RU2554939C1 - Antitumour anthrafurandione and based pharmaceutical compositions - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to a new derivative of anthrafurandione of formula I

or its pharmaceutically acceptable salts possessing high antitumour effect and activity on tumours resistant to other drug preparations. Besides, the invention refers to antitumour pharmaceutical compositions containing the compound of formula I, a pharmacologically acceptable carrier and one or mode excipients specified in co-solvents, solubilisers, filling agents, emulsifiers, preserving agents, antioxidants, buffer compounds, substances for maintaining isotonicity.

EFFECT: effective use of anthrafurandione, as it possesses high storage stability as a lyophilisate, and as a solution, and also other improved characteristics, including solubility, efficacy and acceptability.

10 cl, 6 tbl, 13 ex

Description

Field of Invention

The present invention relates to the field of drugs, in particular to a new derivative of anthrafurandion of the formula I, which has a more pronounced antitumor effect and activity against tumor diseases with resistance to other drugs, as well as pharmaceutical compositions based on it and their medical use. More specifically, the invention relates to 3 - [(3-amino-1-pyrrolidinyl) carbonyl] -4,11-dihydroxy-2-methylanthra [2,3-6] furan-5,10-dione, its stereochemical composition and use its 5-isomer as an active base in antitumor pharmaceutical compositions with improved characteristics such as efficacy, tolerability, solubility and stability.

BACKGROUND OF THE INVENTION

Oncological diseases remain one of the main causes of death in most developed countries, and in recent decades there has been a tendency to an increase in the number of diagnosed diseases. The effectiveness of the fight against cancer largely depends on drug therapy. Along with surgical and radiation methods, chemotherapy for malignant tumors is widespread, especially since with a number of localizations of the tumor process, it is the only treatment method.

Despite the high clinical efficacy of antitumor agents, their use is limited by a number of disadvantages. In addition to general toxicity to normal tissues, the effectiveness of antitumor agents is often limited by the development of multidrug resistance (MDR) in tumor cells in response to chemotherapy or acquired due to disease progression [Shtil A.A. J. Hematother. Stem Cell Res., 2002, 11, 437]. Expression of ATP-binding cassette transporters, for example p-glycoprotein (p-gp), on the surface of tumor cells [Ambudkar S.V., et. al. Oncogene, 2003, 22, 7468], mutations or changes in the level of target expression [Heisig P. Mutagenesis, 2009, 24 (6), 465], as well as the loss of a functioning proapoptotic tumor suppressor gene p53 [Vousden K.H., et. al. Cell, 2005, 120, 7] are the most typical resistance mechanisms that develop in tumor cells in response to chemotherapy [Knez L., et. al. Lung Cancer, 2011, 72 (3), 271]. Therefore, one of the ways to increase the effectiveness of chemotherapy is to develop drugs that can affect tumor cells with MDR induced by chemotherapeutic agents.

It is known that natural and synthetic derivatives of anthraquinone have high biological activity, and a number of them (for example, doxorubicin, farmrubitsin, mitoxantrone) are used in the clinic for the treatment of tumor diseases. Therefore, the anthraquinone nucleus is widely used in medical chemistry as the basis for the development of new antitumor agents. Thus, a number of heterocyclic derivatives are patented as antitumor agents with improved chemotherapeutic properties [WO 2006031719, RU 2412166]. In patent RU 2412166 we found an original chemotype of linear heteroarenanthracenedione derivatives with high activity against tumor cells with various mechanisms of multidrug resistance. The present invention is a further development of the invention described in RU 2412166. The aim of the present invention was to increase the efficiency of one of the derivatives of anthra [2,3-b] furan-5,10-dione described in RU 2412166 by optimizing its stereochemistry and salt form . Another objective of the invention was to develop, on the basis of this anthrafurandion, pharmaceutical compositions with improved properties applicable to the treatment of tumor diseases, including neoplasms resistant to other drugs.

Disclosure of invention

Derivatives of heteroarenanthracenediones containing cyclic diamines in the side chain with high antiproliferative activity are known to inhibit the growth of a number of resistant tumor cell lines [Shchekotikhin A.E. et al. Bioorg. Med. Chem., 2005, 13 (6), 2285; RU 2412166]. So, previously 3 - [(3-amino-1-pyrrolidinyl) carbonyl] -4,11-dihydroxy-2-methylanthra [2,3-6] furan-5,10-dione (1) hydrochloride was patented as a potential antitumor agents.

However, the previously described anthrafurandione 1 hydrochloride is poorly soluble in water (<1 mg / ml at 20 ° C), and its solubility in aqueous pharmacologically acceptable environments, for example in an “isotonic solution” or in a “glucose solution for injection”, is even lower. Although the solubility of hydrochloride in water increases with heating, the cooling of its concentrated solutions results in the formation of a gel-like precipitate, apparently due to the formation of intermolecular associates. Thus, further studies and the practical use of anthrafurandione 1 hydrochloride as a parenteral drug are hindered by its low solubility. In this regard, it would be desirable to develop a drug with improved solubility, based on anthrafurandion 1, capable of forming stable aqueous solutions. The authors of the present invention conducted intensive research to find a new salt form of anthrafurandione 1 having better solubility than previously known hydrochloride. As a result of the study of other salts of anthrafurandione 1 with mineral or organic acids, it was found that alkanesulfonic acids or hydroxyalkanesulfonic acids form stable salts with anthrafurandion 1 having better solubility than hydrochloride. Thus, mesylate (methanesulfonate) and isethionate (2-hydroxyethanesulfonate) have the highest solubility in distilled water (3-5 mg / ml at 20 ° C) and high stability both in solid form and in solutions, of all the salts of anthrafurandion 1 tested. Therefore, in preferred embodiments of the present invention, salts of anthrafurandione 1 with alkanesulfonic acids (preferably methanesulfonic acid) or hydroxyalkanesulfonic acids (preferably 2-hydroxyethanesulfonic acid), more preferably methanesulfonic acid, are used.

Anthrafurandion 1 described in the present invention can be obtained in various ways. One of the typical approaches that we developed to obtain racemic anthrafurandione 1 [RU 2412166] includes the sequential activation of 4,11-dihydroxy-5,10-dioxo-2-methylanthra [2,3-b] furan-3-carboxylic acid [Gorelik M.V., Mishina E.V. ZhORKh., 1983, 2185], interaction with exocyclic protected amino group 3-aminopyrrolidine, subsequent removal of the protective group and obtaining the appropriate salt form of the target anthrafurandione 1. Various methods well known to specialists in the field of organic synthesis can be used to activate the initial carboxylic acid, including the conversion of 4,11-dihydroxy-5,10-dioxo-2-methylanthra [2,3-b] furan-3-carboxylic acid to the corresponding acid chloride, mixed anhydride, imidazolid, activated ester or other derivatives data used to obtain amides. So, for activation, you can use the treatment of the starting acid with thionyl chloride, phosphorus oxychloride, phosphorus pentachloride, carbonyldiimidazole, carbodiimide derivatives (e.g. DCC or EDC), diphenylphosphoryl azide (DPPA), Castro reagents (BOP or PyBOP) or other condensing agents (HBT and TBTU) preferably thionyl chloride. Obviously, for regioselective acylation, it is necessary to use the protection of the amino group of the starting 3-aminopyrrolidine, which can be used as various protective groups well known to specialists in the field of organic synthesis, preferably a tert-butoxycarbonyl group (Boc group). In this preferred embodiment, the removal of the protective Boc group of the intermediate amide can be carried out by the action of methanesulfonic acid, which allows combining the steps of removing the protective group and obtaining the preferred water-soluble salt form of the target anthrafurandione methanesulfonate. In alternative embodiments, in order to obtain the desired salt forms of anthrafurandione in the final step, the interaction of the free base of anthrafurandione with the corresponding acids (e.g. methanesulfonic and isethionic acids) or an exchange reaction can be used.

