agents (e.g. acids, bases, ac sodium tat, sorbitan monolaurate), components for regulating osmolarity (e.g. glycerin), thickeners (e.g. aluminum monostearate, stearic acid, cetyl alcohol, stearyl alcohol, guar gum, methyl cellulose, hydroxypropyl cellulose, tristearin, cetolic esters) , pigments, dyes, flow improvers, non-volatile silicones (e.g. cyclomethicone), clays (e.g. bentonites), adhesives, bulking agents, flavors, sweeteners, adsorbents, fillers carriers (e.g. water, saline, electrolyte solutions), binders (e.g. starches, such as corn starch, wheat starch, rice starch or potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, polyvinyl chloride, polyvinyl gum arabic), disintegrating agents (e.g. starches, such as corn starch, wheat starch, rice starch, potato starch or carboxymethyl starch, structured polyvinylpyrrolidone, agar, algae hydrochloric acid or its salt, such as sodium alginate, croscarmellose sodium or crospovidone), lubricants (e.g., silica, talc, stearic acid or its salts, such as magnesium stearate, or polyethylene glycol), coating agents (e.g., concentrated sugar solutions, including gum arabic, talc, polyvinylpyrrolidone, carbopolgel, polyethylene glycol or titanium dioxide) and antioxidants (e.g. sodium metabisulfite, sodium bisulfite, sulfite

SUBSTITUTE SHEET (RULE 26) sodium, dextrose, phenols and thiophenols).

According to a preferred embodiment, the pharmaceutical composition according to the invention comprises at least one non-aqueous pharmaceutically acceptable solvent and an antitumor compound having a solubility therein of at least about 10-60 mg / ml. Without being bound by any particular theory, it is believed that the solubility of the antitumor compound in dimethyl sulfoxide can be directly related to its effectiveness. It is also preferred that the antitumor compound has an ID ₁₀₀ value (i.e., a drug concentration that causes 100% inhibition of colony formation) at least 4 times less than cisplatin when measured according to the procedure described in the book “An Experimental Assessment of Antineoplastic Drugs in the USSR and the United States” edited by Z. P. Sof'ina, A.B. Syrkina (USSR), A. Goldin, A. Klein (USA) M.: “Medicine”, 1979, pp. 71 - 105.

The introduction of the dosage form in these ways can be continuous or periodic, depending, for example, on the physiological condition of the patient, on whether the purpose of the introduction of therapeutic or prophylactic, and other factors known or evaluated by the practitioner.

The dose and administration regimen of the pharmaceutical compositions of the invention can be easily determined by an oncologist. It is understood that the dose of antitumor compounds depends on the age, gender, state of health and weight of the recipient, the type of treatment carried out simultaneously, if any, the frequency of treatment and the nature of the desired effect. For any route of administration, the exact amount of the antitumor compound used, as well as the prescribed dose necessary to achieve the beneficial effects described here, also depends, in particular, on factors such as the bioavailability of the antitumor compound, the human disease being treated, the desired therapeutic dose, and others factors that are obvious to the specialist.

The concentration of the antitumor compound in the liquid pharmaceutical composition is, most preferably, 50-100

SUBSTITUTE SHEET (RULE 26) μm. Relatively low concentrations are generally preferred, since the antitumor compound is most soluble at low concentrations.

Emulsions for parenteral administration can be prepared by dissolving the antitumor compound in any pharmaceutically acceptable solvent capable of dissolving the compound (e.g., DMSO, dichloromethane) to form a solution. An appropriate volume of carrier is added to the solution with stirring, which is an emulsion, such as an emulsion of Lirosup II or Lirosup III, to obtain a pharmaceutically acceptable emulsion for parenteral administration to a patient. If desired, such emulsions may be prepared with or without a minimum amount of a Cemorhor ^® solution.

