Cyclipostins, method for their preparing, pharmaceutical composition based on thereof, method for its preparing and strain streptomyces as producer of cyclipostins

FIELD: organic chemistry, microbiology, pharmacy.

SUBSTANCE: invention relates to new biologically active compounds. Invention describes a compound of the formula (1):

wherein R1 means carbon chain with 2-30 carbon atoms that can be linear, branched, saturated and can be substituted once or twice with the following groups: -OH, =O, -(C1-C6)-alkyl or the group:

; R2 means (C1-C6)-alkyl; E means phosphorus atom (P); each X1, X2 and X3 means independently of one another -O- in its all stereochemical forms and mixtures of these forms taken in any ratio, and its physiologically acceptable salts also. Also, invention describes a method for preparing cyclipostins and the strain Streptomyces HAG 004107, DSM 13381 as a producer of cyclipostins. Invention provides preparing new compounds possessing valuable biological properties.

EFFECT: improved preparing method, valuable properties of substances.

12 cl, 9 tbl, 21 ex

 

The invention relates to new compounds, called cyclopentenone, which is produced by culturing Streptomyces species HAG 004107 (DSM 13381), and their physiologically acceptable salts and their chemical equivalents. The invention relates further to a method for cyclooctanol, the microorganism HAG 004107 (DSM 13381), the use of cyclooctene and their physiologically acceptable salts and chemical equivalents as pharmaceuticals, in particular as inhibitors of lipases, as well as to pharmaceutical compositions containing cycloocten or its physiologically acceptable salt, or its equivalent.

The disease, which is particularly preferably can be treated with inhibitors of lipase is sugar diabetes (Diabetes mellitus). Diabetes mellitus is a disease characterized by elevated concentrations of blood sugar due to chronic metabolic disorders. The metabolism is based on the deficiency of insulin or reduced action of insulin. The lack of insulin leads to suboptimal utilization of somatic cells entering the blood glucose. Through this, and also due to neoplasms of glucose from protein (gluconeogenesis) increases the level of glucose in the blood. Moreover, in the case of reduced insulin action in adipose tissue antagonistical the e to insulin hormones, as glycogen, leading to increased lipolysis and thereby to increased concentrations of fatty acids in the blood. We are talking about ketoacidosis, i.e. the increased formation of ketone bodies (as acetoacetic acid, β-hydroxybutanoic acid, acetone). In acute situations, the lack of biochemical regulation is dangerous to life and leads, without treatment, diabetic coma, finally, to die quickly. Diabetes mellitus is the most frequent chronic diseases of the metabolism of a person, and believe that over 3% of the population have diabetes or prediabetes predisposition and thus are under immediate threat of the disease. Therefore, there is a great need for treatment or cure of diabetes.

Treatment of diabetes is carried out by injection of insulin; in the case of senile diabetes, the so-called non-insulin-dependent diabetes (NIDDM) or type II diabetes, first enter sulfonylureas. The principle of action of the sulfonylureas is to increase the secretion of insulin β-cells in the pancreas to compensate for the hormone deficiency or insulin resistance. In case of disease progression also need to enter the insulin. Insulin can be summarized as follows. This peptide hormone reduces the oxygen the radio glucose in the blood and leads to increased anabolic and simultaneously to the suppression of catabolic processes:

- increases the transport of glucose in somatic cells;

- promotes the formation of glycogen in the liver and in the muscles;

- inhibits lipolysis;

- increases the flow of fatty acids in adipose tissue and

- increases the flow of amino acids into somatic cells, but also enhances protein synthesis.

One of the strongest effects of insulin is the suppression of lipolysis. In the case of patients with diabetes type II to this regulation of lipolysis is more than enough and come to an increased level of free fatty acids in the blood. Free fatty acids in the blood stimulate gluconeogenesis in the liver and reduces glucose utilization in skeletal muscle. Lipolysis, i.e. the release of fatty acids is controlled by the so-called gormonchuvstvitelnoy the lipase (HSL), which is located in fat cells and is inhibited by insulin through a cascade of phosphorylation. Therefore would be desirable inhibitors, that is, the HSL inhibitors, which can mimic the action of insulin and reduce the level of fat in the blood. Such agents suitable for the treatment of patients with type II diabetes for the regulation of fat metabolism, which, however, could also be applied in the case of other diseases of accumulation. For all these reasons, it is strongly required and therefore looking for new HSL inhibitors and other lipases.

Unexpectedly, it was shown that the strain is of microorganism species Streptomyces HAG 004107, DSM 13381, can produce highly efficient new lipase inhibitors, which inhibit gormonchuvstvitelnoy the lipase even at very low concentrations. New natural compounds are organophosphorus compounds that consist of dual cyclic system (Bicycle) and substituted carbon chain and specifically inhibit lipase. Cyclic skeleton - only with a methyl group instead of the carbon chain as an inhibitor of acetylcholinesterase, CGA 134736, was first described by R. Neumann and H.H. Peter in Experientia, 43, 1235-1237 (1987) and later the same connection, called cycloocten, described T. Kurokawa, and others, in J. Antibiotics, 46, 1315-1318 (1993). This structuretone connection does not have any selective, inhibiting the lipase property. Known hitherto substances have shortcomings, which are expressed in unsatisfactory intensity action, high toxicity and/or unwanted side effects.

The present invention relates to compounds of General formula (I):

where

R1means

1. carbon chain with 2-30 carbon atoms, which may be linear, branched, saturated or unsaturated, Carbo - or heterocyclic, and where the carbon chain can be single or twofold substituted with:

1.1-HE;

1.2 =On;

1. -O-(C 1-C6)-alkyl, where alkyl is linear or branched;

1.4-O-(C2-C6-alkenyl, where alkenyl is linear or branched;

1.5 -(C1-C6)-alkyl, where alkyl is linear or branched;

1.6-aryl;

1.7 -(C1-C6)-alkylbenzene;

1.8-diphenyl;

1.9-NH-(C1-C6)-alkyl, where alkyl is linear or branched;

1.10-NH-(C2-C6-alkenyl, where alkenyl is linear or branched;

1.11-NH2;

1.12 =S;

1.13-S-(C1-C6)-alkyl, where alkyl is linear or branched; or

1.14-S-(C2-C6-alkenyl, where alkenyl is linear or branched;

1.15 halogen; and Vice-1.1-1.15 can also be substituted;

2. -[aryl-(CH2)n]mwhere [aryl-(CH2)n]munsubstituted or one - or twofold substituted as described in 1.1-1.15, and n and m, independently of one another, denote integers zero, 1, 2 or 3;

R2means

1. (C1-C6)-alkyl, where alkyl unsubstituted or one - or twofold substituted as described in 1.1-1.15;

2. (C2-C6)-alkenyl, where alkenyl unsubstituted or one - or twofold substituted as described in 1.1-1.15; or

3. (C2-C6)-quinil where quinil unsubstituted or one - or twofold substituted as described in 1.1-1.15;

E. means an atom of phosphorus (P) or sulfur atom (S);

X1, X2and X3independently from each other mean

1. -O-;

2. -NH-;

3. -N=;

4. -S - or

5. -CH2- and-CHR2-;

all their stereochemical forms and mixtures of these forms in any ratio, and their physiologically acceptable salts and their chemical equivalents.

Preferably R1is the length of the chain, consisting preferably of 6-24 carbon atoms, most preferably from 10-18 carbon atoms. The chain may be saturated, such as alkyl, where alkyl may be linear or branched, or unsaturated, such as, for example, alkenyl or quinil, where alkenyl or quinil is a linear or branched. R1may be unsubstituted or mono - or twice, identically or differently substituted with groups 1.1-1.15, as described above. Preferred substituents at the carbon atoms 8'-16'; especially preferred position 10'-14'. Vice-1.1-1.15 may also be substituted by one or more groups selected from alcohol, aldehyde, acetylenic, katalinich, ether, carboxyl, ester, amino-, cyano-, nitro-, oxomnik groups, groups, ethers Akimov and halogen.

