Derived piranha, method thereof and pharmaceutical composition

 

(57) Abstract:

A new connection, which we call Latrotoxin H, has the formula (I)

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and its pharmaceutically acceptable salts. This compound can be prepared by hydrolysis of naturally occurring latrotoxin and can be used for the treatment and prevention of thrombocytopenia. 3 S. and 4 C.p. f-crystals, 2 tab.

The invention provides a new connection, which we called latrotoxin H (Leustroducsin H) and which has the formula (I) listed in the description. The invention also provides a method of obtaining this compound, as well as the composition and methods of treatment using the compounds.

Compounds of the invention are a new compound that stimulates the formation of lymphocytes and, therefore, is useful for the treatment of thrombocytopenia. Trombopenia can be caused by various reasons, such as immune disorders or adverse reactions to chemotherapy or radiotherapy of tumors. This serious disease, which, being burdened, causes bleeding throughout the body and can sometimes cause death. Currently, the only reliable method of treatment thrombopenia SIM is the hematopoietic (blood-forming) activity. They include certain interleukins, such as interleukin 6 (herein abbreviated IL-6), interleukin II (IL-II) and factor inhibiting leukemia (PHIL). Among other properties, it is known that these compounds stimulate the production of platelets and as such are reported to be useful in the clinic (Ishibashi and other Blood 74, 1241-1244 (1989), Asano and other Blood 75, 1602-1605 (1990); Okada other Blood' Tumor 22, 23-31, (1991). It was found that the introduction of these connections to people in different ways leads to a distinct pharmacological effects, which entails the possible use of these compounds in therapy. However, it seems that these compounds are largely produced in vivo by certain types of cells (e.g. lymphocytes, monocytes, fibroblasts, cells endothelia vessels and stromal cells) through a complex regulatory system and that they play a homeostatic role in the development of various types of blood cells. Accordingly, if these connections apply without regard to the delicate balance of this regulatory mechanism may cause side effects caused by imbalance of this regulatory mechanism; examples of such side effects include damage to the liver tissue, poorav, such as muramyldipeptide (TIR), can increase the total number of platelets (R. Nakajima and others Arzneimittel-Forsch/Drug Research 41, 60-65 (1989). Suggest that these compounds stimulate the production of IL-6 indirectly, through activation of monocytes and macrophages. Produced IL-6 then causes an increase in platelet count. However, it is also known that at the same time there are other, perhaps less desirable physiological effects based on the activation of macrophages, for example, the formation of monokines, such as interleukin 1 (IL-1) and tumor necrosis factor (TNF). There are also adverse reactions, such as fever (Jap. J. Radiotherapy 48 (4), 514 (1988).

Thus, it is clear that there remains a need for the agent that can stimulate the formation of platelets and which, therefore, can be used for the treatment of thrombocytopenia, but which does not cause imbalance of internal regulatory mechanism and does not lead to unpleasant effects, such as those described above.

Published application for European Pat. N 506 463 reveals some new derivatives of piranha called latrotoxin A, B and C having the following formula (X):

< / BR>
where R represents myces platensis, have activity in reducing adverse reactions to chemotherapy or radiotherapy for cancer, easily from infections, activation of macrophages, improving cerebral functions, as well as antifungal activity. Japanese patent application Hei 5-213 758, published on August 24, 1993, describes the use latrotoxin A, B, and C to influence trombozitopoez.

European patent publication No. 329 361 reveals some new derivatives of 2-pyranone (Pyranone), which is reminiscent of the compounds of the invention, except that they are replaced in a similar manner with the compounds of European patent publication N 506 463, above. Moreover, these compounds-prototypes declared as agricultural biocides, and in the published literature, there is no evidence that they possess useful therapeutic and prophylactic properties inherent to the compound of the present invention.

Some other compounds, is very similar to the one described in European patent publication No. 329 361 and used mainly also described in Japanese patent application Kokai Hei 2-186.

Journal of Antibiotics, 42 (1989) 1331-1343 reveals a new antitumor compound, which the authors call "izobreteniya, has an amino group and a phosphoric acid residue, but its molecular formula is different. This connection, therefore, clearly different from that of the invention.

The aim of the invention is to create a new latrotoxin having useful pharmacological activity.

In particular, suppose that the connection of the present invention has great potential in the treatment of thrombopenia, especially caused by chemotherapy and radiotherapy for cancer and immune disorders.

Thus, the invention provides a compound of formula (I):

< / BR>
and its pharmaceutically acceptable salts. This connection we call latrotoxin H".

The invention also provides a method of obtaining latrotoxin H, which will be described in more detail below.

The invention also provides a pharmaceutical composition comprising latrotoxin H, or its pharmaceutically acceptable salt, in a mixture with a pharmaceutically acceptable diluent or carrier.