Solutions for parenteral administration can be prepared by dissolving the antitumor compound in any pharmaceutically acceptable solvent capable of dissolving the compound (e.g., DMSO) to form a solution. To the solution, with stirring, add the appropriate volume of the carrier, which is a proton-containing medium (saline solutions, sugars, water-soluble polymers, proteins, electrolyte solutions) for parenteral administration to the patient. For example, for liquid formulations, non-aqueous pharmaceutically acceptable polar solvents are usually used as a carrier, such as oils, alcohols, amides, esters, ethers, ketones, hydrocarbons and mixtures thereof, as well as water, saline solutions, dextrose solutions (e.g. DW5), electrolyte solutions, or any other proton pharmaceutically acceptable medium.

Suitable non-aqueous pharmaceutically acceptable polar solvents include, but are not limited to, amides (e.g. dimethylacetamide (DMA), dimethylacetamide benzylbenzoate, dimethylformamide, N- (β-hydroxyethyl) lactamide, N ₅ N-dimethylacetamide, 2-pyrupolidinone-l-me - 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,

SUBSTITUTE SHEET (RULE 26) alkyl oleate, benzyl benzoate, benzyl acetate, dimethyl sulfoxide (DMSO), glycerol esters such as mono-, di- or triglyceryl citrates or tartrates, ethyl benzoate, ethyl acetate, ethyl carbonate, ethyl lactate, ethyl oleate, sorbitan fatty acid esters and polyethylene ethers ), r-glyceryl monostearate, esters of glycerides, such as mono-, di- or triglycerides, fatty acid esters, such as isopropyl myristate, fatty acid-based esters and PEG, such as PEG-hydroxyoleate and PEG-hydroxystearate, N-methylpyrrolidinone , plupnik-60, p oxyethylenated polyesters based on sorbitol and oleic acid, such as poly (oxyethylen) zo- ₆ ocopbitol (oleate) _2-4 , poly (oxyethylen) ₁₅ . ₂ mono-olone, fields (oxyethylen) _15-20 mono-12-hydroxy-ester and poly (oxyethylen) _{\ 5-2} mono-amino acid; polyoxyethyl sorbitan esters, such as polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monostearate and polysorbate ^® 20, 40, 60 or 80 from ICI Américas, DEmig; polyvinylpyrrolidone; fatty acid esters modified with alkylene oxides, such as hydrogenated polio (40) castor oil and polyoxyethylene castor oils (for example, Cortorhor ^® EL solution or Creamhorr ^® RH 40 solution); fatty acid esters of a saccharide (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, tetrosa, heptoses and octoses), a disaccharide (e.g. sucrose, maltose, lactose and trehalose) or an oligosaccharide or mixtures thereof with a fatty acid (s) 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 saturated fatty acids such as palmitoleic acid, oleic acid, elaidic acid, erucic acid and linoleic acid)), or steroidal esters); alkyl, aryl or cyclic ethers with 2-30 carbon atoms (for example, diethyl ether, tetrahydrofuran, dimethyl isosorbite, diethylene glycol monoethyl ether); glycofurol (simple

SUBSTITUTE SHEET (RULE 26) polyethylene glycol ether and tetrahydrofurfuryl alcohol); ketones with 3-30 carbon atoms (e.g. acetone, methyl ethyl ketone, methyl isobutyl ketone); aliphatic, cycloaliphatic or aromatic hydrocarbons with 4-30 carbon atoms (e.g. benzene, cyclohexane, dichloromethane, dioxolanes, hexane, n-decane, n-dodecane, n-hexane, sulfolane, tetramethylene sulfone, tetramethylene sulfoxide, toluene, dimethyl sulfoxide) tetramethylene sulfoxide); alkyl or aryl halides containing 1-30 carbon atoms and optionally more than one halogen atom as a substituent; dichloromethane; monoethanolamine; petroleum ether; trolamine; omega-3 polyunsaturated fatty acids (for example, alpha-linolenic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid); polyglycol ether of 12-hydroxystearate acid and polyethylene glycol (Solutol® HS-15 from BASF, Ludwigshafep, Hempap); polyoxyethylene glycerol; sodium laurate; sodium oleate; or sorbitan monooleate. Preferably, the proton content of the medium, such as water, saline solutions, water-soluble polymers, proteins, dextrose solutions (eg, DW5), electrolyte or alcohol solutions from the PAA Labotor's catalog (2006) p, is used as a pharmaceutically acceptable carrier in the present invention. 26. In a further aspect of the present invention, there is provided a kit for treating cancer, which comprises: (1) a pharmaceutical composition comprising a binuclear nitrosyl iron complex with the benzazheterocyclic derivatives of the present invention, in an airtight package; and (2) auxiliaries. The kit may contain the composition in the form of a single dosage form or in the form of multiple doses. The kit may include forms for oral or parenteral administration.