One suitable scheme for the synthesis of anthrafurandione, described in detail in the examples of the present invention and shown in Scheme A, includes:

1. Conversion of the starting anthrafuran-3-carboxylic acid a when heated with thionyl chloride to the corresponding acid chloride b ;

2. Acylation of b- 3- (tert-butoxycarbonylamino) pyrrolidine acid chloride b to form amide c ;

3. The splitting of the protective group of the amide with the action of methanesulfonic acid, leading to the methanesulfonate of the target anthrafurandione 1.

It is well known that individual stereoisomers can differ significantly from each other and from their racemic mixture in both physicochemical and biological properties. First of all, substances with different stereoisomeric compositions often differ in specific biological activity, toxicological and pharmacokinetic parameters, as well as side effects [The Practice of Medicinal Chemistry (Third Edition). C.G. Wermuth (ed.), 2008, 537-545]. Therefore, the authors of the present invention conducted research on the synthesis and study of the properties of individual stereoisomers of anthrafurandione 1, previously known as a racemic mixture. Using the commercially available stereoisomers of 3-aminopyrrolidine derivatives in Scheme A, the R- and 5-isomers of anthrafurandione (Formulas 2 and 3) were obtained.

The study of the antiproliferative activity of methanesulfonates of individual stereoisomers of anthrafurandione 2 and 3 revealed their difference in their ability to inhibit the growth of tumor cells. The S-isomer of anthrafurandion 3 is more active than its antipode 2 against tumor cells of various histogenesis, including cells with activated MDR mechanisms (example 7, table 1). So, it is almost equally toxic for human leukemia cells of the K562 line and Pgp-positive subline K.562 / 4. Expression of ABC transporters (eg, p-gp P-glycoprotein) is one of the most common causes of multidrug resistance (MDR) in tumor cells [Shtil AAJ Hematother. Stem Cell Res., 2002, 11, 437]. Another MDR development mechanism associated with mutations leading to inactivation of the p53 proapoptotic tumor suppressor gene is also common in tumor cells and can lead to resistance to many chemotherapeutic agents, including doxorubicin [Vousden KH, et. al. Cell, 2005, 120, 7]. In contrast to doxorubicin, the S-isomer of anthrafurandion 3 acts almost equally on both HCT116 cells (with the p53 ^{+ / +} phenotype) and the HCT116p53KO subline with MDR caused by deletion of the p53 gene (p53 ^{- / -} phenotype).

In addition, the inventors have found that the stereoisomers of anthrafurandione 2 and 3 differ significantly in specific (antitumor) activity in vivo. Thus, the R-isomer 2, when applied five times at a dose of 30 mg / kg, causes an increase in the life expectancy of mice with P388 lymphocytic leukemia at the minimum threshold value (VLP = 22%), while the 5-isomer 3 is 6 times more active (VLP = 140%). Moreover, in terms of antitumor activity, the 5-isomer 3 is 2-3 times more active than the hydrochloride and methanesulfonate of racemic anthrafurandione 1 (example 8, table 2).

Thus, the studies conducted by the authors show that the S-isomer of anthrafurandion 3 more actively inhibits the growth of tumor cells and has significantly higher antitumor activity than its antipode 2 and racemic form 1, therefore, the present invention further relates to pharmaceutical preparations having 5 the isomer of anthrafurandione 3 or a salt thereof, preferably alkanesulfonates or hydroxyalkanesulfonates, more preferably methanesulfonate.

The study of the methanesulfonate of the individual stereoisomer of anthrafurandione 3 showed that its solubility in distilled water under normal conditions (~ 1.0 mg / ml at room temperature) is lower than the solubility of racemate methanesulfonate 1. The dissolution of anthrafurandion 3 in water under normal conditions is slow, and the solubility increases with increasing temperature . In boiling distilled water, the solubility is about 20 mg / ml, however, when the resulting solution is cooled, anthrafurandione 3 crystallizes. These circumstances complicate the practical use of anthrafurandion 3 as a parenterally administered drug, therefore, according to another aspect of the present invention, anthrafurandion 3 can be used for the preparation of pharmaceutical compositions for parenteral use, for example, in the form of a liquid dosage form for injection.

Pharmaceutical compositions for parenteral administration are usually aqueous or non-aqueous sterile isotonic solutions for injection, which, in addition to a therapeutically effective amount of the drug, contain a pharmacologically acceptable carrier (solvent), as well as various excipients (excipients). The pharmaceutical compositions can be either liquid ready-to-use injectable solutions or suspensions, sterile powders or lyophilized compositions, the use of which requires the addition of a sterile solvent (e.g., water for injection) immediately before use. Techniques for preparing parenteral drug compositions are well known in the art.

In pharmacologically acceptable aqueous carriers (solvents or diluents) most commonly used for the preparation of parenteral pharmaceutical compositions, for example, in “isotonic saline solution (0.9% sodium chloride aqueous solution)” or in “isotonic glucose solution (5%) for intravenous injection ", The solubility of anthrafurandione 3 is lower than in distilled water. The authors of the present invention found that the substance of anthrafuradion 3 in "physiological solution" dissolves significantly worse than in "glucose solution". This revealed pharmaceutical incompatibility of anthrafurandione and physiological saline is obviously associated with a high content of chlorine ions in the physiological saline, which cause the transformation of anthrafurandion 3 methanesulfonate into a less soluble salt form - hydrochloride. Therefore, as a carrier (solvent) for the preparation of liquid pharmaceutical compositions, the present invention preferably uses a “glucose solution” in which the substance dissolves better than in “physiological saline”. In addition to the “glucose solution”, other less preferred carriers, including aqueous isotonic saline solutions, electrolyte solutions, as well as non-aqueous pharmaceutically acceptable polar solvents such as oils, alcohols, amides, esters, ethers, can be used to prepare liquid dosage forms. , ketones, hydrocarbons and mixtures thereof.

In addition to the drug and carrier, the pharmaceutical compositions may contain one or more pharmaceutically acceptable excipients (auxiliary components) known in the art. In particular, in the present invention, excipients can be selected from cosolvents, solubilizers, pH and / or isotonicity promoting compounds, fillers, emulsifiers, preservatives, antioxidants and other substances. These well-known components of pharmaceutical compositions help to improve their consumer or beneficial properties by facilitating use, increasing stability, adjusting pH, changing the retention time of the drug compound at the injection site. Thus, in accordance with another aspect of the present invention, the solubility of anthrafurandione 3 methanesulfonate, as well as the stability of its pharmaceutical compositions and their effectiveness, can be enhanced by one or more excipients independently selected from the group of cosolvents, solubilizers, fillers, emulsifiers, preservatives, antioxidants, buffer salts and substances to maintain isotonicity. The amount of the anthrafurandione 3 derivative or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is not limited, but is preferably from 0.1% to 10% (w / v), more preferably from 0.1% to 5% (w / v). The amount of solvent (filler or diluent) is not limited, but can reach up to 99 wt.% Based on the total weight of the composition, as is well known in the technology for the preparation of dosage forms. The amount of cosolvent in the composition is not limited and may vary, for example, from 1% to 60%. The amount of solubilizing agent in the composition is not limited and may vary, for example, from 0.1% to 20%. The amount of stabilizer and antioxidant in the composition is not limited, but can reach, for example, from 0.1 to 1%. The amount of agent to maintain isotonicity in the composition is not limited, but can reach, for example, from 0.5 to 10%. The amount of preservative is not limited and may be, for example, from 0.001 to 5%.

As cosolvents, the pharmaceutical compositions of anthrafurandione 3 may contain one or more pharmacologically acceptable solvents suitable for the preparation of liquid parenteral forms. Such cosolvents are well known to those skilled in the pharmaceutical field (but not limited to): alcohols or polyols (e.g. ethanol, 1,2-propylene glycol, glycerin, 1,3-butylene glycol), polyoxyalkylene-modified alkylene oxides or lipids (e.g. PEG polyethylene glycol), complex polyoxyethylene sorbitan fatty acid esters, polyethylene glycol ethers, polyoxyethylene fatty acid esters, ethylene oxide and propylene oxide copolymers, preferably polyethylene glycol (PEG). Other pharmaceutically acceptable cosolvents that may be used in the present invention are well known in the art and are described in the literature [eg, Modern Pharmaceutics (Fourth Edition). G. Banker et al. (eds.), 2002, 864; Remington's Pharmaceutical Sciences (21th Edition) D B. Troy, P. Beringer (ed.), 2006, 2393, A.J. Spiegel et al. J. Pharm. Sci., 1963, 52 (10), 917-927]. These excipients can be obtained by methods well known in the art or from industrial suppliers.