The invention further provides a method of treatment or prevention thrombopenia, especially caused by cancer treatment by chemotherapy or radiotherapy, including the introduction of e which can be also a man, suffering from thrombopenia, or susceptible.

As can be seen from the above equation, latrotoxin of the present invention contains a number of asymmetric carbon atoms and more double bonds. Therefore, it can form various geometrical and optical isomers. Although they are all presented in this document one molecular formula, the invention includes both the individual isomers and mixtures thereof, including racemates. In the case of application techniques stereospetsifichno synthesis or in the case of using as starting material an optically active compounds of the individual isomers can be prepared by direct; on the other hand, if the mixture of isomers, an individual isomers can be obtained using standard separation techniques.

Latrotoxin of the present invention contains as an acid group, a phosphoric acid residue) and the main group (the amino group) and can, therefore, form salts. There are no particular restrictions regarding the nature of these salts; if they are intended for therapeutic use, they must be pharmaceutically acceptable. When they are dedicated to superior quality products is possible, more active compounds, do not apply even this limitation. Compounds of the invention can form salts with bases. Examples of such salts include: salts of alkali metals such as sodium, potassium or lithium; salts of alkaline earth metals such as calcium or barium, salts of other metals, such as magnesium or aluminum; salts of organic bases, such as salts dicyclohexylamine; and salts of basic amino acids such as lysine or arginine. Compounds of the invention can also form salts with acids. Examples of such salts include: salts of inorganic acids, especially halogenation acids (such as Hydrobromic acid, itestosterone acid or hydrochloric acid), salts of nitric acid, salts of perchloric acid, salts of carbonic acid, salts of sulfuric acid or salts of phosphoric acid; salts of lower alkylsulfonyl, such as methanesulfonate, triftoratsetata or econsultation; salts arylsulfonyl, such as benzosulfimide or p-toluensulfonate; salts of organic carboxylic acids, such as acetic acid, fumaric acid, tartaric acid, oxalic acid, maleic acid, succinic acid or citric acid; and Sania can be obtained by hydrolysis of compounds of formula (II) or its salt

< / BR>
where Z is an acyl group with the following (optional) the formation of salts of the compounds obtained.

The hydrolysis reaction can be performed using gidroliznogo agent commonly used in reactions of this type, for example, by hydrolytic enzyme or substrate.

In the formula (II) above, Z is an acyl group. The exact nature of the acyl group is not essential for the invention, because this group can be removed by hydrolysis to obtain the compounds of formula (I). In General, we have found that compounds in which Z is an aliphatic acyl group with a straight or branched chain or cyclic aliphatic group having from 2 to 16 carbon atoms, are suitable initial products. This acyl group in this case may be, for example, one of the following groups: butyryl, isobutyryl, isovaleryl, 2-methylbutyryl, 4-methylallyl, cyclohexane-carbonyl, 4-methylhexanoic, 5-methylhexanoic, 6-methylheptanoic, cyclohexylcarbonyl, octanoyl, 6-methyloctanoic, or 7-methyloctanoic.

The compounds of formula (II) for use as starting materials in the process of the invention in which Z represents isobutyryl, otics 42, 1019-1036 (1989).

The compounds of formula (II) for use as starting materials in the process of the invention in which Z represents butyryl, isobutyryl, isovaleryl, 2-methylbutyryl, cyclohexanecarbonyl, 4-melengestrol, 6-methylheptanoic, cyclohexylcarbonyl or octanoyl known and disclosed, for example, in European patent publication No. 329 361. The source materials in which Z is one of these groups, can be obtained by cultivation of a strain of the microorganism Streptomyces platensis SAM 0654 and the selection of the desired compounds of the cultural liquid. Streptomyces platensis SAM 0654 is stored under the number FERM BP-1668 in connection with European patent publication No. 329 361 in Fermentation Rearch Institute, Agency of Industrial Science and Technology on January 22, 1988, the Relevant methods of cultivation of the microorganism and highlight the desired compounds are given in European patent publication No. 329 361.

The compounds of formula (II) in which Z represents a 5-methylhexanoic, 6-methyloctanoic or 7-methyloctanoic, also known and described, for example, in European patent publication N 506 403.

The compounds of formula (II) for use as starting materials in the process of the invention in which Z represents 4-METHYLPHENOL, known and can be prepared, for example, by culturing Streptomyces platensis SANK 60191, followed by separation from the culture fluid. Appropriate conditions for cultivation of the microorganism, are, for example, in European patent publication N 506 463. Streptomyces platensis SANK 60191 is stored under the number FERM BP-3288 in Fermentation Rearch Institute, Agency of Industrial Science and Technology, dated February 20, 1991, in connection with European patent publication N 506 463.