The pharmaceutical composition in the kit can be placed in glass or polymer vials, ampoules, vials, metered dose cartridges for injectors, blisters, capsules, sachets with the composition, used respectively for oral or parenteral form.

Aids include reconstitution fluids

SUBSTITUTE SHEET (RULE 26) a composition administered parenterally if it is presented in a kit in concentrated form, for example, in the form of a dry substance, a dried preparation, and the like; Means for the preparation of oral liquid forms and forms for injection ex-troter. Water for injection, physiological saline, lidocaine solution, etc., can be used as a recovery fluid. To restore the composition used in liquid form orally, a solution of glucose, sugars, syrups, etc. can be used.

Optional kit aids include openers for closures, means for sealing opened reusable closures, instruction inserts.

The pharmaceutical composition, which is a solid dosage form for oral administration, can be presented in a kit in the form of tablets, capsules in blisters, ampoules, vials, vials, sachets, and the like. The pharmaceutical composition, which is a liquid dosage form for parenteral or oral administration, may be presented in a kit in bottles, capsules, ampoules, cartridges, and the like.

An example of a kit for parenteral administration includes a package that contains instructions for use, ampoules or vials with a dry composition and ampoules with saline for injection. A device for opening ampoules is inserted into the package. Ampoules are packed in blisters of 10 ampoules.

Other kit options are obvious to a person skilled in the art from the above description.

The following examples are provided only as a further illustration of the invention and should not be construed as limiting the invention.

Examples of the synthesis of complexes

Example 1

Synthesis of complex (D), where R = benzimidazol-2-yl (Fe ₂ S ₂ NRCuHiQO ₄ )

For the synthesis, the starting reagents Na ₂ S ₂ O ₃ -5H ₂ O (Aldrich), NaOH (analytical grade), 2-mercaptobenzimidazole (Aldrich) were used. All operations for the preparation, mixing of solutions and the separation of complexes were carried out in an atmosphere of industrial argon at room temperature. For recrystallization

SUBSTITUTE SHEET (RULE 26) used an organic solvent.

A mixture of 0.992 g (4 mmol) of Na ₂ S ₂ O ₃ -SH ₂ O and 1,074 g (2 mmol) of the thiosulfate nitrosyl iron complex of bis (μ-thiocylphato-S) - bis (dinitrosyl fepat) (2-) sodium Na ₂ [Fe ₂ (S ₂ Os) ₂ (NO) ₄ ] (TNLC) was dissolved in distilled water. A mixture consisting of 1,500 g (10 mmol) of 2-mercaptobenzimidazole 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 intermediate was 0.719 g. After one day, the precipitate was recrystallized. The complex is highly soluble in ethanol, methanol, acetone, DMSO and

DMF. Insoluble in dichloromethane, water, heptane and ether. Yield: 63.50%;

Found for Fe ₂ S ₂ N ₈ C ₁₄ H ₁₀ O ₄ ,%: 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 (cm ⁴ ): 3207, 1787, 1738, 1515, 1467, 1428, 1384, 1272, 1218, 1183,

1002, 742, 713, 603.