Carbocyclic carbon chain with 2-30 carbon atoms means consisting of 2-30 carbon atoms chain with one or NESCO is Kimi, preferably one, two or three cyclic systems, which preferably consist of, respectively, 4, 5, 6 or 7 carbon atoms. These systems can be mono-, di - or tricyclic, preferably monocyclic, and can be located at the beginning, middle and/or end of the carbon chain. Carbocycle can be aliphatic or aromatic nature. Examples are substituted biphenyls or alkyl benzenes.

Heterocyclic carbon chain with 2-30 carbon atoms means consisting of 2-30 carbon atoms chain with one or more, preferably from one to three cyclic systems in which at least one carbon atom replaced by heteroatoms, such as O, S or N. These systems can be mono-, di - or tricyclic, preferably monocyclic, and can be located at the beginning, middle and/or end uglerodnoi chain. Can be preferably 4-, 5-, 6 - or 7-membered cycles of aliphatic or aromatic nature. Examples include substituted or unsubstituted alkylpiperidines.

Aryl means an aromatic cycle or a cyclic system with 6-14, preferably 6-10, carbon atoms, such as, for example, possibly substituted, alkyl phenol or alkylation.

Halogen denotes chloride, bromide, fluoride or pseudohalogen as cyanide (nitrile).

(C1 -C6)-alkyl means a linear or branched alkyl with 1, 2, 3, 4, 5 or 6 carbon atoms, such as methyl, ethyl, isopropyl, tert-butyl and hexyl.

(C2-C6)-alkenyl means a linear or branched alkenyl 2, 3, 4, 5 or 6 carbon atoms, such as allyl, crotyl and pentenyl.

(C2-C6)-quinil means a linear or branched quinil 2, 3, 4, 5 or 6 carbon atoms, such as, for example, PROPYNYL, butynyl and pentenyl.

R1preferably means:

1. -(CH2)15CH3;

2. -(CH2)13CH(CH3)2;

3. -(CH2)11CH(OH)(CH2)3CH3;

4. -(CH2)11CH(OH)CH2CH(CH3)2;

5. -(CH2)12CH(OH)(CH2)2CH3;

6. -(CH2)13CH(OH)CH2CH3;

7. -(CH2)14CH(OH)CH3;

8. -(CH2)15CH2(HE);

9. -(CH2)16CH3or

10.0 -(CH2)13C=co2CH3;

11.0 -(CH2)12C=co2CH2CH3;

12.0 -(CH2)11C=co2CH2CH2CH3;

13.0 -(CH2)13CH3;

14.0 -(CH2)11CH(CH3)2;

15.0 -(CH2)14CH3or

16.0 -(CH2)12CH(CH3)2.

R2predpochtite is correctly means (C 1-C6)-alkyl, in particular methyl, ethyl or propyl.

Preferred compounds according to the invention are listed below:

cyclopenten And formula (II):

cyclopenten A2 of the formula (II A):

cyclopenten In formula (III):

cyclopenten With formula (IV):

cyclopenten D of the formula (V):

cyclopenten E of the formula (VI):

cyclopenten F of the formula (VII):

cyclopenten G of the formula (VIII):

cyclopenten H of the formula (IX):

cyclopenten N of the formula (X):

cyclopenten P of the formula (XI):

cyclopenten P2 of formula (XI A):

cyclopenten Q of formula (XII):

cyclopenten R of the formula (XIII):

cyclopenten R2 of the formula (XIII A):

cyclopenten's formula (XIV):

cyclopenten T of the formula (XV):

cyclopenten T2 formula (XV-A):

all their stereochemical forms and mixtures of these forms in any ratio, and their physiologically acceptable salts and chemical equivalents.

The numbering of carbon atoms for the NMR spectra in the above formulas is the following:

Cyclic system contains only two asimmetricheskii substituted atom, the carbon atom at position 3 (atom C3) and the phosphorus atom. Both atom can be R - or S - configuration. Unexpectedly, it was shown that the strain of the species Streptomyces HAG 004107, DSM 13381, and are able to produce, respectively, several stereoisomers of compounds of General formula (I), i.e. the strain synthesizes compounds in which the atoms C3 and R independently of each other can take the R - or S-configuration. Isomers with the spatial form of carbon-(3) in the R-configuration and the phosphorus atom in the S-configuration are found in cultures of Streptomyces species HAG 004107, DSM 13381, more. Formula (IA):

Along with them, however, are also formed cyclooctene with other configurations, as (R,R), (S,S) or (S,R), which unexpectedly also have significant, any abscopal the lipase activity.

The compound of formula (I) or a physiologically acceptable salt, or its chemical equivalent of get the fact that a microorganism of the species Streptomyces HAG 004107, DSM 13381, or one of it in the variants or mutants fermented in suitable conditions in a culture medium until the accumulation in the culture medium one or more compounds of the formula (I) and then isolated from the culture medium and, if necessary, transferred to the chemical equivalents and physiologically acceptable salts.

Proposed according to the invention cyclooctene can be produced views of Actinomycetales, preferably in the form of Streptomyces HAG 004107, DSM 13381. View Streptomyces HAG 004107, DSM 13381, has colored dentin mycelium (RAL 1014) has a characteristic streptomycete conidiophore.

The selected product is deposited in the German collection of microorganisms and cell cultures GmbH, Mascheroder Weg 1B, D-38124 Braunschweig, Germany, under the rules of the Budapest Treaty, on March 16, 2000, under the following number: Streptomyces species HAG 004107, DSM 13381.

Instead of the strain of the species Streptomyces HAG 004107, DSM 13381, you can use its mutants and variants that produce one or more proposed according to the invention of cyclooctanol. Such mutants can be obtained in itself known, when the use of physical means, for example by irradiation with ultraviolet or x-rays, or chemical mutagens, such as ethylmethanesulfonate (EMS), 2-hydroxy-4-methoxybenzophenone (MOU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

Thus, the invention relates to a method for obtaining compounds of formula (I) or its physiologically acceptable salts, characterized in that the microorganism of the species Streptomyces HAG 004107, DSM 13381, or one of its variant is in or mutants fermented in suitable conditions in a culture medium until the accumulation in the culture medium one or more compounds of General formula (I) and then isolated from the culture medium and, if necessary, transferred to the chemical equivalents and physiologically acceptable salts.

Preferably the strain of the species Streptomyces HAG 004107, DSM 13381, its mutants and/or variants are fermented in a nutrient solution (also called cultural environment) sources of carbon and nitrogen, as well as with the usual inorganic salts until the accumulation of new Cyclopentanol in culture medium, then cyclooctene isolated from the culture medium and, if necessary, divided into separate active components.

The fermentation is preferably carried out under aerobic conditions, it is particularly well at temperatures in the range from 18°to 35°and when the pH value of 6-8.

Proposed according to the invention method can be used for fermentation at laboratory scale (in volume from milliliters to liters) and in industrial scale (volume cubic meter). All the percentage data relate, unless stated otherwise, to the mass. The ratio of mixture components in the case of liquids refer to the volume, if not given other instructions.

As the preferred sources of carbon for aerobic fermentation of suitable assimilated carbohydrates and received by the recovery of sugars alcohols, such as glucose, lactose, sucrose or D-mannitol, as well as containing carbohydrates, natural products, to the to, for example, oat flakes, soy flour and malt extract. As the nitrogen-containing nutrient use amino acids, peptides and proteins and their cleavage products like gelatin or triptone, then, meat extracts, yeast extracts (Hefeextrakte), ground seeds, such as corn, wheat, beans, soybean or cotton, a residue from the distillation in obtaining alcohol, meat fodder flour, but also ammonium salts and nitrates. As inorganic salts nutrient solution may contain, for example, chlorides, carbonates, sulfates or phosphates of alkali or alkaline earth metals, iron, zinc, cobalt and manganese.