As excipients that increase the solubility of anthrafurandione 3, its pharmaceutical compositions may contain one or more pharmacologically acceptable solubilizing agents well known to specialists in the field of pharmaceuticals, such as (without limitation listed) polyvinylpyrrolidone (PVP), dextran, polysorbate 80 (tween 80), Cremophor EN, hydroxyalkylated β-cyclodextrins and γ-cyclodextrins, etc. The addition of PVP, Polysorbate 80, or Cremophor EN to the “glucose solution” as auxiliary components, although they do not accelerate the dissolution of the substance at room temperature, however, 0.5% solutions obtained after dissolving the substance when heated are stable when stored in a refrigerator (+ 5 ° C ) for 3 months. Polyethylene glycols (PEG400 or PEG200), as well as β- or γ-hydroxypropylcyclodextrins not only stabilize the solution of the substance, but also contribute to its dissolution at room temperature. Therefore, in preferred embodiments of the present invention, PVP, Tween 80, Cremophor EH, polyethylene glycols and β- or γ-hydroxypropylcyclodextrins are used to prepare stable liquid pharmaceutical compositions for parenteral use of the anthrafurandione 3 of the present invention.

As preservatives protecting the composition from exposure to microorganisms, the present invention can use various antimicrobial agents well known to those skilled in the pharmaceutical field, such as (but not limited to) sulfites, benzyl alcohol, chlorobutanol, sorbitol, xylitol, sorbic acid, benzoic acid , thimerosal, parabens and their various salts.

As auxiliary buffering components in the pharmaceutical compositions of the present invention, acids (for example, citric, acetic, phosphoric, etc.) or their salts with alkali metals (for example, citrate, acetate or sodium dihydrogen phosphate, etc.), or combinations thereof, can be preferably used. (e.g. citric acid and sodium citrate). The ionic strength of the buffer solution used as a component of the liquid composition in the present invention is not limited and may be, for example, at a level of 0.01-0.6.

As antioxidants, for example, glucose, ascorbic acid, sodium metabisulfite, sodium bisulfite, sodium sulfite, phenols and thiophenols can be used.

In one embodiment of the preparation of liquid pharmaceutical compositions, the pharmaceutically acceptable salt of anthrafurandione 3 and the necessary excipients (eg, solubilizing or stabilizing agents, buffers, antioxidants, etc.) are dissolved or suspended in a suitable pharmacologically acceptable carrier (for example, “water for injection” or in a “glucose solution”, or PEG) with maintaining the mixture at a temperature of from 20 to 100 ° C until all ingredients are completely dissolved. After the formation of the solution, the pH of the composition is adjusted to a value of 5-8, more preferably pH 6-7, using a suitable buffer (e.g. citric acid and sodium citrate, etc.). If necessary, the solution is diluted with water for injection to obtain the desired concentration of anthrafurandione 3 (preferably 0.1% to 5%), after which, to remove insoluble materials and pyrogen, the solution is filtered through a membrane filter and then the solution is filled with a sealed glass vessel (ampoule or vial) then sterilized, corked and stored in a place protected from light.

Thus, the present invention describes ready-made parenteral liquid pharmaceutical compositions containing anthrafurandion 3. In addition, in some embodiments, the composition is a dry (such as lyophilized) composition that can be reconstituted, resuspended or rehydrated to provide a stable liquid composition pharmaceutical substance. Thus, the authors of the present invention by lyophilization of liquid compositions obtained a number of lyophilized preparations that have excellent drug stability during preparation and storage and are easily restored to the liquid composition with the addition of an aqueous solvent. Accordingly, the present invention also includes lyophilized anthrafurandione 3 compositions. In some embodiments, the composition can be an intermediate liquid (such as, for example, aqueous) composition that can be dried (such as, for example, lyophilized) or a liquid (such as, for example, aqueous) composition, obtained by reconstituting or resuspending the dry composition.

In order to obtain lyophilized compositions of the present invention, the liquid anthrafurandione 3 composition prepared as described above is poured into a hard vessel, such as a sterile ampoule, vial or vial, and lyophilized in a conventional manner. The amount of liquid preparation with which the vessel is filled is, for example, from 5 to 50% (v / v) of the volume of the vessel, particularly preferably 10-25% (v / v). The external temperature during lyophilization is preferably maintained from -70 to 0 ° C, particularly preferably from -60 to -40 ° C, and the applied pressure for sublimation of the solvent is preferably from 0.01 to 0.2 mm Hg. Art., more preferably from 0.01 to 0.1 mm RT. Art. Preferably, the lyophilization rate is controlled so that the solvent (based on the solution) sublimates at a rate of from 10 μl to 100 μl per 1 cm of surface area with which the solvent sublimates for 1 hour.

In the case of lyophilization of a liquid composition, especially containing dextran, mannitol, and / or sodium carbonate, etc., protection against destruction of a vessel containing a lyophilized solution is provided by the introduction of additives, for example, alkali metal sulfates (e.g. sodium sulfate, potassium sulfate, etc.) in the specified liquid preparation. In addition, lyophilized compositions may contain additional cryoprotective components that prevent the deposition of the drug compound, such as surfactants, wetting or emulsifying agents (such as lecithin, polysorbate 80, polyoxyethylene stearate). The content of these excipients may be 0.01-10% by weight of the drug.

Lyophilized compositions of the present invention, prepared as described above, have excellent properties with respect to the stability of the active substance (anthrafurandione 3) during preparation or storage. The lyophilized forms described above, as well as liquid compositions for parenteral use of anthrafurandione 3, can be packaged in sterile vials, ampoules or bags, vials for single or multiple use. Pharmaceutical compositions in dry (lyophilized) or liquid concentrated form, as is known in the art, can be reconstituted or diluted before use by adding a sterile pharmaceutically acceptable solvent to obtain the desired concentration of therapeutic agent. This makes it possible, if necessary, to easily prepare a sterile liquid composition for parenteral use of anthrafurandione 3, which can be directly administered to the patient.

Anthrafurandion 3 and liquid pharmaceutical compositions containing from 0.1 to 5% of a compound of formula 3 or a salt thereof can be administered parenterally (e.g., intravenously, intraarterially, intramuscularly, subcutaneously, rectally, intraspinally, intraperitoneally, intracavitary) for the treatment of neoplastic diseases, including diseases complicated by resistance to other drugs.

The term "anthrafurandion" as used herein means 3 - [(3-amino-1-pyrrolidinyl) carbonyl] -4,11-dihydroxy-2-methylanthra [2,3-6] furan-5,10-dione .

The term "protective group" in the present invention refers to a group suitable for blocking a functional group under the conditions of the reactions, as described in the literature [Green T.W .; Wuts P.G. M. Protective Groups in Organic Synthesis. 1991, 351]. An example of such groups for blocking an amino group includes, but is not limited to, tert-butoxycarbonyl (Boc), adamantyloxycarbonyl (Adoc), fluorenylmethoxycarbonyl (Fmoc), acetyl, carbonibenzyloxy (Cbz), methoxycarbonyl, ethoxycarbonyl.

The term "pharmaceutical composition", as used herein, means, for example, a mixture containing a certain amount of a therapeutic agent, for example, a therapeutically effective amount of a drug compound with at least one pharmaceutically acceptable carrier, for administration to a mammal, for example a human, for treatment of tumor diseases.

The term "pharmaceutically acceptable", as used in the present description, refers to compounds, compositions and / or dosage forms which, when in contact with mammalian tissues, especially human tissues, do not cause allergic reactions, irritation, complications or other toxic manifestations, and compounds are characterized by a rather high benefit / risk ratio.