Example 2

For the synthesis, the starting reagents Na ₂ S ₂ O ₃ -5H ₂ O (Aldrich), NaOH (analytical grade), 2-mercapto-5-methylbenzimidazole (Aldrich) were used. All operations for the preparation, mixing of solutions and the separation of complexes were carried out in an atmosphere of industrial argon at room temperature. An organic solvent was used for recrystallization. Distilled water was prepared for the synthesis by passing a stream of technical argon through it for 30 minutes.

A mixture of 0.4960 g (2 mmol) of Na ₂ S ₂ O ₃ -5H ₂ O and 0.5740 g (1 mmol) of the thiosulfate nitrosyl iron complex of bis (μ-thiocylphato-S) - bis (dinitrosylphosphate) (2-) sodium Na ₂ [Fe ₂ (S ₂ O ₃ ) ₂ (NO) ₄ ] (TNLC) 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 intermediate was 2.2017 g. After one day, the precipitate

SUBSTITUTE SHEET (RULE 26) recrystallized.

The complex is well soluble in acetone, DMSO and DMF. Insoluble in dichloromethane, water, heptane and ether.

Yield: 45.53%; Found for Fe ₂ S ₂ N ₈ C ₁₆ H ₁₄ O ₄ ,%: 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, 0, 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

For the synthesis, the starting reagents Na ^ S ₂ O ₃ -SH ₂ O (Aldrich), NaOH (bhd), 2-mercaptobenzthiazole (Aldrich) were used. All operations for the preparation, mixing of solutions and the separation of complexes were carried out in an atmosphere of industrial argon at room temperature. An organic solvent was used for recrystallization. Distilled water was prepared for the synthesis by passing a stream of technical argon through it for 30 minutes.

A mixture of 0.4960 g (2 mmol) of Na ₂ S ₂ O ₃ -5H ₂ O and 0.5740 g (1 mmol) of the thiosulfate nitrosyl iron complex of bis (μ-thiocylphato-S) - bis (dinitrosylphosphate) (2-) sodium Na ₂ [Fe ₂ (S ₂ O ₃ ) ₂ (Nθ) ₄ ] (TNLC) was dissolved in distilled water. A mixture of 0.8402 g (5 mmol) of 2-mercaptobenzthiazole 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 intermediate was 1.1182 g. After a day, the precipitate was recrystallized.

The complex is well soluble in ethanol, methanol, acetone, methylene chloride, acetonitrile, ether, THF, DMSO and DMF. Not soluble in water, heptane.

Found for Fe ₂ S ₄ N ₆ C ₁₄ H ₈ O ₄ ,%: 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 ₅ 79, 0, 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.

SUBSTITUTE SHEET (RULE 26) IR spectra of all samples were recorded on a SPECTRUM BX-II Fourier spectrometer. A sample was prepared in the form of tablets with KBr (1 mg of the analyte per 300 mg of KBr).

X-ray diffraction analysis of the Fe ₂ S ₂ NgCi ₄ H _1O O ₄ (I) complex was carried out on a BRUKER P-4 automatic four-circle diffractometer,

(graphite monochromator, λ (Mo-K _α ) = 0.71073 A, temperature 200K, Θ / 2Θ scan). The experiment used a black single crystal in the form of a parallelepiped with dimensions of 0.35x0.20x0.15 mm. The crystals are unstable at room temperature and are destroyed at a rate of 10% in 1 hour. The structure is deciphered by the direct method. The positions and thermal parameters of non-hydrogen atoms are refined in the isotropic and then anisotropic approximation by the full-matrix least-squares (MHK) method. Hydrogen atoms were detected from spaced Fourier syntheses and refined in the isotropic approximation.

The molecular structure of complex I is shown in FIG. The complex has a centrosymmetric dimeric binuclear structure. In the dimer, two tetrahedrally coordinated by two NO groups and two benzazheterocyclic thiols, iron atoms are connected by a bridge: Fe-S-C-

N-Fe '. The complex contains two solvent molecules - acetone.