Method 1. The use of a hydrolytic enzyme.

In this method, the compound of formula (I) are prepared by reaction of compounds of formula (II) with a hydrolytic enzyme.

The exact nature of this enzyme is not essential for the invention, and equally you can use any hydrolytic enzyme that is commonly used in reactions of this type. In General we prefer to use such enzymes as the pork liver esterase (ESP), lipase, acetylacetonate, Takadiastase (not available in the Nomenclature of enzymes: recommendations of the International Union of biochemistry) or cholesterolaemia.

This reaction is normally and preferably proceeds in the presence of a solvent. Any special restrictions on prir on the reaction or on the reagents and is capable of dissolving the reactants, at least partially. Examples of suitable solvents include a mixture of alcohol, such as methanol or ethanol with the buffer solution, preferably a phosphate buffer with a pH from about 6 to 8, or a mixture of a ketone, such as acetone or methyl ethyl ketone with a buffer solution, preferably a phosphate buffer with a pH from about 6 to 8.

We, in particular, it is preferred to carry out this reaction using ESP or lipase as a hydrolytic enzyme in a solvent comprising a mixture of phosphate buffer pH 6 to 8 with acetone and methanol.

The reaction may proceed in a wide range of temperatures, and the precise reaction temperature is not essential for the invention, although the preferred temperature may vary depending on the nature of the initial compounds and enzyme, as well as from the nature of the solvent. In General, we find it convenient to carry out this reaction at a temperature of from about 10 to 40oC, preferably approximately from 20 to 40oC.

The time required for the reaction may also vary widely, depending on many factors, particularly the reaction temperature and the nature of the starting compound, enzyme and solvent. However, about is from 12 hours to 30 days, preferably from 3 to 15 days.

Upon completion of the reaction, the compound of formula (I) can be isolated from the reaction mixture and purified by standard methods. One example of a suitable method includes removing miscible with water solvent, such as acetone, by distillation under reduced pressure, followed by extraction of the resulting aqueous layer with an organic solvent such as ethyl acetate. Then may be followed by fractionation and purification of the aqueous layer, for example, using an open column Cosmosil (registered trademark).

Method 2. The application of Foundation.

In this method, the compound of formula (I) are obtained by reaction of compounds of formula (II) with a base.

In regard to the nature of the used grounds, significant there are no limits; you can use any base that is usually used in reactions of this type, provided that it has no adverse effect on any portion of the molecules or reagents. Examples of preferred bases include: inorganic base, for example, a carbonate of an alkali metal such as sodium carbonate, potassium carbonate, cerium carbonate ilikecereal lithium; the alkali metal hydroxide, such as sodium hydroxide, potassium hydroxide or lithium hydroxide; or alkali earth metal hydroxide such as barium hydroxide, magnesium hydroxide or calcium hydroxide. Among these reagents, we prefer to use a carbonate of an alkali metal or hydrogen carbonate of an alkali metal, most preferably sodium carbonate, potassium carbonate or sodium bicarbonate.

This reaction is normally and preferably proceeds in the presence of a solvent. There is no specific limitation regarding the nature of the solvent, it is sufficient if it does not have an adverse effect on the reagents and are able to dissolve, at least partially. Examples of suitable solvents include a mixture of alcohol, such as methanol or ethanol, with water; or a mixture of a ketone, such as acetone or methyl ethyl ketone, water.

The reaction can proceed at a wide range of temperatures, and the precise reaction temperature is not essential for the invention, however, the preferred temperature may vary depending on the nature of the initial compounds and bases, as well as from the nature of the solvent. In General we find an adequate flow reaction> The time required for completion of the reaction may also vary widely, depending on many factors, namely the reaction temperature and on the nature of the parent compound, base and solvent. However, if the reaction proceeds under optimal conditions listed above are usually sufficient period of time from about 3 hours to 5 days, preferably from 3 hours to 2 days.

Upon completion of the reaction, the compound of formula (I) can be extracted from the reaction mixture and purified using standard techniques. One example of a suitable method includes removing miscible with water solvent, such as acetone by distillation under reduced pressure, followed by extraction of the resulting aqueous layer with an organic solvent such as ethyl acetate. Then you can take the fractionation and purification of the aqueous layer, for example, using an open column Cosmosil (registered trademark).

Biological activity.

The following study demonstrates the activity of the compounds of the invention as tromboticheskoe agent. We use the term "thrombopoiesis agent" to refer to an agent that, Boguchany thrombocytopenia, due to various reasons, such as immune disorders and adverse reactions to radiotherapy or chemotherapy of cancer.

Experiment 1.

Strengthening thrombocytopoiesis in mice after intravenous latrotoxin H.

Thrombopoetin activity of the compounds of the invention can be studied using the methodology developed by Ishibashi and others described in Blood 74, 1241-1244 (1989).