"Pharmaceutically acceptable salt" in the present invention means common salts, obtained by adding acid to the base, which retain the biological effectiveness and properties of anthrafurandione 3 and are formed from its free base and suitable organic or inorganic acids. Examples of salts obtained by the addition of acid include salts derived from organic acids, for example methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic acids or inorganic acids, for example, hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfamic acids and the like. In addition, the term “pharmaceutically acceptable salt” also includes pharmaceutically acceptable solvates, preferably hydrates. The chemical conversion of anthrafurandione 3 to a salt is carried out by a method well known to pharmaceutical chemists, providing improved physical and chemical stability, hygroscopicity, flowability and solubility of the compounds [see, for example, Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Edition). R.C. Rowe (Ed.), 1995, 1456-1457].

The term "pharmacologically acceptable carrier" in the present invention means one or more compatible liquid or solid diluents or excipients that are suitable for administration to a mammal, preferably a human. Preferably, proton-containing media, more preferably aqueous media, are used as the pharmaceutically acceptable carrier in the pharmaceutical compositions of the invention.

The concentration of the therapeutic agent in the pharmaceutical composition is a certain value, ensuring the introduction of a therapeutically effective amount of the drug, which depends on the rate of absorption, inactivation and excretion of the drug from the body, as well as on the severity of the patient's condition or other factors known to specialists in this field of technology.

An "effective amount" or "therapeutically effective amount" of a compound of the present invention means an amount of anthrafurandion 3 or a pharmaceutically acceptable salt thereof that significantly inhibits the proliferation of tumor cells and is effective in preventing, alleviating or alleviating the symptoms of the disease, or increasing the life span of the subject being treated. The determination of a therapeutically effective amount is within the competence of a person skilled in the art. A therapeutically effective amount or dosage of a compound of the present invention can vary widely and can be determined by a method known in the art.

For a particular recipient, the appropriate course of treatment is selected taking into account the individual needs and opinions of the doctor who administers the pharmaceutical composition to the patient or prescribes the introduction of pharmaceutical compositions. The daily dose of the therapeutic agent can be administered once as a single dose or repeatedly as divided doses that are administered over certain periods of time, or, with parenteral administration, it can be administered by continuous infusion. Thus, the required amount of a therapeutic agent, for example the necessary therapeutically effective amount, is determined by a person skilled in the art. For example, the dose of a therapeutic agent may vary depending on age, body weight, or conditions in the range of from about 1 mg to about 100 mg, based on anthrafurandion 3 or a pharmaceutically acceptable salt thereof per kilogram of recipient body weight per day.

The following non-limiting examples are given to demonstrate preferred embodiments of the present invention. Those skilled in the art will readily understand that various excipients and embodiments of the present invention can be used to stabilize anthrafurandion 3-based drug compositions.

Example 1

Synthesis of (R, S) -3 - [(3-amino-1-pyrrolidinyl) carbonyl] -4,11-dihydroxy-2-methylanthra [2,3-b] furan-5,10-dione methanesulfonate (1)

A mixture of 0.5 g (1.6 mmol) of 4,11-dihydroxy-5,10-dioxo-2-methylanthra [2,3-b] furan-3-carboxylic acid ( a , Scheme A ), benzene (25 ml) and 0.6 ml (8.5 mmol) of thionyl chloride is boiled under vigorous stirring for 60 minutes, and the volatile components of the mixture are distilled off in vacuo. The resulting acid chloride b (Scheme A ) was suspended in chloroform (25 ml), and a solution of 0.7 g (3.3 mmol) of (R, S) -3- (tert-butoxycarbonylamino) pyrrolidine in a mixture of chloroform (10 ml) and pyridine ( 0.6 ml). The reaction mixture is boiled for 10 minutes, transferred to a separatory funnel, diluted with warm chloroform (50 ml) and washed with dilute HCl solution, water, dried with Na ₂ SO ₄ , and the solvent is distilled off in vacuo. The residue was dissolved in chloroform, the solution was added to a silica gel column chromatography, and the product was eluted with chloroform-methanol (20: 1). The product containing fractions were combined and the solvent was distilled off in vacuo. The residue was recrystallized from dioxane, washed with dioxane and dried. The yield of the Boc derivative c (Scheme A ) was 0.65 g (83%) as a red powder. HPLC (Kromasil 100-5 C18, 4.6 × 250 mm column (Akzo Nobel); eluents: A - H ₃ PO ₄ (0.01 m), B - MeCN; gradient B 20 → 60% (30 min); LW = 260 nm ): t _R = 8.9 min, 96% purity. ¹ H NMR Spectrum (400 MHz, DMSO-d ₆ ), δ, ppm: 8.18 (2H, m, H-6.7); 7.90 (2H, m, H-8.9); 7.19 (1H, m, NH); 4.08 (1H, m, NCH ₂ ); 3.95 (1H, m, NCH ₂ ); 3.69 (2H, m, NCH ₂ ); 3.69 (1H, m, CH); 2.51 (3H, s, Me); 2.08 (1H, m, CH ₂ CH ₂ CH); 1.80 (1H, m, CH ₂ CH ₂ CH), 1.28 (9H, s, OBu ^t ). Found: m / z (ESI), 507.1769 [M + H] ⁺ . C ₂₇ H ₂₇ N ₂ O ₈ . Calculated: 507.1762.

In a round-bottom flask equipped with a reflux condenser with a calcium chloride tube, 0.6 g (1.2 mmol) of carboxamide with in warm chloroform (25 ml) was dissolved, and a solution of methanesulfonic acid (0.15 ml) in chloroform (2 ml) was added with stirring. The mixture is stirred for 1.5-2 hours, and the red precipitate is filtered off on a Buchner funnel, washed with acetone (10 ml), ether (10 ml) and dried. Yield 0.5 g (80%) of methanesulfonate (R, S) -3 - [(3-amino-1-pyrrolidinyl) carbonyl] -4,11-dihydroxy-2-methylanthra [2,3-6] furan-5.10 -dione-3-carboxamide (1) in the form of a brick-red powder with T. pl. > 250 ° C. TLC (SilicaGel 60 F ₂₅₄ (Merck)): Rf = 0.1, chloroform-methanol-water (10: 10: 1). HPLC (Kromasil 100-5 C18, 4.6 × 250 mm column (Akzo Nobel); eluents: A - H ₃ PO ₄ (0.01 m), B - MeCN; gradient B 20 → 60% (30 min); LW = 260 nm ): t _R = 17.2 min, purity 99.2%. ESP (EtOH), λ _max. nm, (logε): 261 (4.4), 479 (4.0). IR spectrum, ν _max , cm ^-1 : 1615, 1584, 1462, 1419, 1351, 1301, 1166, 1044, 1021, 968. ¹ H NMR spectrum (400 MHz, DMSO-d ₆ ), δ, ppm .: 8.21 (2H, m, H-6.7); 7.92 (2H, m, H-8.9); 3.89 (1H, m, NCH ₂ ); 3.80 (1H, m, NCH ₂ ); 3.55 (3H, m, NCH ₂ , CH); 2.53 (3H, s, Me); 2.32 (3H, s, Me); 2.22 (1H, m, CH ₂ CH ₂ CH); 1.98 (1H, m, CH ₂ CH ₂ CH). Found,%: C 51.64, H 4.67, N 5.13. C ₂₂ H ₁₈ N ₂ O ₆ * CH ₄ O ₃ S * 2H ₂ O. Calculated,%: C 51.30, H 4.87, N 5.20. Mass spectrum found, m / z (ESI): 407.1221 [M + H] ⁺ . C ₂₂ H ₁₉ N ₂ O ₆ . Calculated: 407.1238.