Education proposed according to the invention of cyclooctanol formulas (II)-(XVA) proceeds particularly well in culture medium, which contains about 0.1-5%, preferably 0.3 to 3%, oat flakes and trace elements. Data in percent, respectively, related to the mass of the entire culture medium.

Preferably, according to the invention the formation of cyclooctanol formulas (VIII)-(XVA) occurs in nutrient solutions which contain about 0.1-5%, preferably 0.3 to 2%, glycerol and 0.2-5%, preferably 0.5 to 3%, soy flour and 0.05-1.0 g/l, preferably 0.1 to 1.0 g/l of sodium chloride.

In the culture medium of Streptomyces species HAG 004107, DSM 13381, produces a mixture of cyclooctene is. Depending on the composition of the culture medium can change the mass percent of one or more proposed according to the invention of cyclooctanol. In addition, due to the composition of the environments, you can accelerate the synthesis of certain cyclooctanol, so that one or several of cyclooctanol not produced by the microorganism or produced in quantities below the detection limit.

Culture, preferably, contain detectable cyclopenten. Preferably formed cyclooctene a or P, or P2.

Along with cyclooctene And-T2 (compounds of formulas (II)-(XVA)) in the culture medium of the species Streptomyces HAG 004107, DSM 13381, produces other related compounds, which differ modified residues R1and R2in the presented formulas (II)-(XVA) connections. In smaller quantities find cyclooctene, which have a shorter or more branched residue R1. In cultures of Streptomyces HAG 004107, DSM 13381, also find products of oxidation (hydroxylation) of these additional components.

Cultivation of the microorganism is aerobic, for example, submerged by shaking or stirring in shake flasks or fermenters, if necessary, by passing air or oxygen. Cultivation can be done in the temperature range of the sample is about 18° With up to 35°C, preferably at about 25-32°With, especially when 26-30°C. the range of values of pH should be 6 to 8, preferably 6.5 to about 7.8. The microorganism is cultivated under these conditions, in General during the period of time from 24 hours up to 300 hours, preferably 30-90 hours.

Preferably cultivated in several stages, i.e. first in the liquid culture medium receive one or more preculture, which are then subcultured directly into the production culture medium (primary culture), for example, in a volume ratio of 1:10. Preculture receive, for example, the fact that the mycelium subcultured in nutrient solution and cultured for approximately 36-120 hours, preferably lasts for 48-96 hours. The mycelium can be obtained, for example, the fact that the strain is cultured for about 3-40 days, preferably 4-10 days on solid or liquid nutrient medium, for example saltatoria agar or agar oat flakes.

Over the course of the fermentation can be monitored by pH values of the cultures or the volume of the mycelium, as well as by chromatographic methods, such as thin layer chromatography or high performance liquid chromatograhy (HPLC), or by testing biological activity. Cyclooctene according to the invention are contained in the mycelium, to a lesser extent also in the cult of the Central fluid. The following allocation method clears proposed according to the invention of cyclooctanol, preferably for treatment of cyclooctene and R.

Selection, respectively, cleanup proposed according to the invention of cyclooctanol of culture medium carried out by known methods with respect to chemical, physical and biological properties of natural substances. To determine the concentration cyclooctene in culture medium or in the individual stages of the selection, you can use thin-layer chromatography, for example on silica gel using mixtures of dichloromethane and ethyl acetate or chloroform and methanol (for example, in the quantitative ratio of 98:1) as solvents, or HPLC. Detection in the case of separation by thin-layer chromatography can be performed, for example, by using a coloring reagent, as phosphomolybdenum acid or vapours of iodine, and the number of formed substances, it is advisable compared to the reference solution.

To highlight proposed according to the invention of cyclooctanol first separate the mycelium from the culture medium by conventional means and then cyclooctene extracted from the cell mass by using, if necessary, miscible with water and organic solvent. The organic phase solvent contains cyclopenten is according to the invention, if necessary, it was concentrated in vacuo and purified further as described below.

The culture fluid, if necessary, combined with the concentrate of the extract of the mycelium and extracted using suitable, is not miscible with water, an organic solvent, for example using n-butanol or ethyl acetate. Separated then the organic phase, if necessary, concentrated in vacuo and dissolved in 1/30 of the original volume of the mixture of water and methanol.

Further purification of one or more proposed according to the invention of cyclooctanol carried out by chromatography on suitable materials, preferably, for example, molecular sieves, in a conventional stationary phases, such as silica gel, alumina, ion exchangers or adsorbent resins, respectively, reversed phase (RP). Using these chromatographic methods cyclooctene share. Chromatography of cyclooctanol carried out using organic solvents or mixtures of aqueous and organic solutions.

Under mixtures of aqueous or organic solutions understand all miscible with water and organic solvents, preferably methanol, propanol and acetonitrile, at a concentration of 10-100% solvent, preferably 60-90% of the solvent, or all buffered aqueous solutions, to the which are mixed with organic solvents. Used buffers are as specified above.

The separation of cyclooctanol on the basis of their different polarity is carried out using chromatography with reversed phases, for example, MCI®(resin-adsorbent by Mitsubishi, Japan) or Amberlite XAD®(TOSOHAAS), on the other hydrophobic materials, such as, for example, on the phase RP-8 or RP-18. In addition, the separation can be performed using chromatography on a conventional stationary phases, for example, on silica gel, aluminum oxide and the like.

Chromatography of cyclooctanol carried out using a buffered or acidified aqueous solutions or mixtures of aqueous solutions with alcohols or other, miscible with water and organic solvents. As the organic solvent preferably used propanol and acetonitrile.

Under buffered or acidified aqueous solutions, see, for example, water, phosphate buffer, ammonium acetate, citrate buffer at a concentration of from 1 mm to 0.5 M, as well as formic acid, acetic acid, triperoxonane acid or all commercially available well-known specialist acid preferably in a concentration of from 0.01 to 3%, in particular 0.1 percent.

Chromatographic using a gradient that starts with a 100% aqueous buffer and ends with 100% of solvent, preferably using linear the initial gradient from 50% to 100% propan-2-ol or acetonitrile.

Alternative you can also gel chromatography or chromatography on hydrophobic phases.

Gel chromatography is carried out on polyacrylamide or copolymer gels, such as Biogel-P 2®(company Biorad), Fractogel TSK HW 40®(Merck, Germany, or Toso Haas, USA) or Sephadex®(firm Pharmacia, Uppsala, Sweden).

The sequence of the above chromatography may vary.

Further very effective stage of purification of Cyclopentanol is crystallization. Cyclooctene crystallized from solutions in organic solvents and mixtures of water with organic solvents. The crystallization is carried out in itself known, for example, by concentration or cooling of saturated solutions cyclooctene.

Proposed according to the invention cyclooctene are in solid or liquid state and is stable in solutions in the field of pH 4 to 8, in particular 5 to 7, and thus they can be administered in conventional herbal composition.

The invention relates also to the chemical equivalents of the compounds of formula (I), which have a slight chemical difference, therefore, have the same efficiency or in mild conditions can turn into a proposed according to the invention the connection. To these equivalents include nab, the emer, simple and complex esters, as well as the oxidation products, recovery and hydrogenation proposed according to the invention compounds.

Simple and complex esters as derivatives, the products of oxidation, hydrogenation, and recovery can be obtained are described in the literature of ways, as, for example, in "Advanced Organic Synthesis", fourth edition, J. March, John Wiley and Sons, 1992.

The present invention relates to all stereoisomeric forms of the compounds of formulas (I)-(XV-A). All asymmetric centers contained in the compounds of formulas (I)-(XV A), independently of one another can have the S-configuration or R-configuration. The invention includes all possible enantiomers and diastereoisomers, and also mixtures of two or more stereoisomeric forms, for example mixtures of enantiomers and/or diastereomers in any ratio. The object of the invention is, therefore, the enantiomers in the form of pure enantiomers in the form of levogyrate, and programada antipodes R - and S-configurations, in the form of racemates and in the form of mixtures of both enantiomers in any ratio. In the case of CIS/TRANS-isomerism object of the invention are CIS-form and TRANS form and mixtures of these forms in any ratio.