In more detail, the test compound of the invention was dissolved in physiological solution containing 1,25 about. aqueous ethanol. A sample of this mixture was injected intravenously 7-week old female C57BL with 24-hour intervals for 5 days. Control mice received only saline solution containing 1,25 about. aqueous ethanol. After 72 h after the last injection took blood samples from the orbits of the eyes of experimental animals and were counting the number of platelets. The study was performed by the method of electric resistance with the use of an automatic blood analyser (K-1000, Toa-Iyo Denshi Co.) The results are shown in table. 1.

The exact conditions of the experiment, listed above, are not essential to determine the number of platelets, and opportunities that may exist about animals who rata. For example, animals can be mice, rats, dogs or monkeys, as test connection, you can enter parenterally, administered intraperitoneally, intramuscularly or subcutaneously.

Experiment 4.

The study of toxicity.

BALBIC mice intravenously injected with 4 mg/kg latrotoxin H. After 10 days has not been a single death.

From the above data it is obvious that latrotoxin H of the invention shows excellent thrombopoetin activity in vivo and low toxicity and is therefore useful as a therapeutic agent for the treatment of thrombocytopenia due to various reasons, in particular, immune disorders and adverse reactions after chemotherapy or radiotherapy for cancer.

For the purposes of such treatment, the compounds of formula (I) can be administered orally in the form of tablets, capsules, granules, powders or syrups, or parenterally, by intravenous, subcutaneous or intramuscular injection, use of suppositories, etc., These pharmaceutical compositions can be prepared by mixing the compounds of the invention with one or more adjuvants such as fillers (for example, organic fillers, such as with hmal crops mashed potato-starch, dextrin or carboxymethyl-starch; cellulose derivatives such as crystalline cellulose, cellulose with hydroxypropyl Deputy, hypromellose, carboxymethylcellulose, connection carboxymethylcellulose calcium, compound with sodium carboxymethylcellulose; Arabian gum; dextran; Pullulan; and inorganic excipients including silicate, such as light silicic acid anhydride, synthetic aluminum silicate or magnesium silicate/magnesium aluminate; phosphates such as calcium phosphate; carbonates such as calcium carbonate; and sulfates such as calcium sulfate), lubricants (e.g., metallic stearates, such as calcium stearate or magnesium stearate; talc; colloidal silica; waxes such as beeswax or spermaceti; boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL-leucine; sodium salts of aliphatic acids; laurilsulfate, such as sodium lauryl sulfate or lauryl sulfate, magnesium silicates, such as silicon dioxide or silicon hydroxide; and the above-mentioned starch derivatives); binders (e.g., polyvinylpyridine, Macrodol, and substances, padanaplast and chemically modified starch-cellulose, such as croscarmellose-sodium, sodium-carboximetilkrahmal or polyvinylpyrrolidone with a bridge connection), stabilizers (for example, p-hydroxybenzoate, such as methylparaben or propylparaben; alcohols such as chlorobutanol, benzyl alcohol or phenethyl alcohol; Chlorobenzilate alcohol; phenols such as phenol or cresol; deshidratada acid and sorbic acid); coregency (for example, sweeteners, vinegar, or perfumes, which are usually used for such purposes); solvents, etc.

Doses vary depending on the condition and age of the patient, but also on the method and type of drug, however, for example, compounds of the invention can be administered in a daily dose of from 0.01 to 10 mg/kg (preferably from 0.01 to 1 mg/kg) as a single dose or as divided doses.

Getting some compounds of the invention is further illustrated by the examples. In the section "Preparation" describes how to obtain some of the original materials used in the examples below.

Example 1. 5,61 g of crude oily substance obtained in stage B of preparation 1 (see below), was dissolved in 130 ml of acetone. Then added 1400 ml of 0.05 M phosphate buffer solution (NaH2PO4 is, the company's product Amano Pharm. Co. Ltd.) and the mixture was stirred at a temperature of 35oC. the Source material had a retention time of 8.8 min in liquid chromatography high resolution, and the course of the reaction, therefore, was controlled using this technique. ESP was added in the amount of 0.82 g, 1.52 g, 1,02 g and 0.9 g of the mixture over a period of time in two weeks at the 35oC and the resulting mixture was stirred for two weeks. At the end of this time the reaction solution was filtered using a filtration system Celite (registered trade mark) to remove the policy. The filtrate was then extracted with ethyl acetate, and the aqueous layer was fractionally and was purified column chromatography through a 400 g Cosmosil 75C18-OPN (registered trademark for a product company Nakaraitesque, Inc. using aqueous methanol as the eluent, to obtain 1.73 g latrotoxin H.

Liquid chromatography high resolution performed under the following conditions.