Example 2

Synthesis of (R) -3 - [(3-amino-1-pyrrolidinyl) carbonyl] -4,11-dihydroxy-2-methylanthra [2,3-b] furan-5,10-dione methanesulfonate (2)

Obtained by the method similar to that described in example 1, from 4,11-dihydroxy-5,10-dioxo-2-methylanthra [2,3-6] furan-3-carboxylic acid ( a , Scheme A ) and (R) - 3- (tert-butoxycarbonylamino) pyrrolidine. The yield of anthrafurandion 2 is 85%. Mp > 250 ° C. TLC (SilicaGel 60 F ₂₅₄ (Merck)): Rf = 0.1, chloroform-methanol-water (10: 10: 1). HPLC (Kromasil 100-5 C18, 4.6 × 250 mm column (Akzo Nobel); eluents: A — H ₃ PO ₄ (0.01 m), B — MeCN; gradient B 20 → 60% (30 min); LW = 260 nm ): t _R = 17.2 min, purity 99.2%. ESP (EtOH), λ _max. nm, (logε): 261 (4.4), 479 (4.0). IR spectrum, ν _max , cm ^-1 : 1615, 1584, 1462, 1419, 1351, 1301, 1166, 1044, 1021, 968. ¹ H NMR spectrum (400 MHz, DMSO-d ₆ ), δ, ppm .: 8.21 (2H, m, H-6.7); 7.92 (2H, m, H-8.9); 3.89 (1H, m, NCH ₂ ); 3.80 (1H, m, NCH ₂ ); 3.55 (3H, m, NCH ₂ , CH); 2.53 (3H, s, Me); 2.32 (3H, s, Me); 2.22 (1H, m, CH ₂ CH ₂ CH); 1.98 (1H, m, CH ₂ CH ₂ CH). Found,%: C 51.32, H 4.60, N 5.07. C ₂₂ H ₁₈ N ₂ O ₆ * CH ₄ O ₃ S * 2H ₂ O. Calculated,%: C 51.30, H 4.87, N 5.20. Mass spectrum found, m / z (ESI): 407.1221 [M + H] ⁺ . C ₂₂ H ₁₉ N ₂ O ₆ . Calculated: 407.1238.

Example 3

Synthesis of methanesulfonate (5) -3 - [(3-amino-1-pyrrolidinyl) carbonyl] -4,11-dihydroxy-2-methylanthra [2,3-6] furan-5,10-dione (3)

Obtained by the method similar to that described in example 1, from 4,11-dihydroxy-5,10-dioxo-2-methylanthra [2,3-6] furan-3-carboxylic acid ( a , Scheme A ) and (S) - 3- (tert-butoxycarbonylamino) pyrrolidine. The yield of anthrafurandion 3 is 83%. T. pl. > 250 ° C. TLC (SilicaGel 60 F ₂₅₄ (Merck)): Rf = 0.1, chloroform-methanol-water (10: 10: 1). HPLC (Kromasil 100-5 C18, 4.6 × 250 mm column (Akzo Nobel); eluents: A - H ₃ PO ₄ (0.01 m), B - MeCN; gradient B 20 60% (30 min); LW = 260 nm) : t _R = 17.2 min, purity 99.8%. ESP (EtOH), λ _max. , nm, (logω): 261 (4.4), 479 (4.0). IR spectrum, ν _max , cm ^-1 : 1615, 1584, 1462, 1419, 1351, 1301, 1166, 1044, 1021, 968. ¹ H NMR spectrum (400 MHz, DMSO-d ₆ ), δ, ppm .: 8.21 (2H, m, H-6.7); 7.92 (2H, m, H-8.9); 3.89 (1H, m, NCH ₂ ); 3.80 (1H, m, NCH ₂ ); 3.55 (3H, m, NCH ₂ , CH); 2.53 (3H, s, Me); 2.32 (3H, s, Me); 2.22 (1H, m, CH ₂ CH ₂ CH); 1.98 (1H, m, CH ₂ CH ₂ CH). Found,%: C 51.45, H 4.60, N 5.01. C ₂₂ H ₁₈ N ₂ O ₆ * CH ₄ O ₃ S * 2H ₂ O. Calculated,%: C 51.30, H 4.87, N 5.20. Mass spectrum found, m / z (ESI): 407.1221 [M + H] ⁺ . C ₂₂ H ₁₉ N ₂ O ₆ . Calculated: 407.1238.

The resulting (S) -3 - [(3-amino-1-pyrrolidinyl) carbonyl] -4,11-dihydroxy-2-methylanthra [2,3-b] furan-5,10-dione (3) methanesulfonate having excellent stability, used as a therapeutic agent for the preparation of the pharmaceutical compositions described below in examples 5, 6. The specified therapeutic agent is characterized by limited solubility in an aqueous medium. The examples describe the preparation of sterile metered-dose injectable compositions of anthrafurandion 3 and auxiliary components of pharmaceutical purity suitable for use in parenteral dosage forms. The preparation of dosage forms of anthrafurandion 3 was carried out by the weight-volume method according to the instructions of the State Pharmacopoeia of the Russian Federation.

Example 4

The study of the stability of anthrafurandion 3

The study of samples of methanesulfonate (S) -3 - [(3-amino-1-pyrrolidinyl) carbonyl] -4,11-dihydroxy-2-methylanthra [2,3-6] furan-5,10-dione (3) (obtained by the method described in example 3), stored for 1 year under natural conditions (25 ° C, in a darkened place) and 2 months under accelerated aging (64 days at 60 ° C), no significant changes were revealed in any of the indicators, including elemental and stereochemical composition of the substance. The study of anthrafurandione 3 samples stored at 95% relative humidity (chamber with 10% sulfuric acid) and in conditions of zero humidity (chamber with phosphoric anhydride) showed that after 3 months of storage, the weight change of the samples does not exceed 0.5-1 %, which allows us to consider the substance of anthrafurandion 3 stable. The quality of the samples according to the indicators “Appearance”, “Color” and “Impurities” stored under the indicated conditions has not changed compared to the original samples. Thus, the obtained data indicates the non-hygroscopic nature of the substance of anthrafurandion 3 and the high stability of its composition during storage.

Example 5

Preparation of parenteral pharmaceutical formulations of anthrafurandion 3

To maintain isotonicity, the liquid pharmaceutical compositions of anthrafurandione 3 are prepared in an “isotonic glucose solution (5%) for intravenous injection” in a sterile container. 3/4 of the required volume of solvent is poured into a measuring vessel, a weighed amount of anthrafurandione 3 and auxiliary components are dissolved in it, heating and stirring the mixture if necessary. Solutions of the substance are pre-sterilized by heating to 95-98 ° C and keeping at this temperature for 10 minutes. After cooling, bacterial contamination and solids are removed by filtering the solution through a microporous filter (pore size 35 μm) under aseptic conditions. The resulting solution is transferred into a measuring vessel and adjusted with a sterile solvent to the required volume. After checking the content of anthrafurandion 3 by HPLC (required concentration 2 ± 0.1 mg / ml), the solutions are dosed with 10 ml in sterile neutral glass vials. Vials with solutions are closed with sterile parchment paper, re-sterilized for 10 min at 95 ° C and, after cooling, they are sealed with sterile rubber stoppers, wrapped in aluminum caps and marked. Thus, 0.2% solutions of anthrafurandion 3 for parenteral use were prepared (each vial contains 10 ml of solution), illustrative formulations of which are given below (only concentrations of anthrafurandion 3 and auxiliary components are presented).

Composition 1: 2 mg / ml anthrafurandione 3; 200 mg / ml polyethylene glycol (PEG 400); 0.5 mg / ml benzyl alcohol.

Composition 2: 2 mg / ml anthrafurandione 3; 200 mg / ml polysorbate 80 (tween 80); 0.5 mg / ml sodium benzoate, 0.2 mg / ml ascorbic acid.

Composition 3: 2 mg / ml anthrafurandione 3; 10 mg / ml 3-hydroxypropyl-β-cyclodextrin (degree of substitution 5); 0.5 mg / ml potassium sorbate, 0.1 mg / ml sodium citrate.

The obtained pharmaceutical compositions of anthrafurandione 3 are stable under storage in natural conditions for 6 months (at 25 ° C, in a dark place). Monitoring the composition of injectable solutions of anthrafurandion 3 (formulations 1-3), stored for 2 months under accelerated aging (64 days at 60 ° C), showed that the decrease in the content of the active substance in them is 2.7% (composition 1), 1.2% (composition 2) and <0.5% (composition 3). Changes in other indicators in compositions 1-3 were not identified.