Crystallographic data and the main refinement parameters are presented in table 1.

The interatomic distances and angles are presented in Table 2. All calculations were performed using the SHELXTL software package (G.M. Sheldriсk, SHELXTL v.6.14, Stätter Döthermäti Söftwäré Suite, Wacker AXS, Madisop, Wisconsip, USA, 2000). The distance Fe (I) Fe (I a) is 4,057 A cm (table 2).

Table 1 Crystallographic data and analysis characteristics for complex I

SUBSTITUTE SHEET (RULE 26)

table 2

SUBSTITUTE SHEET (RULE 26)

Matrices of generation of equivalent atomic positions: # 1-x, -y, -z

The crystal structure of complex I is shown in FIG. 2.

The Mössbauer absorption spectra were recorded on a WissEl setup operating in a constant acceleration mode. Co ⁵⁷ in the Rh matrix served as a source. Spectra were measured at low temperatures using a flow-controlled helium cryostat CF-506 (Oxford Istrumepts) with a controlled temperature. The Mössbauer spectra were processed using the least squares method under the assumption of the Lorentzian shape of individual spectral components.

The results are presented in table 3.

Table 3

The NGR parameters of the spectra of Fe, 57 complexes I – III at T = 290K

SUBSTITUTE SHEET (RULE 26) Investigation of the NO-donor activity of binuclear nitrosyl iron complexes with benzazheterocyclic derivatives

The NO-donor activity of complexes I – III was first established by studying their reactions with hemoglobin (Hb). The research method is based on the reaction of the formation of Hb-NO in the interaction of hemoglobin with free NO in solution [R. Sassolu, Q.N. Gibsop, Compormatiop, co-reactivit ap ligapd ipgip humap hemoglobip, J. MoI. Biol. 91 (1975) 301-313; TJ. MsMahop, JS Stalmer, Soppertit pitris okhide / okhuhep to a distributor, ip: L. Rasker (Ed.), Methods In Publisher, Academis Resource, 301 (1999) 99 Pa. The binding constant of Hb with NO is 3 ^{10 10} M ^"1 , which is three orders of magnitude higher than the corresponding constant for CO, and six orders of magnitude higher than the binding constant for O ₂ [E. Pharmacy, M. Všyuri, Hemoglopp apd muoglobiip térethiops with ligapds, ip: A. Neuberger, E.L. Tatum (Ed.) Nort-Hellapd resec-mopogrhs. Froptіs оf biologogu. VoI. 21, North-Hoplapul boplopt 21, 197 Amst, 21, 197 Amp. the interaction of Hb with NO is close to diffusion: 1.02-10 ⁸ M ^“1 -s ^{” 1} in 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 an NO donor ktivnosti compounds, generating nitric oxide. Thus the optical absorption spectra of free Hb and Hb-NO vary greatly, making it easy to detect the formation of nitrosyl adduct.

The results of the study are presented for clarity in Fig.Z for complex I.

A homogeneous preparation of bovine Hb was obtained from bovine hemoglobin from MP Biomedicals, which is a mixture of metHb and HbO ₂ . At all stages of Hb production and in all experiments with Hb, 0.05 M phosphate buffer, pH 7.0, was used. To convert a mixture of metHb with HbO ₂ into Hb, a 2x15 cm column with Sephadex G-25 was preliminarily prepared and transferred to an anaerobic state. At the outlet from the column was collected with 5 ml Hb concentration 6-10 ^{"mol L".} Hb was stored frozen in the form of balls in liquid nitrogen. Before use, Hb was thawed in 5 ml vessels in a stream of nitrogen. In an anaerobic experimental cuvette with a volume of 4 ml with an optical path length of 1 cm