Column: Cosmosil 5C18-ARIM4.6250 mm (product Nakaraitesque, Inc.);

eluent: 20% (vol.) acetonitrile: 0.5 to about. the triethylamine; 79,5 about. phosphate buffer (pH 3.0);

flow rate: 1.0 ml/min;

wavelength: 230 nm.

Mass spectroscopy when BOM (1H, doublet of doublets, J 5.4 and 9.6 Hz); 6,21 (1H, doublet of doublets, J 11.7 and 20.5 Hz); 6,12 (1H, doublet of doublets, J 12.2 and 20.5 Hz); 5,93 of 5.84 (2H, multiplet); 5,71 (1H, doublet, J 16.6 Hz); 5,32-a 5.25 (2H, multiplet); of 3.94 (1H, doublet of triplets, J 3,4 and 10.3 Hz); 3,48 (1H, multiplet); of 2.93 (2H, triplet, J 7.8 Hz); 2,53-to 2.40 (2H, multiplet); 2,03 (1H, multiplet); 1,80-0,73 (13H, multiplet); to 0.73 (3H, triplet, J 7.8 Hz).

Example 2. 20 mg of the compounds of formula (II) (see above), which is 4-methylhexanoic (prepared as described in stage B of preparation 1, see below) was dissolved in a small amount of methanol. The resulting solution was diluted with phosphate buffer (pH 6,7), after which was added 10 mg of ESP (product of Amano Pharm. Co. Ltd.) and the mixture was stirred for 6 days at 30oC. after this time the reaction solution was filtered. The remaining methanol was removed from the filtrate by distillation under reduced pressure, and the resulting aqueous solution was fractionally and was purified on a column of C18-Cosmosil using aqueous methanol as the eluent. The fraction was suirable about 20. aqueous methanol and obtained 13 mg connection with the same physical properties as the compound obtained in example 1, above.

Example 3. Was performed the same procedure that is described in the above PR is th, as described in stage 3 preparation of 1, see below), and was obtained 30 mg latrotoxin H. Following the procedure similar to that described in example 2, above, latrotoxin H with the same properties as that described in example 1, above, also were prepared from the compounds of formula (II) in which Z is represented the following groups. Amount of starting material and the compounds of the invention are shown below.

Example 8. 20 mg of the compounds of formula (II) in which Z is 6-methylheptanoic (obtained as described in stage B of preparation 1, see below) was dissolved in a small amount of aqueous methanol. Then to the mixture was added saturated aqueous sodium hydrogen carbonate solution and the resulting solution was stirred for one day. After this time the pH of the reaction solution was brought to 2 by addition of an appropriate quantity of aqueous hydrochloric acid and the resulting mixture was fractionally and was purified on column Cosmosil C18, resulting received 3 mg latrotoxin H.

Preparation 1. Culture and isolation of the parent compounds.

1 (A) Culture. One platinum loop of spores of Streptomyces platensis SANK 60191 was inoculable in a 500 ml Erlenmeyer flask, equipped with a separation lane is croorganisms were cultured for 3 days at 28oC and the rotation of 200 rpm (rotation radius of 7 cm), by using a rotary shaker.

Cultural environment

Soluble starch 30 grams

Crude yeast 10 grams

The soybean powder 7 g

Fish meal 5 grams

Tincture corn 2 grams

Beef extract 1 g

Calcium carbonate 1 g

Water Up to 1000 ml

pH 7 (before sterilization)

15 l of the same medium used for cultivation, were loaded into four 30-liter fermenter stainless steel and sterilized by heating to 120oC for 30 minutes Then added 150 ml of culture medium prepared as described above. The mixture was cultured for 3 days at 28oC with aeration with air flow rate of 15 l/min with constant stirring. In order to maintain the oxygen concentration in the liquid, equal to 5 parts per million (ppm), the mixing speed is automatically controlled and remained in the range of 100 to 300 rpm