Example 6

Obtaining lyophilized pharmaceutical compositions for parenteral use of anthrafurandione 3

Solutions of anthrafurandion 3 for the preparation of lyophilized pharmaceutical compositions are prepared in distilled water in a sterile container. 3/4 of the required volume of water is poured into a measuring vessel, a weighed amount of anthrafurandione 3 and auxiliary components are dissolved in it, if necessary, heating and stirring the mixture. The solutions of anthrafurandion 3 are pre-sterilized by heating to 95-98 ° C, and maintained at this temperature for 10 minutes. After cooling, bacterial contamination and solids are removed by filtering the solution through a microporous filter under aseptic conditions. The resulting solution is transferred into a measuring vessel and brought with sterile water to the required volume. After checking the content of anthrafurandion 3 by HPLC (the required concentration is 10 ± 0.5 mg / ml), the solutions are metered in 2 ml in sterile neutral glass vials. The solution bottles are closed with sterile gauze swabs, re-sterilized for 10 minutes at 95 ° C and, after cooling to room temperature, kept at -70 ° C for 12 hours. The bottles with frozen solution are introduced into the freeze-drying unit and kept for 12 hours at a pressure of 0.01 mm Hg. . Art. After removal from the installation, the vials are sealed with sterile rubber stoppers and wrapped in aluminum caps.

Illustrative formulations of lyophilized pharmaceutical compositions of anthrafurandione 3 for the preparation of injection solutions are given below (the composition of the compositions of anthrafurandion 3 is calculated per one vial with a volume of 10 ml).

Composition 4: 20 mg of anthrafurandione 3; 200 mg of polyvinylpyrrolidone; 1 mg sorbitol.

Composition 5: 20 mg of anthrafurandione 3; 200 mg of 3-hydroxypropyl-γ-cyclodextrin; 1 mg polysorbate 80.

Composition 6: 20 mg of anthrafurandione 3; 80 mg of 3-hydroxypropyl-β-cyclodextrin (degree of substitution 5), 1 mg of sodium citrate.

The prepared compositions are stable during storage both in natural conditions (1 year, 25 ° C, in a darkened place), and in conditions of accelerated aging (64 days at 60 ° C). Lyophilized compositions of compositions 4-6 are easily restored to the original homogeneous solution at room temperature in less than 5 minutes with the addition of sterile water or "isotonic glucose solution (5%) for intravenous injection". To prepare 10 ml of a 0.2% solution of anthrafurandion 3, 10 ml of “isotonic glucose solution (5%) for intravenous injection” is added to the contents of the vial, periodically stirring the contents of the vial until the composition is completely dissolved.

Example 7

Comparative study of the antiproliferative activity of stereoisomers of anthrafurandione

A comparative study of the antiproliferative activity of the R- and S-isomers of anthrafurandione 2 and 3 was carried out on the methanesulfonates described in examples 2 and 3 in vitro tests on an expanded panel of mouse and human tumor cells, including lines with activated mechanisms of multidrug resistance (MDR). Data on antiproliferative activity (IC ₅₀ - concentration inhibiting cell growth by 50%) are given in table 1, where the type is indicated for each line. The determination of IC ₅₀ was carried out using the MTT test according to the method described in the literature [T. Mossman, Immunol. Methods, 1983, 65, 55]. As can be seen from the data presented, both stereoisomers of anthrafurandione 2 and 3, described in examples 2, 3, inhibit the growth of tumor cells in micromolar and submicromolar concentrations. For all tested tumor cell lines, the antiproliferative activity of S-anthrafurandione 3 described in example 3 is higher than its antipode 2 obtained in example 2.

It is important to note that S-anthrafurandion 3 is more active than its antipode 2 in relation to the tested tumor cell lines with activated MDR mechanisms. So, it is almost equally toxic for human leukemia cells of the K562 line and Pgp-positive resistant subline K562 / 4. In addition, S-anthrafurandion 3 acts almost equally on both the HCT116 cell line (with the p53 ^{+ / +} phenotype) and the resistant subline HCT116p53KO with the p53 gene deletion (with the p53 ^{- / -} phenotype).

Thus, the results obtained indicate that (S) -3 - [(3-amino-1-pyrrolidinyl) carbonyl] -4,11-dihydroxy-2-methylanthra [2,3-b] furan-5,10-dione (3), which is the subject of the present invention, inhibits the proliferation of tumor cells, including a number of resistant lines, and more actively than its enantiomer 2.

Example 8

A comparative study of the antitumor activity of various salt and stereoisomeric forms of anthrafurandion in mice with intraperitoneally inoculated P388 lymphocytic leukemia

In the experiments, female mice of BDF ₁ hybrids with a body weight of 20-21 g were used. Before the experiment, mice were ranked in groups, in each group of 6 individuals. One group of mice with a tumor (n = 5) was left without specific effects and was considered a control. The main groups received treatment with various salt and stereoisomeric forms of anthrafurandione. For the study used a strain of lymphocytic leukemia P388. In experiments, 2–9 passages of the tumor in vivo were used on female mice of the DBA ₂ line. The inoculum was prepared ex tempore in 199 nutrient medium and transplanted into mice in the abdominal cavity, 1 million cells per mouse. A therapeutic experiment with the evaluation of the results of the study was performed in accordance with the recommended methodology [Guidelines for preclinical studies of drugs. Part One, 2012, 642-657].

The main groups of animals received a solution of methanesulfonates of various stereoisomeric forms of anthrafurandione (examples 1-3) or racemic anthrafurandion 1 hydrochloride (obtained by the method described in patent RU 2412166). All test solutions contained 2 mg / ml of active ingredient. Salts of anthrafurandione were dissolved in the required volume of “isotonic glucose solution (5%) for intravenous injection” when heated to 50-60 ° C and after cooling the solution was administered to animals. The mice of the control group received a b / w 5% glucose solution in an adequate volume and treatment regimen. The introduction of solutions to animals was carried out in the appropriate individual doses in volumes once a day. Treatment was carried out on the 2nd-6th day after tumor inoculation.

Evaluation of the antitumor effect was performed to increase the life expectancy (VL) of mice in the treated groups compared with the control. For this, the individual and average life expectancy of the mice (RV, RV) in the group were determined, which were then used to calculate RV. The indicator making up the difference in the LSS of the control and treated groups was expressed as a percentage of the control group. The data obtained were processed statistically using confidence intervals of the average compared values according to the standard student method in the modification of RB Strelkova. The differences were considered significant at p≤0.05.

The results of a comparative study of various salt and stereoisomeric forms of anthrafurandione (examples 1-3), presented in table 2, show that with five times daily intraperitoneal administration in optimal single doses of 20 mg / kg (racemic hydrochloride of anthrafurandione 1) and 30 mg / kg ( anthrafurandione methanesulfonates 1-3) all the investigated forms of anthrafurandion have a reliable antitumor effect. Anthrafurandion 1 hydrochloride and methanesulfonate in optimal conditions of use showed a significant (more than minimal criterion of effectiveness) antitumor effect without significant difference (ALC = 71 and 51%, respectively). The stereoisomers of anthrafurandione 2 and 3 methanesulfonate differ significantly in their ability to inhibit the development of P388 leukemia. So, antitumor activity. R-anthrafurandione 2 (example 2) is at the level of the minimum performance criterion (25%), while the activity of 5-isomer 3 (example 3) is significantly higher. Moreover, the antitumor activity of the S-isomer of anthrafurandion 3 methanesulfonate 3 is significantly (2-2.5 times) significantly higher than the activity of various salts of the racemic form of anthrafurandion 1. As can be seen from the presented data, S-anthrafurandion 3, which is the subject of the present invention, significantly exceeds the antitumor activity antipode 2 and racemic anthrafurandion 1.

Example 9

A comparative study of the antitumor activity of the pharmaceutical compositions of anthrafurandion 3 in mice with intraperitoneally inoculated P388 lymphocytic leukemia

In the experiments, female mice of BDFi hybrids with a body weight of 20-21 were used. Before the experiment, the mice were ranked in groups, in each group 4-6 individuals. One group of mice with a tumor (n = 5) was left without specific effects and was considered a control. The main groups received treatment with one of the variants of liquid pharmaceutical compositions of anthrafurandione 3 (formulations 1, 2, example 5) or with the substance of anthrafurandion 3, the preparation of which is described in example 3. For the study, the P388 lymphocytic leukemia strain was used. In experiments, 2–9 passages of the tumor in vivo were used on female mice of the DBA ₂ line. The inoculum was prepared ex tempore in 199 nutrient medium and transplanted into mice in the abdominal cavity, 1 million cells per mouse. A therapeutic experiment with the evaluation of the results of the study was performed in accordance with the recommended methodology [Guidelines for preclinical studies of drugs. Part One, 2012, 642-657].