Because of their valuable pharmacological properties proposed according to the invention compounds suitable for use in quality is the firmness of the medicinal products in human medicine and/or animal. They inhibit lipase and possess favorable properties for the treatment of metabolic diseases, the cause of which is the violation of lipid metabolism. Proposed according to the invention compounds of General formula (I) possess an unexpected inhibitory effect on gormonchuvstvitelnoy the lipase, HSL, an allosteric enzyme in adipocytes, which is inhibited by insulin and is responsible for the breakdown of fats in fat cells and thus for the transfer of the fatty components in the bloodstream. Inhibition of this enzyme corresponds, therefore, insulin action proposed according to the invention compounds, which ultimately leads to the reduction of free fatty acids in the blood and blood sugar. Therefore, they can be used in case of deviation from normal metabolism, such as, for example, in the case of non-insulin dependent diabetes mellitus, diabetic syndrome in the case of direct damage to the pancreas.

The invention relates, therefore, to pharmaceutical compositions that contain one or more proposed according to the invention of cyclooctene and/or cash equivalents. Preferred is the use of in mixture with suitable excipients or carriers. As the carrier in the case of a person can use the -- all pharmacologically acceptable carriers and/or excipients.

The invention relates further to a method for proposed according to the invention medicines, characterized in that at least one of the suggested according to the invention compounds together with a pharmaceutically suitable and physiologically acceptable carrier and, if necessary, other suitable biologically active substances, additives or auxiliary substances brought to a suitable form of application.

Proposed according to the invention the drug is injected in General, orally, locally or parenterally, however, also possible in principle rectal application.

Suitable solid or liquid galenovye forms of the compositions are, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, aerosols, drops or injectable solutions in ampoule form and also preparations with prolonged release of biologically active ingredients, which are used typically the carriers and additives and/or auxiliary means as porofor, binder, means for coating, means for swelling, which imparts lubricity (tablets) of the substance or lubricants, flavorings, sweeteners or contributing to the dissolved substances. As commonly used media or in pologitelnui substances should be called, for example, magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols.

If necessary single dose for oral administration can be microencapsulate to slow down or extend the time of release, as, for example, by coating or the introduction of biologically active substances in the form of particles in a suitable polymers, waxes or the like.

The pharmaceutical preparations are preferably prepared and administered as single doses, each dose contains as an active ingredient a certain amount of one or more of cyclooctanol according to the invention and/or their chemical derivatives. In the case of a solid single doses as tablets, capsules and suppositories, this quantity may be up to about 200 mg, preferably, however, about 0.1-100 mg, and in the case of injection solutions in ampoule form up to about 200 mg, preferably, however, about 0.1-100 mg per day.

Enter daily dose depends on body weight, age, sex and condition of the patient. Depending on the circumstances, however, you can also enter a higher or Beletskii daily dose. The introduction of the daily dose can be achieved by one-time injection in the form of a unit dose, or as multiple smaller doses, as well as by repeated administration of divided doses at certain intervals of time.

The invention relates also to pharmaceutical compositions that contain one or more proposed according to the invention of cyclooctene and/or their chemical derivatives. Preferred is the use of in mixture with suitable excipients or carriers. As the carrier in the case of a person can use all pharmacologically acceptable carriers and/or excipients.

The action proposed according to the invention compounds of formula (I) tested according to the following enzymatic test system:

Receiving enzyme:

Obtaining partially purified HSL:

Isolated fat cells of rats derived from adipose tissue of the epididymis untreated male rats (Wistar, 220-250 g) by treatment with collagenase according to published methods (e.g., S. Nilsson, and other, Anal. Biochem.,158, 399-407 (1986); G. Fredrikson and others, J. Biol. Chem.,256, 6311-6320 (1981); H. Tornquist, etc., J. Biol. Chem.,251, 813-819 (1976)). Fat cells from 10 rats washed three times by flotation using each time 50 ml of buffer for homogenization (25 ml Tris/HCl, pH=7,4; 0.25 M sucrose; 1 mm EDTU, 1 mm di is iatrical, 10 µg/ml leupeptin, 10 μg/ml antipain, 20 μg/ml of pepstatin) and, finally, treated with 10 ml of buffer for homogenization. The fat cells are homogenized in a Teflon cups in a homogenizer (Braun-Melsungen) by 10 moves at a speed of 1500 rpm and at a temperature of 15°s Homogenized centrifuged tubes Sorvall SM-24, 5000 rpm, 10 minutes, 4°). Select the supernatant, located between the upper fatty layer and the sediment after centrifugation, and repeat the centrifugation. Derived from supernatant centrifuged again (tubes Sorvall SM-24, 20000 rpm, 45 minutes, 4°). Take the supernatant and mixed with 1 g heparinase (Pharmacia-Biotech, CL-6B, washed 5 times with 25 mm Tris/HCl, pH to 7.4; 150 mm NaCl). After incubation for 60 minutes at a temperature of 4° (in intervals of 15 minutes shaken), the mixture centrifuged tubes Sorvall SM-24, 3000 rpm, 10 minutes, 4°). The supernatant was adjusted to pH=5,2 by adding glacial acetic acid and incubated for 30 minutes at a temperature of 4°C. Precipitates obtained by centrifugation (Sorvall SS34, 12000 rpm, 10 minutes, 4°C), harvested and suspended in 2.5 ml of 20 mm Tris/HCl, pH=7,0; 1 mm EDTU, 65 mm NaCl, 13% sucrose, 1 mm dithiothreitol, 10 μg/ml leupeptin/pepstatin/antipain. Suspension cialiswhat over night at a temperature of 4°about the Yves 25 mm Tris/HCl, pH=7,4; 50% glycerol, 1 mm dithiothreitol, 10 μg/ml leupeptin, pepstatin, antipain, and then applied to a column of hydroxyapatite (0.1 g in 1 ml of suspension, equilibrated with 10 mm potassium phosphate, pH=7,0; 30% glycerol, 1 mm dithiothreitol). The column is washed with four volumes of buffer for equilibration at a flow rate of 20-30 ml/HR. HSL elute with one volume of buffer used for equilibration, which contains 0.5 M potassium phosphate, then cialiswhat (see above) and concentrate 5-10-fold by ultrafiltration (Amicon filter Diaflo PM 10) at a temperature of 4°C. Partially purified HSL can be stored for 4-6 weeks at -70°C.

Analysis:

To prepare substrate mix 25-50 µci of [3H]-triological (in toluene), 6.8 mmol of unlabeled triological and 0.6 mg of phospholipids (phosphatidylcholine/phosphatidylinositol in the ratio 3:1 weight/volume), dried in an atmosphere of nitrogen and then dissolved in 2 ml of 0.1 M CUi(pH=7,0) by ultrasonic treatment (Branson 250, microjava load, volume 1-2, 2×1 min with 1 min interval). After adding 1 ml KPiand a new ultrasonic treatment (4×30 sec on ice at intervals of 30 seconds) injected 1 ml of 20%bovine serum albumin (KPi) (final concentration of triological is 1.7 mm). For the reaction of 100 μl of substrate solution piped the th added to 100 μl of a solution HSL (HSL receive as described above, diluted in 20 mm CUipH=7,0; 1 mm EDTU, 1 mm dithiothreitol, 0.02% of bovine serum albumin, 20 μg/ml of pepstatin, 10 μg/ml leupeptin) and incubated for 30 minutes at a temperature of 37°C. After the addition of 3.25 ml of a mixture of methanol, chloroform and heptane (10:9:7) and 1.05 ml of 0.1 M2CO3, 0.1 M boric acid (pH=10,5) mix well and finally centrifuged (800 g, 20 minutes). After separation of the phases selected one equivalent of the upper phase (1 ml), and determine the radioactivity by measuring the scintillation fluid.