1 (B) Allocation. To 60 l of culture liquid obtained as described above in paragraph 1 (A), was added as a filtering system 2.4 kg Celite 545 (trade name of product of the company Johns and Manville Corporation Project USA) and the mixture was filtered. After filtering the culture of Jedi 20 l 80 about. aqueous acetone. These extracts were combined and the organic solvent evaporated using a rotary evaporator. To the residue was added an aqueous solution of hydrochloric acid in a quantity sufficient to obtain a pH of 2.0, and then the mixture was extracted twice, each time for 10 l of ethyl acetate. The extracts were combined and added to 10 l of 1% (weight/volume) aqueous solution of sodium bicarbonate. Active fractions were transferred into the aqueous layer, and the ethyl acetate layer was removed. The ethyl acetate layer again was extracted with 5 l of 1% (weight/volume) aqueous solution of sodium bicarbonate. Solutions of sodium bicarbonate were combined and the pH of the combined solution was brought to 2 by addition of an aqueous solution of hydrochloric acid. This solution was extracted twice, each time for 10 l of ethyl acetate. The organic extracts were combined, washed with water, and then saturated aqueous solution of sodium chloride and then dried over anhydrous sodium sulfate. After the gradual addition of methanol and the solution so obtained are condensed by evaporation under reduced pressure using a rotary evaporator, resulting in received 10 ml of oily substances. This oily substance was dissolved in 100 ml of 60 about the company's Water Co. USA). Contamination has suirable 30 ml of 60 rpm. aqueous methanol. Then suirable latrotoxin 15 ml 100% methanol, and the eluate are condensed, resulting in a received 600 mg of an oily substance. This oily substance directly, without further purification used in example 1 (see above). In order to clear this material was dissolved in 10 ml of methanol and subjected to liquid chromatography high resolution. Fractions with peaks at about 13 min, 19 min and 24 min was collected and designated as "crude fraction A", "crude fraction B" and "crude fraction C", respectively. Conditions of chromatography is shown below.

Preparative liquid chromatography.

Column: Radial-Pak 2510 (Waters, USA);

eluting solution: 50% by volume aqueous acetonitrile containing 0.5% triethylamine-phosphate buffer, pH 3.0;

flow rate: 9 ml/min;

wavelength: 230 nm.

After condensation of all these peaks obtained fractions were subjected to preparative liquid chromatography high resolution. Crude fraction A was subjected to preparative chromatography to collect peaks about 53 and 56 minutes under the following conditions; it was then desalted and condensed using a Sep-Pak, resulting received 22,03 mg saedinaiushaia crude fraction A.

Column: Cosmosil 5C 18-AR 20250 mm (Nakaraitesque, Inc.);

eluting solution: about 42. aqueous acetonitrile containing 0.5% triethylamine-phosphate buffer, pH 3.0;

flow rate: 9 ml/min;

wavelength: 230 nm.

Crude fraction B was subjected to preparative chromatography to collect peaks about 74 min 79 min 82 min under the following conditions, then it was absoluely and are condensed by using Sep-Pak, resulting received 26,16 mg of the compounds of formula (II) in which Z represents cyclohexylcarbonyl, and 3.24 mg of the compounds of formula (II), which represents octanol.

Preparative conditions for crude fraction B.

Column: Cosmosil 5C 18-AR 20250 mm (Nakaraitesque, Inc.);

eluting solution: about 47. aqueous acetonitrile containing 0.5% triethylamine-phosphate buffer, pH 3.0;

flow rate: 9 ml/min;

wavelength: 230 nm.

Crude fraction C was subjected to preparative chromatography to collect peaks around 47 minutes and 51 min under the following conditions; then it was absoluely and are condensed by using Sep-Pak, resulting received 9,83 mg latrotoxin B and 5,22 mg latrotoxin C.

Preparative conditions for crude fraction C.

Column: Cosmosil 5C 18-AR 20250 mm (Nakaraitesque, Inc.);

Luka: 9 ml/min;

wavelength: 230 nm.

Preparation 2. Culture and isolation of the parent compounds. Streptomyces platensis SAN-0654 (FERM BP-1668) was cultured and the compounds of formula (II) in which Z represents butyryl, isobutyryl, isovaleryl, 2-methylbutyryl, cyclohexanecarbonyl, 4-methylhexanoic, 6-methylheptanoic, cyclohexylcarbonyl and octanol, were isolated from the culture fluid in accordance with the methods described in European patent publication No. 329 361.

Recipe 1.

Cooking capsules

Latrotoxin H 100 mg

Lactose 100 mg

Corn starch 148, 8 persons mg

Magnesium stearate 1.2 mg

The total number of 350 mg

The above ingredients were mixed, the resulting powder was missed through screening machine, the mesh 20 (Tyles standard) and then filled them with capsules.

1. Derived piranha General formula I

< / BR>
or its pharmaceutically acceptable salt.

2. The pharmaceutical composition exhibiting thrombopoetin activity, containing the active ingredient and a pharmaceutically acceptable diluent or carrier, wherein the active component is used as a compound of formula I under item 1, in an effective amount.

3. ASS="ptx2">

4. The method of obtaining the compounds of formula I, wherein is exercised by the hydrolysis of compounds of formula II

< / BR>
where Z acyl group,

or its salt with subsequent optional formation of salts of the compounds obtained.

5. The method according to p. 4, characterized in that the hydrolysis is carried out with the use of a base or hydrolytic enzyme.

6. The method according to p. 4, wherein Z is an aliphatic acyl group with a straight or branched chain or cyclic aliphatic acyl group with 2 to 16 carbon atoms.