The main groups of animals received a solution of the substance of anthrafurandion 3 (example 3) or liquid compositions based on it (compositions 1, 2). All test solutions of anthrafurandion 3 contained 2 mg / ml of active ingredient. The substance of anthrafurandion 3 was dissolved in the required volume of “isotonic glucose solution (5%) for intravenous injection” when heated to 50-60 ° C and after cooling the solution was administered to animals. The mice of the control group received a b / w 5% glucose solution in an adequate volume and treatment regimen. The introduction of solutions to animals was carried out in the appropriate individual doses in volumes once a day. Treatment was carried out on the 2nd-6th day after tumor inoculation.

Evaluation of the antitumor effect was performed to increase the life expectancy (VL) of mice in the treated groups compared with the control. For this, the individual and average life expectancy of the mice (RV, RV) in the group were determined, which were then used to calculate RV. The indicator making the difference in the LSS of the control and treated groups was expressed as a percentage of the control group. At autopsy of the mice that died in the experiment, the presence of leukemic ascites in the abdominal cavity was determined. The number of ascites-free mice was used as one of the parameters of treatment effectiveness. The data obtained were processed statistically using confidence intervals of the average compared values according to the standard student method in the modification of RB Strelkova. The differences were considered significant at p≤0.05.

The results of a comparative study of the antitumor activity of the pharmaceutical compositions of anthrafurandion 3 (formulations 1, 2), presented in table 3, show that with five times daily intraperitoneal administration in a single dose of 30 mg / kg (a total of 150 mg / kg), all compositions have significant (more than minimal effectiveness criterion) with a high antitumor effect at the level of the substance of anthrafurandion 3, the preparation of which is described in example 3: UPZ = 144-148% (without significant differences, p <0.05). The tolerance of the substance and both dosage forms is good, their use does not cause mouse death from toxicity, however, dosage forms differ in their ability to inhibit the development of leukemic ascites: the use of composition 1 completely blocks the development of ascites in all treated animals, while when using composition 2 it was noted the formation of ascites in one animal. As can be seen from the data presented, the examples of liquid pharmaceutical compositions of anthrafurandione 3, which are the subject of the present invention, have a high antitumor activity that advises the active substance (5-anthrafurandione 3), but is more convenient for practical use.

Example 10

Comparative study of the antitumor activity of lyophilized pharmaceutical compositions of anthrafurandion 3 in mice with intraperitoneally inoculated P388 lymphocytic leukemia

In the experiments, female mice of BDF ₁ hybrids with a body weight of 20-21 were used. Before the experiment, mice were ranked in groups, in each group 5-6 individuals. One group of mice with a tumor (n = 5) was left without specific effects and was considered a control. The main groups received treatment with one of the variants of lyophilized pharmaceutical compositions of anthrafurandione 3 (formulations 4-6, example 6) or with the substance of anthrafurandion 3 described in example 3. For the study, the P388 lymphocytic leukemia strain was used. In experiments, 2–9 passages of the tumor in vivo were used on female mice of the DBA ₂ line. The inoculum was prepared ex tempore in 199 nutrient medium and transplanted into mice in the abdominal cavity, 1 million cells per mouse. A therapeutic experiment with the evaluation of the results of the study was performed in accordance with the recommended methodology [Guidelines for preclinical studies of drugs. Part One, 2012, 642-657].

The main groups of animals received a solution of the substance of anthrafuradion 3 (example 3) or solutions of anthrafurandion 3 obtained by reconstitution of lyophilized formulations 4-6 (note 6). All test solutions of anthrafurandion 3 contained 2 mg / ml of active ingredient. The substance of anthrafurandion 3 (Example 3) was dissolved in the required volume of “isotonic glucose solution (5%) for intravenous injection” when heated to 50-60 ° C and after cooling the solution was administered to animals. A solution of lyophilized formulations 4-6 (Example 6) was prepared by adding the required amount (10 ml per 1 vial) of an “isotonic glucose solution (5%) for intravenous injection” with periodic stirring of the contents of the vial until the composition was completely dissolved. The mice of the control group received a b / w 5% glucose solution in an adequate volume and treatment regimen. The introduction of solutions to animals was carried out in the appropriate individual doses in volumes once a day. Treatment was carried out on the 2nd-6th day after tumor inoculation.

Evaluation of the antitumor effect was performed to increase the life expectancy (VL) of mice in the treated groups compared with the control. For this, the individual and average life expectancy of the mice (RV, RV) in the group were determined, which were then used to calculate RV. The indicator making the difference in the LSS of the control and treated groups was expressed as a percentage of the control group. At autopsy of the mice that died in the experiment, the presence of leukemic ascites in the abdominal cavity was determined. The number of ascites-free mice was used as one of the parameters of treatment effectiveness. The data obtained were processed statistically using confidence intervals of the average compared values according to the standard student method in the modification of RB Strelkova. The differences were considered significant at p≤0.05.

The results of a comparative study of the antitumor activity of lyophilized pharmaceutical compositions of anthrafurandion 3 (formulations 4-6), presented in table 4, show that with five times daily intraperitoneal administration in a single dose of 30 mg / kg (150 mg / kg in total) all drug compositions give significant ( more than a minimum criterion of effectiveness) a high antitumor effect at the level of the substance of anthrafurandion 3 obtained in example 3: UPZ = 115-162%, without significant differences (p <0.05). The tolerance of the substance and both dosage forms is good, their use does not cause mouse death from toxicity, however, the dosage forms differ in their ability to inhibit the development of leukemic ascites: the use of the substance anthrafurandione 3 and compounds 4, 6 completely blocks the development of ascites in all treated animals, while as when using composition 5, the formation of ascites in a number of animals was noted. As can be seen from the data presented, the examples of lyophilized pharmaceutical compositions of anthrafurandion 3, which are the subject of the present invention, have a high antitumor activity corresponding to the active substance (S-isomer of anthrafurandion 3), but are more convenient for practical use.

Example 11

Investigation of the antitumor activity of the lyophilized pharmaceutical composition of anthrafurandion 3 in mice with intraperitoneally inoculated P388 / DOX multidrug-resistant lymphocytic leukemia

In the experiments, female mice of DBA ₂ hybrids with a body weight of 18-22 g were used. Before the experiment, the mice were ranked in groups, in each group 5-7 individuals. One group of mice with a tumor (n = 5) was left without specific effects and was considered a control. The main groups (n = 7) received treatment with a lyophilized pharmaceutical composition of anthrafurandion 3 (composition 6, example 6) or doxorubicin to confirm the resistance of the strain. For the study, we used the P388 lymphocytic leukemia line with multidrug resistance induced by doxorubicin (P388 / DOX). As a result of the activation of the resistance mechanism, the tumor cell line P388 / DOX is insensitive not only to doxorubicin, but also to most traditional antitumor agents, including rubomycin, carminomycin, vincristine, vinblastine, mitomycin C, bleomycetin [Donenko F.V. and other Bulletin of experimental biology and medicine, 1993, 116 (No. 9), 309-311]. The strain was maintained in vivo in adult DBA ₂ female mice with the administration of doxorubicin in a maintenance dose of 0.1 mg / kg in 0.05 ml of sterile saline. In the experiments, leukemic ascites obtained from the 3rd passage in vivo was used. The inoculum was prepared ex tempore in 199 nutrient medium and transplanted into female mice DBA ₂ in the abdominal cavity, 1 million cells per mouse. A therapeutic experiment with the evaluation of the results of the study was performed in accordance with the recommended methodology [Guidelines for preclinical studies of drugs. Part One, 2012, 642-657].