SUBSTITUTE SHEET (RULE 26) 2.8 ml of buffer, 0.1 ml of Hb 6-10 ^"4 mol-l ^{" 1} were added and the absorption spectrum was recorded. Anaerobic absolute DMSO was added to the weighed portion of the nitrosyl complex in the vessel filled with nitrogen, so as to obtain a solution of the complex with a concentration of 6-10 ³ mol- ¹ . Next, the solution was stirred for 3-5 minutes until completely dissolved and 0.1 ml of the resulting solution was introduced into an experimental Hb cuvette and into a comparison cuvette containing 2.9 ml of anaerobic buffer. The final concentration of the nitrosyl complex was 2-10 ^"4 mol-l ^{" 1} , DMSO - 3.3%. Next, difference absorption spectra were recorded, the first one after 1 minute from the start of the reaction (shown in FIG. 3), then at intervals of 3 minutes during the first 30 minutes of the reaction, and then at intervals of 15, 30, 60 minutes (in FIG. 3) only part of these spectra is shown for clarity). Spectra were recorded until complete conversion of Hb to HbNO, when the spectrum ceased to change. The amount of NO formed was estimated spectrophotometrically by the amount of Hb-NO formed. To determine the concentration of HbNO, the absorption spectrum of the reaction system containing Hb and HbNO was decomposed by computer processing using the MatCad program into components — the spectrum of Hb and HbNO. The calculation was carried out in the wavelength range 450–650 nm using 200 experimental points. For registration of the absorption spectra, we used a Sеrd M-40 spectrophotometer equipped with an interface for computer recording of spectra and a thermostatically controlled cuvette compartment. Spectra were recorded at 25 ° C.

As a result of these studies, it was found for the first time that the complexes generate NO in aqueous solutions of dimethisulfoxide spontaneously (i.e., in the absence of chemo, photo, or enzymatic activation). Study of cytotoxicity of tetranitrosyl iron complexes with benzazheterocyclic thiolyls on human tumor cells in vitro

Experimental technique

Before the experiment, all compounds were dissolved in 200 μl of DMSO, and then brought to the desired concentration with RPMIl 640 nutrient medium. The final concentration of DMSO in the samples did not exceed 0.2% and did not affect cell growth.

The cell line is SKO V3 human ovarian cancer. Cells were grown in

SUBSTITUTE SHEET (RULE 26) monolayer in RPMIl 640 medium containing 10% fetal calf serum at 37 ^° C and 5% CO ₂ . For experiments, cells were seeded in 96-well plates and grown under the same conditions.

The cytotoxic effect was tested using the MTT test based on the ability of living cell dehydrogenase to restore the unpainted tetrazolium salt into blue formazan crystals soluble in dimethyl sulfoxide (DMSO).

All compounds were added to the wells in a volume of 20 μl at 4 final concentrations (μM) of 100, 50, 25, and 10. The total incubation volume was 200 μl. Cells with the preparations were incubated under the above conditions for

72 hours. At the end of the incubation, MTT reagent was added to the cells and incubated under the same conditions for 2 hours. Then, the formed crystals of formazan were dissolved in 100 μl of DMSO at 37 ° C for 20 minutes.

The optical absorption of DMSO solutions was measured on an optical counter for multi-well plates at a wavelength of 540 nm. The results were expressed as mean values for 4 parallel measurements. The cytotoxic effect was evaluated by cell survival in experimental samples relative to the control in%. A compound was considered active if, at a concentration of 100 μM, the number of living cells was 50% or less (IK50 <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, containing thiobenzimidazole and thiobenzothiazole ligands, showed cytotoxic activity on the SCOV ₃ cell line. For I and III, the IR ₅₀ was 57 μM and 25 μM, respectively.

The studied nitrosyl iron complexes I and III showed a different cytotoxic effect on human tumor cells in vitro. The maximum activity was shown by complex III (HK ₅₀ = 25 μM).