Evaluation:

Compounds are usually tested in four independent trains. Inhibition of the enzymatic activity of HSL at the expense of the test substance is determined by comparison with neighborhool control reaction. The calculation of the IC50(inhibiting 50% concentration) are carried out by the curve of inhibition of at least 10 concentrations of test substance. For data analysis using the software package GRAPHIT, Elsevier-BIOSOFT. In this test compounds showed the following action: cyclooctene A, P, P2 and R inhibit lipolysis in adipocytes of rats withthey inhibit human gormonchuvstvitelnoy lipase (HSL) creoleinthecity.com as substrate:HSL rats with NBD (4-chloro-7-nitrobenzo-2-oxa-1,3-diazol) as with the of strata inhibited at concentrations of 4 to 10 nmol.

Cyclooctene inhibit as gormonchuvstvitelnoy lipase (HSL)and monoacylglycerides extract from rats in submicromolar concentrations.

In the following examples the invention is illustrated hereinafter. The percentages relate to weight. The ratio of mixture components in the case of liquids refer to the volume, if not given other instructions.

Examples

Example 1

Getting glycerol culture of Streptomyces species HAG 004107, DSM 13381.

100 ml of nutrient solution (2,0% malt extract, 0.2% of yeast extract, 1.0% glucose, 0.05% of (NH4)2HPO4pH=6,0) in a sterile Erlenmeyer flask with a capacity of 300 ml produce a crop strain of the species Streptomyces HAG 004107, DSM 13381, incubated for 7 days at a temperature of 28°and speed 180 rpm on a rotating device for shaking. Then 1.5 ml of this culture is diluted with 1.5 ml of 99%glycerol and stored at -20°C.

Example 2

Getting in Erlenmeyer flask of preculture species Streptomyces HAG 004107, DSM 13381.

In a sterile Erlenmeyer flask with a capacity of 300 ml with 100 ml of the following nutrient solution: 15 g/l glucose, 15 g/l soybean flour, 5 g/l soaked corn kernels, 2 g/l caso3and 5 g/l NaCl produce a crop grown in a test tube with a slanted agar (same nutrient solution, but with 2% agar) culture or 1 ml glycerol culture (see example 1) is incubated in a device for shaking at a speed of 180 rpm and a temperature of 28° C. For seed fermenters with a capacity of 10 l and 200 l sufficiently cultivated in the course lasts for 48-96 hours submerged culture (number of seed about 10%) from the same nutrient solution.

Example 3

Getting in Erlenmeyer flask culture of the species Streptomyces HAG 004107, DSM 13381.

In a sterile Erlenmeyer flask with a capacity of 300 ml with 100 ml of the following nutrient solution:

20 g/l of oatmeal,

2.5 ml of trace element solution

produce a crop 10% of the quantity of seed from preculture (example 2) and incubated in a device for shaking at a speed of 180 rpm and a temperature of 28°C. two days Later, the culture is used to produce Cyclopentanol or seed fermentors. Trace element solution has the following composition:

3 g/l CaCl2×2 H2O;

1 g/l of citrate Fe(III);

0.2 g/l MnSO4×H2O;

0.1 g/l ZnCl2;

0.025 g/l CuSO4×5 H2O;

0.02 g/l of sodium tetraborate;

0.004 g/l CoCl2×6 H2O;

0.01 g/l of sodium molybdate.

Example 4

Getting cyclooctanol formulas (II)-(IX)

The fermenter with a capacity of 200 l with 90 l of nutrient solution operates in the following conditions:

nutrient medium:

20 g/l of oatmeal in water;

2.5 ml/l of trace elements;

pH=7,8 (before sterilization).

Nutrient solution for 30 minutes and subjected to heat sterili the purpose after cooling, 5% of the total sown with the seed material, obtained according to example 3. Trace element solution:

3 g/l CaCl2×2 H2O;

1 g/l of citrate Fe(III);

0.2 g/l MnSO4×H2O;

0.1 g/l ZnCl2;

0.025 g/l CuSO4×5 H2O;

0.02 g/l of sodium tetraborate;

0.004 g/l CoCl2×6 H2O;

0.01 g/l of sodium molybdate;

the duration of the process:72 hours;
incubation temperature:28°C;
speed stirrer:90 revolutions per minute;
aeration:6 m3of air per hour.

The fermentation is carried out without the addition of non. Maximum production reached after about 40-76 hours.

Example 5

Getting cyclooctanol formula (X)to(XV A)

The fermenter with a capacity of 200 l with filling 100 l operates in the following conditions:

nutrient medium:

5 g/l glucose;

20 g/l glycerol;

20 g/l soybean flour;

5 g/l yeast extract;

3 g/l NaCl;

2.5 ml/l of trace element solution;

pH=7,0 (before sterilization);

the duration of the process:72 hours;
incubation temperature:27°C;
speed stirrer:65 revolutions per minute;/td>
aeration:6 m3of air per hour.

The fermentation is carried out without the addition of means to suppress foaming. Maximum production reached after about 48 hours.

Example 6

The selection of the mixture of cyclooctene from the culture fluid of the species Streptomyces HAG 004107, DSM 13381.

Upon completion of the fermentation of Streptomyces species HAG 004107, DSM 13381 100 l of culture broth from the fermenter, obtained according to example 4, filtered adding about 2% auxiliary filtering means (as, for example, Celite®) and cell mass (10 l) was extracted with 40 l of methanol. Containing a biologically active substance methanol solution released from the mycelium by filtration and concentrated in vacuo. Concentrate contribute to the prepared 7 l®MCI GEL, SNRR-column. Elute using a gradient from water to propane-2-ol. Flow from the column (20 l per hour) collect fractions (10 l) containing cyclobutene fraction (19-21), respectively, concentrated in vacuo. Fractions analyzed by HPLC (see example 7). Fraction 19 contains cyclooctene a-E, as well as their isomers; fraction contains 20 cyclopenten F and its isomers; fraction 21 contains inhibitors: cyclopenten N, P, P2, Q, R, S and T, as well as their isomers.

Example 7

Analysis of cyclooctene by HPLC

Anal is C by high performance liquid chromatography (HPLC) of cyclooctene carried out in the device, HP 1100 ®with column YMC-Pack Pro C18®[AS-303, 250×4.6 mm, S - 5 μm, 120]. The flow is 1 ml/min, column temperature 40°C. Use the gradient from 0.05% triperoxonane acid to acetonitrile. 100% acetonitrile as the eluting means reach after 11 minutes and then elute without further changes (isocratically) using this solvent. Detection is carried out by measuring the absorption in the ultraviolet region of the spectrum at 210 nm. Using this method, you determine get the following retention times for cyclobutanol:

titleposted:12.7 minutes;
cyclopenten A2:12.6 minutes;
cyclopenten F:13.2 mins;
cyclopenten N:15.9 minutes;
cyclopenten R:17.7 minutes;
cyclopenten P2:the 17.3 minutes;
cyclopenten Q:of 18.3 minutes;
cyclopenten R:16.7 minutes;
cyclopenten R2:16,4 minutes;
cyclopenten S:18.5 minutes;
cyclopenten T:19,1 minutes and
cyclopenten T2:18.7 minutes.

P the emer 8

Getting clean cyclobutanol As and A2

Fraction 19, obtained according to example 6, was concentrated in vacuo and 1 g of the concentrate dissolved in a mixture of water and methanol (1:1 ratio), making the column Nucleoprep 100-5 C18AB®(21×250 mm). Elute with a gradient from 50% acetonitrile in 0.01%triperoxonane acid to 100% acetonitrile. The flow rate of 50 ml per minute. Flow from the column is monitored by measuring light absorption at 210 nm, and by testing the properties of inhibiting lipase. Selected fractions in 60 ml. In fractions 34 and 35 is cyclopenten And, in fractions 41-44 is cyclopenten A2. These fractions, respectively, are pooled, concentrated in vacuo and consistently separated on a column of SP 250/10 Nucleosil 100-5 C18HD®. As the gradient was chosen from 50% to 66% acetonitrile in 0.01%triperoxonane acid; the pH value of the solutions was set equal to 4.0, using drops of ammonium hydroxide solution. The fractions containing the pure compounds, respectively, were combined and dried by freezing. One of them received a 5.4 mg of pure cyclooctene And in the form of a waxy substance and 3 mg cyclooctene A2 in the form of oil.