7. The method according to p. 4, characterized in that the Z butyryl, isobutyryl, isovaleryl, 2-methylbutyryl, 4-methylallyl, cyclohexanecarbonyl, 4-methylhexanoic, 5-methylhexanoic, 6-methylheptanoic, cyclohexylcarbonyl, octanoyl, 6-methyloctanoic or 7-methyloctanoic.

 

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FIELD: organic synthesis.

SUBSTANCE: invention provides compounds of general formula I:

, where R1 represents -CO-Ra, -SO2-Rb, or aryl optionally substituted by lower alkoxy, wherein Ra represents cycloalkyl, cycloalkyl(lower)alkyl, cycloalkyloxy, aryl, aryloxy, aryl(lower)alkyl, aryl(lower)alkoxy, aryloxy(lower)alkyl, aryl-S-(lower)alkyl, aryl(lower)alkenyl, provided that aryl group can be optionally substituted by halogen, lower alkyl, hydroxy, nitro, cyano, lower alkoxy, phenyl, CF3, cyano(lower)alkyl, lower alkyl-C(O)NH, lower alkyl-CO, and lower alkyl-S; heteroaryl, heteroaryl(lower)alkyl, or heteroaryl(lower)alkoxy, provided that heteroaryl group is 5- or 6-membered ring or bicyclic aromatic group constituted by two 5- or 6-membered rings including 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur and that heteroaryl group can be optionally substituted by lower alkoxy; Rb represents aryl, aryl(lower)alkyl, or heteroaryl, aryl group optionally substituted by halogen, cyano, or lower alkyl-C(O)NH; R2 and R3 represent hydrogen atoms; R4 representshydrogen or lower alkyl; R5 represents hydrogen, lower alkyl, cycloalkyl, benzodioxyl, or aryl optionally substituted by lower alkyl, halogen, lower alkoxy, hydroxy, or (lower)alkyl-C(O)O; n is 1 or 2; and pharmaceutically acceptable salts thereof and/or pharmaceutically acceptable esters thereof. Invention also provides a pharmaceutical composition exhibiting inhibitory activity with regard to cysteine proteases of the cathepsin family, which composition comprises compound of formula I, pharmaceutically acceptable recipient, and/or adjuvant.

EFFECT: increased choice of cysteine protease inhibitors.

34 cl, 1 tbl, 13 ex

FIELD: organic chemistry, chemical technology, medicine, biochemistry, pharmacy.

SUBSTANCE: invention relates to new derivatives of sulfonamides of the formula (I) or their pharmaceutically acceptable salts wherein R1 means -OH or -NHOH; R2 means hydrogen atom; R3 means alkyl, alkoxyalkyl, arylalkyl, pyridylalkyl or morpholinylalkyl; A means piperidyl or tetrahydrofuranyl; n = 0; E means a covalent bond; (C1-C4)-alkylene, -C(=O)-, -C(=O)O- or -SO2-; X means hydrogen atom, alkyl, aryl, arylalkyl, alkoxyalkyl, morpholinyl or tetrahydropyranyl; each among G and G' means -C(R5)=C(R5') wherein R5 and R5' mean hydrogen atom; M means the group -CH-; z means the group -(CR7R7')a-L-R8 wherein a = 0 and each among R7 and R7' means hydrogen atom; L means a covalent bond; R8 means halogen atom or alkoxy-group. Compounds of the formula (I) are inhibitors of metalloproteases and can be used for treatment of arthritis, cancer tumors and other diseases.

EFFECT: valuable medicinal properties of compounds.

15 cl, 7 tbl, 56 ex

FIELD: medicine.

SUBSTANCE: before applying substitute hormonal therapy (SHT) on should evaluate antithrombogenic activity of vascular wall in women. For this purpose one should determine quantitative values of ADP-induced aggregation of thrombocytes, activity of antithrombin III in blood and fibrinolytic blood activity both before and after "cuff"-test. Then one should detect the indices calculated as the ratio of mentioned values both before and after carrying out the mentioned test. If mentioned indices are decreased against the norm by 20-40% women should be prescribed to undergo SHT at additional introduction of aspirin and supradin. The method provides prophylaxis of cardio-vascular diseases in this category of female patients due to correcting affected functional activity of vascular endothelium.

EFFECT: higher efficiency of prophylaxis.

1 cl, 1 ex, 4 tbl

FIELD: medicine, natural compounds.

SUBSTANCE: larch wood is saturated with water and extraction with ethyl acetate is carried out. Prepared extract is treated with hot water and this process is combined with distilling off a solvent. Then water-insoluble resin impurities are separated and crude product is isolated by crystallization and recrystallized. Invention provides simplifying the process.

EFFECT: improved preparing method.

2 ex

FIELD: medicine, pharmaceutical industry, pharmacy.