The main group of animals received daily five times in a single dose of 40 mg / kg a solution of anthrafurandione 3 (containing 2 mg / ml of the active substance) obtained by reconstituting the lyophilized composition 6 (Example 6). A solution of lyophilized composition 6 was prepared by adding the required amount (10 ml per 1 vial) of an “isotonic glucose solution (5%) for intravenous injection” with periodic stirring of the contents of the vial until the composition was completely dissolved. Treatment was carried out on the 2nd-6th day after tumor inoculation. The mice of the control group received a b / w 5% glucose solution in an adequate volume and treatment regimen. The introduction of solutions to animals was carried out in the appropriate individual doses in volumes once a day. To control the stability of the strain, we used a group of mice (n = 7), which were administered intraperitoneal therapy with doxorubicin (Lance-Farm, RF, in 10 mg vials) in a single effective dose of 7.5 mg / kg in 0.3 ml of physiological saline for injection.

Evaluation of the antitumor effect was performed according to the standard indicator of increasing life expectancy (VL), calculated by the difference in average life expectancy (LN) of mice in the treated and control groups as a percentage of the control. The degree of leukemia was judged by the presence of ascites and nodes in the abdominal cavity (+/-). The data obtained were processed statistically using confidence intervals of the average compared values according to the standard student method in the modification of RB Strelkova. As a minimum criterion of effectiveness, an LSS of ≥25% was used [Guidelines for preclinical studies of drugs. Part One, 2012, 642-657]. The differences were considered significant at p≤0.05.

As can be seen from the results shown in table 5, the pharmaceutical composition of anthrafurandione 3, which is the subject of the present invention, unlike doxorubicin, has a high significant specific (antitumor) activity against multidrug-resistant P388 / DOX lymphocytic leukemia. Therapy with composition 6 in an effective regimen (five times in a single dose of 40 mg / kg) leads to a significant prolongation of the life of mice (ALC = 36%, p <0.05). Moreover, the accumulation of ascites was not observed in animals. In the group of mice treated with doxorubicin, a significant increase in life expectancy was not obtained, and ascites accumulated in all animals.

Example 12

The study of the antitumor activity of the pharmaceutical compositions of anthrafurandion 3 in mice with intraperitoneal inoculant melanoma B16 / F10

In the experiments, female mice of BDF ₁ hybrids with a body weight of 18-22 g were used. Before the experiment, mice were ranked in groups, in each group 11 individuals. One group of mice with a tumor (n = 5) was left without specific effects and was considered a control. The second group received treatment with a lyophilized pharmaceutical composition of anthrafurandione 3 (composition 6, example 6). For the study, a strain of highly metastatic to lung melanomas B16 / F10 was used. The strain was maintained in vivo in mature C57B16j mice of both sexes. In the experiments, a tumor inoculum obtained from 2 or 3 passages in vivo was used. The inoculum was prepared ex tempore in 199 nutrient medium and transplanted into BDF ₁ hybrid mice into the abdominal cavity, 1 million cells per mouse. The study was performed in accordance with the recommended methodology [Guidelines for preclinical studies of drugs. Part One, 2012, 642-657].

The main groups of animals received a solution of anthrafurandione 3 containing 2 mg / ml of the active substance obtained by reconstituting the lyophilized composition 6 (Example 6). A solution of lyophilized composition 6 was prepared by adding the required amount (10 ml per 1 vial) of an “isotonic glucose solution (5%) for intravenous injection” with periodic stirring of the contents of the vial until the composition was completely dissolved. The mice of the control group received a b / w 5% glucose solution in an adequate volume and treatment regimen. The solution was administered to animals in volumes corresponding to individual doses once a day. Treatment was carried out on the 2nd-6th day after tumor inoculation.

Evaluation of the antitumor effect was performed to increase the life expectancy (VL) of mice in the treated groups compared with the control. For this, the individual and average life expectancies of the mice (RV, RV) in the group were determined, which were then used to calculate RVR. The indicator making the difference in the LSS of the control and treated groups was expressed as a percentage of the control group. The data obtained were processed statistically using confidence intervals of the average compared values according to the standard student method in the modification of RB Strelkova. As a minimum criterion of effectiveness, an ULC of ≥25% or T / C≥125% was used (in the control, T / C = 100%). The differences were considered significant at p≤0.05.

The results obtained indicate that the lyophilized pharmaceutical composition of anthrafurandion 3 (composition 6), which is the subject of the present invention, has a reliable high antitumor activity, giving at a five-fold daily intraperitoneal administration in a single dose of 30 mg / kg (total 150 mg / kg) UPZ = 50% (p <0.001), which is above the minimum performance criterion. Tolerance of the pharmaceutical composition is good and its use does not cause the death of mice from toxicity.

Example 13

The study of acute toxicity of anthrafurandione 3 and a pharmaceutical composition based on it

The acute toxicity of anthrafurandion 3 and a pharmaceutical composition based on it were studied with a single intraperitoneal injection using the Litchfield-Wilcoxon method on mice of BDF ₁ females weighing 20-24 g. Animals divided into groups of 6 individuals received a solution of the substance of anthrafurandione 3 (example 3) or solutions obtained by reconstituting the lyophilized composition 6 (Example 6). The substance of anthrafurandion 3 (Example 3) was dissolved in the required volume of “isotonic glucose solution (5%) for intravenous injection” when heated to 50-60 ° C and after cooling the solution was administered to animals. A solution of the lyophilized composition of composition 6 (Example 6) was prepared by adding the required amount (10 ml per 1 vial) of “isotonic glucose solution (5%) for intravenous injection” and the contents of the vial were periodically stirred until the composition was completely dissolved. Anthrafurandion 3 solutions were administered to mice at 0.2% concentration once intraperitoneally in a dose range of 20 to 90 mg / kg. Calculation of doses characterizing toxicity was performed using the StatPlus 2006 program.

The results of the study of acute toxicity with intraperitoneal administration, presented in table 6, show that according to the main toxicological parameters, LD ₅₀ (dose that causes the death of 50% of animals) and MID (maximum tolerated dose) toxicity for mice of the pharmaceutical composition 6 (example 6) is significantly lower toxicity of the substance of anthrafurandion 3 (example 3).

Table 6 The toxicity parameters of anthrafurandion 3 and a pharmaceutical composition based on it A drug LD ₅₀ , mg / kg MTD (LD ₁₀ ) Example 3 52.5 ± 5.4 39.4 ± 3.9 Composition 6 68.3 ± 6.2 47.6 ± 2.3

Claims (10)

1. The compound of formula I having antitumor activity and representing (S) -3 - [(3-amino-1-pyrrolidinyl) carbonyl] -4,11-dihydroxy-2-methylanthra [2,3-6] furan-5 , 10-dione, or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, in which the pharmaceutically acceptable salt is methanesulfonate. 3. A pharmaceutical composition having antitumor activity, comprising a therapeutically effective amount of a compound according to claims 1, 2, a pharmacologically acceptable carrier and one or more excipients selected from cosolvents, solubilizers, excipients, emulsifiers, preservatives, antioxidants, buffer compounds, substances to maintain isotonicity. 4. The pharmaceutical antitumor composition of claim 3, wherein the pharmacologically acceptable carrier comprises water. 5. The pharmaceutical composition according to claims 3, 4, wherein the excipient is one or more cosolvents selected from polyethylene glycol, ethanol, glycerol, 1,2-propylene glycol, 1,3-butylene glycol. 6. The pharmaceutical composition according to claims 3-5, wherein the excipient is one or more solubilizers selected from β-cyclodextrin or its derivative, γ-cyclodextrin or its derivative, polyvinylpyrrolidone, polysorbate, cremophor, dextran. 7. Lyophilized composition obtained by lyophilization of the antitumor composition according to claims 3-6. 8. The pharmaceutical composition obtained by dissolving the composition according to claim 7 in an aqueous medium. 9. A method of treating tumor diseases, comprising parenteral administration to a patient in need of a therapeutically effective amount of a substance according to claims 1, 2 or pharmaceutical compositions according to claims 2-8. 10. A method for treating a patient with a tumor disease according to claim 9, having drug resistance to other drugs.