Example 9

Description cyclooctene And

Appearance: soluble in oxygenated organic solvents, however, only is slightly soluble in water and petroleum ether, neutral, colorless, waxy substance.

Maximum absorption in the ultraviolet region of the spectrum: 228 nm in methanol.

IR bands: 1752 and 1671 cm-1.

By mass spectrometry with ionization by fast atom bombardment (FAB-mass spectrometry) high resolution when using matrix nitrobenzyloxy alcohol/LiCl get the following molecular weight: 467,2757 relative mass units, which corresponds to the total formula for cyclooctene A-Li:23H41About7PLi. From here get the total formula for cyclooctene:23H41About7P, molecular weight: 460. By mass spectrometry with ionization by electronic sputtering according to the method of ionization with the formation of positive ions (positive ESI) found peak at the value 461 relative mass units, respectively (M+N)+; moreover, the characteristic peak at 221 relative mass units, respectively, With7H10About6R. According to the method of mass spectrometry with negative ESI find peaks at 459 relative mass units (M-N)-; 337 relative mass units (C16H34About5P) and 219 relative mass units (C7H8About6P). To determine the position of an alcohol group derivatized using N-methyl-N-trimethylsilyl-Tr is forcefed and the sample is examined using mass spectrometry with ionization by electronic sputtering. Formed trimethylsilyl derived:

mass 554 relative mass units. The position Siciliano hydroxyl group indicate intense ions at 497 relative mass units (α-splitting) and 159 relative mass units (α-splitting). NMR signals: see table 1.

TABLE 1
1The h and13C-chemical shifts cyclooctene And in methanol-d4at 300 K
1H13C
1-171,08 (1,4 Hz)b)
2-114,61 (3,4 Hz)b)
3a 3.8740,75
44,46/3,8666,04
5or 4.31/4,25at 69,39 (6,0 Hz)b)
6-161,47 (8,0 Hz)b)
72,4017,89 (4,6 Hz)b)
1'4,2571,61 (6,6 Hz)b)
2'1,7331,16 (6,6 Hz)b)
3'1,4126,39
n'3,4972,45
n±1of 1.46 and 1.3338,44, 38,15
4'-14' (a)1,37-1,2630,85-30,58
15'of 1.3423,84
16'0,9114,43
a): in addition to n and n±1;

b): in parentheses are the constant interaction13C/31R.

Example 10

Description cyclooctene

Cyclopenten To describe in example 9 in the case cyclooctene And allocate by repetition chromatographic stages and characterized as in example 9.

Appearance: soluble in oxygenated organic solvents but only slightly soluble in water and petroleum ether, neutral, colorless, waxy substance.

Maximum absorption in the ultraviolet region of the spectrum: 228 nm in methanol.

By mass spectrometry with ionization by electronic sputtering according to the method of ionization with the formation of positive ions (positive ESI) found peak at the value 461 relative mass units, respectively (M+N)+; moreover, the characteristic peak at 221 relative mass units, corresponding With7H10About6R. According to the method of mass spectrometry with negative ESI find peaks at 459 classifies the selected mass units (M-N) -; 337 relative mass units (C16H34About5P) and 219 relative mass units (C7H8About6P). To determine the position of an alcohol group derivatized using N-methyl-N-trimethylsilyltrifluoroacetamide and the sample is examined using mass spectrometry with ionization by electronic sputtering. Formed trimethylsilyl derived mass 554 relative mass units:

The position Siciliano hydroxyl group indicate intense ions at 511 relative mass units (α-splitting) and 145 relative mass units (α-splitting).

Total formula cyclooctene In:23H41About7R; malcolma weight: 460.

Example 11

Description cyclooctene

Cyclopenten To describe as in example 8 in the case cyclooctene And allocate by repetition chromatographic stages and characterized as in example 9.

Appearance: soluble in oxygenated organic solvents but only slightly soluble in water and petroleum ether, neutral, colorless, waxy substance.

Maximum absorption in the ultraviolet region of the spectrum: 228 nm in methanol.

By mass spectrometry with ionization by electronic sputtering agreement is but the way ionization with the formation of positive ions (positive ESI) found peak at the value 461 relative mass units, the corresponding (M+N)+; moreover, the characteristic peak at 221 relative mass units, respectively, With7H10About6R. According to the method of mass spectrometry with negative ESI find peaks at 459 relative mass units (M-N)-; 337 relative mass units (C16H34About5P) and 219 relative mass units (C7H8About6P). To determine the position of an alcohol group derivatized using N-methyl-N-trimethylsilyltrifluoroacetamide and the sample is examined using mass spectrometry with ionization by electronic sputtering. Formed trimethylsilyl derived mass 554 relative mass units:

The position Siciliano hydroxyl group indicate intense ions at 525 relative mass units (α-splitting) and 131 relative mass units (α-splitting).

Total formula cyclooctene With:23H41About7R; molecular weight: 460.

Example 12

Description cyclooctene F

Fraction 20, obtained according to example 6, separated as described in example 8, and cycloocten F emit by repetition chromatographic stages and characterized as in example 9.

Retention time: 13.2 minutes.

Appearance: soluble to saradapitha organic solvents, however, only slightly soluble in water and petroleum ether, neutral, colorless, waxy substance.

Maximum absorption in the ultraviolet region of the spectrum: 228 nm in methanol.

By mass spectrometry with ionization by electronic sputtering according to the method of ionization with the formation of positive ions (positive ESI) found peak at the value 459 relative mass units, respectively (M+N)+; moreover, the characteristic peak at 221 relative mass units, respectively, With7H10About6R. According to the method of mass spectrometry with negative ESI find peaks when 457,6 relative mass units (M-N)-; 336 relative mass units (C16H32About5P) and 219 relative mass units (C7H8About6R.).

Total formula cyclooctene F: C23H39About7R; molecular weight: 458.

Example 13

Description cyclooctene P

Fraction 21, obtained according to examples 5 and 6, separated as described in example 8, and cycloocten P allocate (210 mg) by repetition chromatographic stages and characterized as in example 9. By dissolving 210 mg cyclooctene P in 3 ml of propan-2-ol and 13 ml of acetonitrile and additives 8 ml of water crystallized cyclopenten R. After filtering off, washing Ho is one acetonitrile get 135 mg cyclooctene. TPL 58-59°C.

Retention time: 17.7 minutes.

Appearance: soluble in oxygenated organic solvents but only slightly soluble in water and petroleum ether, neutral, colorless, waxy substance.

Maximum absorption in the ultraviolet region of the spectrum: 228 nm in methanol.

IR-bands: 2917, 2852, 1753, 1671, 1471, 1214, 996 and 832 cm-1.

By mass spectrometry with ionization by fast atom bombardment (FAB-mass spectrometry) high resolution when using matrix-based nitrobenzyl alcohol get the following molecular weight: 445,2717 relative mass units, respectively (M+N)+for cyclooctene R23H42About6R. Hence get the total formula for cyclooctene P: C23H41About6R; molecular weight: 444. By mass spectrometry with ionization by electronic sputtering according to the method of ionization with the formation of positive ions (positive ESI) found peak at the value 445 relative mass units, respectively (M+N)+; moreover, the characteristic peak at 221 relative mass units, respectively, With7H10About6R. According to the method of mass spectrometry with negative ESI find peaks at 443 relative mass units (M-N)-; 321 relative to the nutrient mass units (C 16H34About4P) and 219 relative mass units (C7H8About6R.).

Data of NMR are presented in table 2.