As a reference drug, cisplatin (cis-DDP), an antitumor drug used in clinics in Russia and abroad, was used

(HK ₅₀ = lb μM). It was found that, when the concentration is halved, complex III retains the same activity as at a concentration of 100 μM

SUBSTITUTE SHEET (RULE 26) (tumor cell survival 12.5% (table 4). The fact that the toxicity of complex III is significantly lower (its total toxicity index LD ju is 50 mg / kg) than for cisplatin (LD ₁₀₀ = 16 mg / kg) may have importance in high-dose chemotherapy, accompanied by severe side effects, as a way to increase the sensitivity of tumor cells to therapeutic effects, and will reduce the concentration of cytostatics used.

Table 4

Cytotoxic activity of nitrosyl iron complexes I-III on SKO V3 cell line

In accordance with the guidelines for studying the antitumor activity of pharmacological substances, studies were conducted on transplantable tumors of mice on Ca-755, lymphocytic leukemia P-388 and melanoma B-16 (on first-generation hybrid mice BDF ₁ (C ₅ - ₇ BI / 6 x DBA / ₂ ) and DBA / ₂ weighing 18-25 g, obtained from the department of laboratory animals of the State Research Center named after N.N. Blokhin RAMS). A statistically significant antitumor effect of the claimed drugs was revealed.

SUBSTITUTE SHEET (RULE 26)

Claims

CLAIM 1. Binuclear nitrosyl complex of iron with benzazheterocyclic derivatives of the general formula [Fe ₂ (SR) ₂ (NO) ₄ ], where R represents

where X represents NH, S or O, R ¹ represents lower alkyl. 2. The binuclear nitrosyl iron complex according to claim 1, wherein R is benzimidazol-2-yl, 5-methylbenzimidazol-2-yl or benzyl thiazol-2-yl. 3. The method of producing binuclear nitrosyl iron complexes according to claim 1, wherein the thiosulfate nitrosyl iron complex is treated with the corresponding benzazheterocyclic thiol in a stoichiometric ratio in the presence of a reducing agent, and the process is carried out in an alkaline medium, followed by isolation of the target product by known methods. 4. The method of claim 3, characterized in that the process is carried out at room temperature, mainly at 18-25 ° C. 5. The method according to p. 3, characterized in that the process is conducted in an oxygen-free atmosphere. 6. The method according to claim 3, wherein benzazheterocyclic thiol is used benzimidazole-2-thiol, 5-methylbenzimidazole-2-thiol or benzyl thiazole-2-thiol. 7. The method according to p. 3, characterized in that hydrogen, metal thiosulfates, hydrogen sulfide and aliphatic thiols are used as reducing agent. 8. The donor of nitrogen monoxide, which is a binuclear nitrosyl iron complex according to claim 1. 9. The use of binuclear nitrosyl iron complex according to claim 1 as an antitumor drug. 10. The use of binuclear nitrosyl iron complex according to claim 1 for SUBSTITUTE SHEET (RULE 26) obtaining an antitumor drug. I. A pharmaceutical composition comprising an effective amount of a binuclear nitrosyl iron complex with a benzazheterocyclic derivative according to claim 1, a pharmaceutically acceptable carrier. 12. The pharmaceutical composition according to claim 1, wherein a proton-containing medium is used as a pharmaceutically acceptable carrier. 13. The pharmaceutical composition according to claim 1, wherein a mixture of proton-containing medium and dimethyl sulfoxide is used as a pharmaceutically acceptable carrier. 14. The pharmaceutical composition according to any one of claims 12 or 13, wherein water, physiological saline, and water-soluble biopolymers are used as the proton-containing medium. 15. The pharmaceutical composition according to claim ll, wherein the binuclear nitrosyl iron complex with the benzazheterocyclic derivative is present in an amount of 50-100 μM. 16. A kit used for the treatment of cancer, including: (1) a pharmaceutical composition comprising a binuclear nitrosyl iron complex with benzazheterocyclic derivatives according to claim 1, in an airtight package; and (2) auxiliary components. SUBSTITUTE SHEET (RULE 26)