SUBSTANCE: invention relates to compositions used for treatment and/or prophylaxis of chlamydium infections caused by C. pheumoniae. Pharmaceutical composition used for treatment and/or prophylaxis of chlamydium infection caused by C. pneumoniae comprises the taken phenolic compound, or extract, or fraction, or incomplete fraction comprising the taken phenolic compound or corresponding synthetic compound, or mixture of indicated compounds obtained from plants. An anti-chlamydium effect of phenolic compound or extract, or fraction, or incomplete fraction obtained from plants and comprising indicated compound or corresponding synthetic compound on C. pneumoniae represents the definite percent of inhibition for formation of inclusions. The composition useful for health eliciting an anti-chlamydium effect with respect to C. pneumoniae comprises the taken phenolic compound or extract, or fraction, or incomplete fraction containing indicated compound or corresponding synthetic compound, or mixture of indicated compounds obtained from plants. An anti-chlamydium effect of phenolic compound or extract, or fraction, or incomplete fraction comprising indicated compound or corresponding synthetic compound obtained from plants on C. pneumoniae represents the definite percent for inhibition in formation of inclusions. Also, invention relates to applying the composition useful for health in preparing foodstuffs or as supplements for nutrition for every day. Also, invention relates to applying phenolic compound or extract, or fraction, or incomplete fraction comprising indicated compound or corresponding synthetic compound or mixture of indicated compounds obtained from plants in manufacturing a medicinal agent used for treatment and/or prophylaxis of chlamydium infections caused by C. pneumoniae. An anti-chlamydium effect of phenolic compound or extract, or fraction, or incomplete fraction comprising indicated compound or corresponding synthetic compound obtained from plants on C. pneumoniae represents the definite percent in inhibition in formation of inclusions. Compositions promote to effective prophylaxis and treatment of chlamydium infections caused by C. pneumoniae.

EFFECT: valuable medicinal properties of compounds.

21 cl, 1 dwg, 1 tbl, 6 ex

FIELD: organic chemistry, medicine.

SUBSTANCE: invention relates to new compounds of coumarone class, namely, to 6-nitro-2-iminocoumarin 3-carboxylic acid 4-toluidide silver salt of the formula (1): that elicits an antibacterial effect and can be used in medicine. Invention provides preparing a new compound eliciting an antibacterial effect with respect to S. aureus, E. coli, and C. albicans with mononuclear cells values 0.25; 0.5 and 7.8 mcg/ml, respectively, and with acute toxicity value LD50 for these compounds 2 460 230 mg/kg.

EFFECT: valuable properties of compound.

1 cl, 1 tbl, 2 ex

FIELD: organic chemistry, medicine, pharmacology.

SUBSTANCE: invention relates to new derivatives of carbamic acid esters of the general formula (I):

and their pharmaceutically acceptable salts eliciting activity with respect to metabotropic glutamate receptors mGlu of group I that can be used for treatment of acute and/or chronic neurological disorders. In the general formula (I) R1 means hydrogen atom or (C1-C7)-alkyl; R2 and R2' mean independently of one another hydrogen atom, (C1-C7)-alkyl, (C1-C7)-alkoxy-group, halogen atom or trifluoromethyl; X means oxygen (O), sulfur (S) atom or two hydrogen atoms not forming a bridge; A1/A2 mean independently of one another phenyl or 6-membered heterocycle comprising 1 or 2 nitrogen atom; B represents group of the formula:

wherein R3 means (C1-C7)-alkyl and others; Y means -O-, -S- or a bond; Z means -O- or -S-; or B means 5-membered heterocyclic group of formulae: (a) , (b) , (c) or (d) . Also, invention relates to methods for preparing compounds and to a medicinal agent based on thereof.

EFFECT: improved preparing methods, valuable medicinal properties of compounds.

22 cl, 1 tbl, 2 sch, 78 ex

FIELD: medicine.

SUBSTANCE: the present innovation deals with phospholipid complexes of proanthocyanidine A2 and pharmaceutical compositions upon their basis as antiatherosclerotic agents, those for preventing and treating myocardial and cerebral infarction. Phospholipids of the above-mentioned complex should be preferably chosen out of lecithins, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine. The innovation provides the preparation to treat the above-mentioned diseases due to decreasing the quantity and burden of atheromatous plaque, decreased obstruction of carotid arteries and decreased thickness of vascular walls.

EFFECT: higher efficiency of prophylaxis and therapy.

9 cl, 11 dwg, 6 ex, 2 tbl

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention relates to treatment of patients suffering from diseases associated with pathologic activity of matrix proteases. Treatment involves administration of compounds depicted by general formula (I).

EFFECT: increased treatment efficiency.

136 cl, 448 ex

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