TABLE 2
Chemical shifts cyclooctene P in MeOD at 300 K
1H13C
1-171,08
2-114,60 (3,0 Hz)a)
3a 3.8740,74
44,47/3,8566,05
54,30/4,2569,40 (6,0 Hz)a)
6-161,47 (8,0 Hz)a)
72,4017,90 (4,6 Hz)a)
1'4,2471,62 (6,9 Hz)a)
2'1,7331,16 (6,3 Hz)a)
3'1,4126,38
4'-13'1,34-1,2930,76-30,11
14'1,34-1,2933,07
15'1,3123,72
16'0,8914,42
a): in brackets the decree is in constant interaction 13C/31R.

Example 14

Description cyclooctene P2

Fraction 21, obtained according to examples 5 and 6, separated as described in example 8, and cycloocten P2 allocate (130 mg) by repetition chromatographic stages and characterized as in example 9.

Retention time: 17.1 minutes.

Appearance: soluble in oxygenated organic solvents but only slightly soluble in water and petroleum ether, neutral, colorless, oily substance.

Maximum absorption in the ultraviolet region of the spectrum: 228 nm in methanol.

By mass spectrometry with ionization by fast atom bombardment (FAB-mass spectrometry) high resolution when using matrix-based nitrobenzyl alcohol get the following molecular weight: 445,2721 relative mass units, respectively (M+N)+for cyclooctene R23H42About6R. Hence get the total formula for cyclooctene P2: C23H41About6P, molecular weight: 444. By mass spectrometry with ionization by electronic sputtering according to the method of ionization with the formation of positive ions (positive ESI) found peak at the value 445 relative mass units, respectively (M+N)+; moreover, the characteristic peak of priznanie 221 relative mass units, respectively With7H10About6R. According to the method of mass spectrometry with negative ESI find peaks at 443 relative mass units (M-N)-; 321 relative mass units (C16H34About4P) and 219 relative mass units (C7H8About6R.).

Data of NMR for cyclooctene P2 are presented in table 3.

TABLE 3
Chemical shifts cyclooctene P2 in CD3OD at 300 K
1H13C
1-171,05
2-114,60 (3,2 Hz)a)
3a 3.8740,74
44,46/3,8566,02
54,30/4,2569,38 (6,0 Hz)a)
6-161,46 (8,0 Hz)a)
72,4017,90 (4,6 Hz)a)
1'4,2471,60 (6,9 Hz)a)
2'1,7331,16 (6,3 Hz)a)
3'1,4126,39
4'-11'1,34-1,29 31,04-30,11
12'1,2928,53
13'1,1740,25
14'1,5229,15
15',16'0,8723,04
a): in parentheses are the constant interaction13C/31R.

Example 15

Fabrication and characterization of cyclooctene N

Fraction 21, obtained according to examples 5 and 6, separated as described in example 8, and cycloocten N allocate (2 mg) by repetition chromatographic stages and characterized as in example 9.

Retention time: 15.9 minutes.

Appearance: soluble in oxygenated organic solvents but only slightly soluble in water and petroleum ether, neutral, colorless, oily substance.

Maximum absorption in the ultraviolet region of the spectrum: 228 nm in methanol.

By mass spectrometry high resolution in terms of ionization by fast atom bombardment (FAB -) see quasimolecular ion (M+N) when 417,2405 relative mass units, which corresponds to the total formula C21H38About6P (theoretically: 417,2406). The characteristic fragment according to the method of ionization by electron spray with positive education is ones: 221 relative mass units.

TABLE 4
Chemical shifts cyclooctene N in MeOD at 300 K
1H13C
1-171,07
2-114,60 (3,1 Hz)a)
3a 3.8740,74
44,45/3,8466,03
54,30/4,25at 69,39 (5,9 Hz)a)
6-161,47 (8,0 Hz)a)
72,4017,90 (4,9 Hz)a)
1'4,2471,60 (6,6 Hz)a)

2'1,7331,16 (6,2 Hz)a)
3'1,4126,38
4'-11'1,35-1,2630,76-30,11
12'1,35-1,2633,06
13'1,3123,72
14'0,8914,41
a): in parentheses are the constant interaction13C/31R.

Example 16

Fabrication and characterization of titlepost is at R

Fraction 21, obtained according to examples 5 and 6, separated as described in example 8, and cycloocten R emit (8 mg) by repetition chromatographic stages and characterized as in example 9.

Retention time: 16.7 minutes.

Appearance: soluble in oxygenated organic solvents but only slightly soluble in water and petroleum ether, neutral, colorless, crystalline substance.

Maximum absorption in the ultraviolet region of the spectrum: 228 nm in methanol.

By mass spectrometry high resolution in terms of ionization by fast atom bombardment (FAB -) see quasimolecular ion (M+N) when 431,2561 relative mass units, which corresponds to the total formula C22H40About6P (theoretically: 431,2562). The characteristic fragment according to the method of ionization by electron spray with the formation of positive ions: 221 relative mass units.

3
TABLE 5
Chemical shifts cyclooctene R in MeOD at 300 K
1H13C
1-171,06
2-114,58 (3,2 Hz)a)
a 3.8740,75
44,45/3,8566,04
54,30/4,2569,40 (6,0 Hz)a)
6-161,48 (8,0 Hz)a)
72,4017,90 (5,0 Hz)a)
1'4,2471,61 (7,0 Hz)a)
2'1,7331,16 (6,2 Hz)a)
3'1,4126,38
4'-12'1,37-1,2530,74-30,10
13'1,1733,06
14'1,3023,71
15'0,8914,40
a): in parentheses are the constant interaction13C/31R.

Example 17

Fabrication and characterization of cyclooctene R2

Fraction 21, obtained according to examples 5 and 6, separated as described in example 8, and cycloocten R2 release (8 mg) by repetition chromatographic stages and characterized as in example 9.

Retention time: 16,4 minutes.

Appearance: soluble in oxygenated organic solvents but only slightly soluble in water and petroleum ether, neutral, colorless, oil is NISTO substance.

Maximum absorption in the ultraviolet region of the spectrum: 228 nm in methanol.

By mass spectrometry high resolution in terms of ionization by fast atom bombardment (FAB -) see quasimolecular ion (M+N) when 431,2564 relative mass units, which corresponds to the total formula C22H40About6P (theoretically: 431,2562). The characteristic fragment according to the method of ionization by electron spray with the formation of positive ions: 221 relative mass units.

31,16 (6,6 Hz)a)
TABLE 6
Chemical shifts cyclooctene R2 in MeOD at 300 K
1H13C
1-171,06 (1,7 Hz)a)
2-114,58 (3,1 Hz)a)
3a 3.8740,75
44,46/3,8566,03
54,30/4,25at 69,39 (6,0 Hz)a)
6-161,47 (8,0 Hz)a)
72,4017,90 (4,9 Hz)a)
1'4,2471,60 (6,9 Hz)a)
2'1,73
3'1,4126,38
4'-10'1,37-1,2531,02-30,10
11'1,2928,51
12'1,1640,24

13'1,5129,15
14', 15'0,8723,02
a): in parentheses are the constant interaction13C/31R.

Example 18

Fabrication and characterization of cyclooctene S

Fraction 21, obtained according to examples 5 and 6, separated as described in example 8, and cycloocten S allocate (0.7 mg) by repetition chromatographic stages and characterized as in example 9.

Retention time: 18.5 minutes.

Appearance: soluble in oxygenated organic solvents but only slightly soluble in water and petroleum ether, neutral, colorless, solid substance.

Maximum absorption in the ultraviolet region of the spectrum: 228 nm in methanol.

By mass spectrometry high resolution in terms of ionization by fast atom bombardment (FAB -) see quasimolecular ion (M+N) when 459,2883 relative mass units, which is suitable for the em total formula C 24H44About6P (theoretically: 459,2575). The characteristic fragment according to the method of ionization by electron spray with the formation of positive ions: 235 relative mass units.

16'
TABLE 7
Chemical shifts cyclooctene S in MeOD at 300 K
1H13C
1-170,87 (1,4 Hz)a)
2-113,66 (3,1 Hz)a)
33,8540,77
44,45/3,8566,04
54,29/4,2469,17 (6,0 Hz)a)
6-165,80 (8,3 Hz)a)
72,98/2,8225,05 (4,6 Hz)a)
81,1610,86
1'4,2571,57 (6,9 Hz)a)
2'1,7431,19 (6,3 Hz)a)
3'1,4226,41
4'-13'1,34-1,2930,76-30,11
14'1,34-1,2933,07
15'1,3123,73
0,8914,43
a): in parentheses are the constant interaction13C/31R.

Example 19

Fabrication and characterization of cyclooctene T

Fraction 21, obtained according to examples 5 and 6, separated as described in example 8, and cycloocten T allocate (5 mg) by repetition chromatographic stages and characterized as in example 9.

Retention time: 19,1 minutes.

Appearance: soluble in oxygenated organic solvents but only slightly soluble in water and petroleum ether, neutral, colorless, solid substance.

Maximum absorption in the ultraviolet region of the spectrum: 228 nm in methanol.

By mass spectrometry high resolution in terms of ionization by fast atom bombardment (FAB -) see quasimolecular ion (M+N) when 473,3030 relative mass units, which corresponds to the total formula C25H46About6P (theoretically: 473,3032). The characteristic fragment according to the method of ionization by electron spray with the formation of positive ions: 249 relative mass units.

TABLE 8
Chemical shifts cyclooctene T in MeOD at 30 K
1H13C
1-170,98 (1,7 Hz)a)
2-114,39 (3,1 Hz)a)
3a 3.8740,78
44,46/3,8566,02
54,29/4.26 deaths69,23 (5,9 Hz)a)
6-164,69 (8,7 Hz)a)
72,89/2,8333,35 (4,5 Hz)a)
81,6520,63
90,98at 13.84
1'4,2571,57 (6,6 Hz)a)
2'1,7431,18 (6,2 Hz)a)
3'1,4226,42
4'-13'1,34-1,2930,78-30,11

14'1,34-1,2933,06
15'1,3123,72
16'0,8914,42
a): in parentheses are the constant interaction13C/31R.

Example 20

Fabrication and characterization of cyclooctene T2

Fraction 21, obtained according to the SNO examples 5 and 6, share as described in example 8, and cycloocten T2 allocate (4 mg) by repetition chromatographic stages and characterized as in example 9.

Retention time: 18.7 minutes.

Appearance: soluble in oxygenated organic solvents but only slightly soluble in water and petroleum ether, neutral, colorless, solid substance.

Maximum absorption in the ultraviolet region of the spectrum: 228 nm in methanol.

By mass spectrometry high resolution in terms of ionization by fast atom bombardment (FAB -) see quasimolecular ion (M+N) when 473,3035 relative mass units, which corresponds to the total formula C25H46About6P (theoretically: 473,3032). The characteristic fragment according to the method of ionization by electron spray with the formation of positive ions: 249 relative mass units.

TABLE 9
Chemical shifts cyclooctene T2 in MeOD at 300 K
1H13C
1-170,98 (1,7 Hz)a)
2114,40 (3,1 Hz)a)
3a 3.87 40,78
44,46/3,8566,02
54,29/4,2569,23 (5,9 Hz)a)
6-164,69 (8,7 Hz)a)
72,90/2,8333,35 (4,5 Hz)a)
81,6520,63
90,98at 13.84
1'4,2471,57 (6,9 Hz)a)
2'1,7431,18 (6,2 Hz)a)
3'1,4226,42
4'-11'1,37-1,2531,03-30,11
12'1,2928,52
13'1,1740,25
14'1,5229,15
15, 16'0,87to 23.03
a): in parentheses are the constant interaction13C/31R.

Example 21

Inhibition gormonchuvstvitelnoy lipase (HSL)

Gormonchuvstvitelnoy the lipase in rats inhibited with creoleinthecity.com as substrate in the following concentrations (IC50):

titleposted:20 nmol;
cyclopenten N:450 n is ol;
cyclopenten R:30 nmol;
cyclopenten P2:40 nmol;
cyclopenten R:10 nmol;
cyclopenten R2:220 nmol;
cyclopenten S:20 nmol;
cyclopenten T:200 nmol;
cyclopenten E2:60 nmol.

1. The compound of General formula (I)

where R1means a carbon chain with 2-30 carbon atoms, which may be linear, branched, saturated, and the carbon chain may be single or twofold substituted with-IT, =O, -(C1-C6)-alkyl or

R2means (C1-C6)-alkyl;

E. means a phosphorus atom (P);

X1X2and X3independently of one another denote-O-,

all stereochemical forms and mixtures of these forms in any ratio, and its physiologically acceptable salt.

2. The compound of formula (I) or a physiologically acceptable salt according to claim 1, wherein R1means a carbon chain with 10-18 carbon atoms, which may be linear, branched, saturated, and the carbon chain is unsubstituted or one - or double the IDT replaced.

3. The compound of formula (I) or a physiologically acceptable salt according to claim 1 or 2, wherein R1means

1.0 -(CH2)15CH3;

2.0 -(CH2)13CH(CH3)2;

3.0 -(CH2)11CH(OH)(CH2)3CH2;

4.0 -(CH2)11CH(OH)CH2CH(CH3)2;

5.0 -(CH2)12CH(OH)(CH2)2CH3;

6.0 -(CH2)13CH(OH)CH2CH3;

7.0 -(CH2)14CH(OH)CH3;

8.0 -(CH2)15CH2(OH);

9.0 -(CH2)16CH3or

10.0 -(CH2)13C=co2CH3;

11.0 -(CH2)12C=co2CH2CH3;

12.0 -(CH2)11C=co2CH2CH2CH3;

13.0 -(CH2)13CH3;

14.0 -(CH2)11CH(CH3)2;

15.0 -(CH2)14CH3or

16.0 -(CH2)12CH(CH3)2.

4. The compound of formula (I) or a physiologically acceptable salt according to claims 1, 2 or 3, wherein R2means (C1-C6)-alkyl.

5. The compound of formula (I) or a physiologically acceptable salt according to claim 4, wherein R2means-CH3, -CH2CH3or-CH2CH2CH3.

6. The compound of formula (I) or a physiologically acceptable salt of the resultant or more of claims 1 to 5, obtained by fermentation of a strain of Streptomyces HAG 004107, DSM 13381 in suitable conditions in a culture medium until the accumulation in the culture medium one or more compounds of General formula (I) and subsequent separation from the culture medium and, if necessary, put in a physiologically acceptable salt.

7. The method of obtaining the compounds of formula (I) or its physiologically acceptable salts according to one or more of claims 1 to 6, characterized in that a strain of Streptomyces HAG 004107, DSM 13381 fermented in suitable conditions in a culture medium until the accumulation in the culture medium one or more compounds of the formula (I) and then isolated from the culture medium and, if necessary, transferred to a physiologically acceptable salt.

8. The method according to claim 7, characterized in that the fermentation is carried out in aerobic conditions at a temperature of from 18 to 35°and when the pH value of 6-8.

9. The compound of formula (I) or a physiologically acceptable salt of one or more of claims 1 to 6, suitable as pharmaceuticals for the inhibition of lipases.

10. The pharmaceutical composition intended for the inhibition hormonecontaining lipase (HSL), containing at least one compound of formula (I) or a physiologically acceptable salt of one or more of claims 1 to 6.

11. A method of obtaining a pharmaceutical is oppozitsii of claim 10, characterized in that at least one compound of formula (I) or an acceptable salt on one or more of claims 1 to 6, together with suitable excipients and/or carriers brought to a suitable form of application.

12. The strain Streptomyces HAG 004107, DSM 13381, which is the producer of cyclooctanol.



 

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