SK15602002A3 - Cyclipostins, a method for their production and the use of the same - Google Patents

Abstract

The invention relates to compounds of the general formula (I), wherein R<1>, R<2>, E, X1, X2 and X3 have the definitions in this application, obtained by the cultivation of the Streptomyces species HAG 004107 (DSM 13381) and to the physiologically compatible salts and chemical equivalents of said compounds. The invention also relates to a method for producing the cyclipostins and their physiologically compatible salts and chemical equivalents as medicaments, in particular as inhibitors of lipases, and to pharmaceutical preparations containing cyclipostin or a physiologically compatible salt or equivalent thereof.

Description

Cyclipostine, a process for its preparation and a pharmaceutical composition containing it

Technical field

The invention relates to a novel compound, called cyclipostin, obtainable by culturing Streptomyces HAG 004107 (DSM 13381) and its physiologically acceptable salts and chemical equivalents thereof. The invention further relates to a process for the production of cyclipostine by HAG 004107 (DSM 13381), to a process for the use of cyclipostine and its physiologically acceptable salts and chemical equivalents as medicaments, in particular as lipase inhibitors, as well as pharmaceutical compositions comprising cyclipostin or physiologically acceptable salts thereof.

BACKGROUND OF THE INVENTION

The disease that can be particularly advantageously treated with lipase inhibitors is diabetes mellitus. Daabetes mellitus is a disease characterized by increased blood sugar levels resulting from impaired metabolism. The metabolism disorder is caused by a deficiency of insulin or by insufficient action of insulin. The lack of action of insulin leads to under-utilization of blood glucose by body cells. This, as well as the new formation of glucose from proteins (gluconeogenesis), increases blood glucose levels. In addition, the reduced action of insulin leads to an increase in lipolysis in adipose tissue by means of the insulin antagonist glucagon hormone, thereby increasing the fatty acid concentration in the blood. Ketoacidosis, i.e., increased formation of ketone compounds (acetic acid, β-hydroxybutyric acid, acetone) occurs. Under acute conditions ¹ , poor biochemical regulation of life is dangerous and, if not treated, leads to diabetic coma and ultimately to rapid death. Diabetes is a common metabolic disorder in humans, and it is estimated that up to 3% of the population show diabetic or pre-diabetic disposition and thus an acute threat. Thus, there is a great need for means to treat diabetes mellitus.

Treatment of diabetes is carried out with the help of insulin doses. insulin-independent diabetes mellitus (NIDDM), or type II diabetes, are mainly administered by the sulfonylureas. The principle of action of sulfonylureas is based on an increase in insulin secretion by β-cells in the pancreas, which balances the amount of insulin or insulin resistance. However, insulin must also be given in advanced disease. Thus, the action of insulin can be summarized as follows. This peptide hormone lowers the blood glucose concentration and leads to anabolic and catabolite processes:

- increases glucose transport in body cells,

- increases the production of glycogen in the liver and muscles,

- suppresses lipolysis,

increases fatty acid production in adipose tissues, and

- increases amino acid production in body cells as well as protein synthesis.

One of the strongest effects of insulin is the suppression of lipolysis. In type II diabetes, this regulation of lipolysis does not go further and there is an increased level of free fatty acids in the blood. Free fatty acids in the blood stimulate gluconeogenesis in the liver and reduce glucose utilization in skeletal muscle. Controlled lipolysis, i.e. the release of free fatty acids via so-called &quot; The hormone-sensitive lipase (HSL) found in body cells is reduced by insulin through a phosphorylation cascade. Accordingly, there is a need for inhibitors, i.e., anti-HSL agents, that simulate the action of insulin and allow a reduction in blood fat concentration. Such agents would be useful for the treatment of type II diabetes, for controlling fat metabolism, but also for treating diseases associated with other stocks. For all these reasons, new inhibitors of HSL and other lipases are needed and are therefore in search of.

Surprisingly, it has been found that a strain of microorganisms of the genus Streptomyces HAG 004107, DSM 13381, can produce novel, highly potent lipase inhibitors that block the action of hormone-sensitive lipase at very low concentrations. The novel natural compounds are organophosphates, which consist of a double cyclic system (bicycl) and a substituted hydrocarbon chain, and which specifically block lipases. A ring base having only a methyl group instead of a hydrocarbon chain was first described as an acetylcholine esterase inhibitor, CGA 134 736, R. Neumann and H.H. Peter in Experientia, vol. 43, pages 1235-1237, 1987. The same compound was later described as cyclophostin (T. Kurokawa et al., In J. Antibiotics, 46, 1315-1318, 1993). This structurally related compound did not exhibit any properties, such as selective lipase blocking. Previously known substances exhibit disadvantages such as insufficient effect, higher toxicity and / or undesirable side effects.

SUMMARY OF THE INVENTION

The present invention provides compounds of formula I

where

R ¹ is

1. a hydrocarbon chain of 2 to 30 carbon atoms, which may be straight, branched, saturated or unsaturated, carbocyclic or heterocyclic, the hydrocarbon chain optionally substituted once or twice:

1.1 -OH group,

1.2 = 0 group,

1.3 -O-C 1 -C 8 -alkyl, wherein the alkyl is straight or branched,

1,4-O-C 2 -C 6 -alkenyl, wherein the alkenyl is straight or branched,

1.5 -C 1 -C 8 -alkyl, wherein the alkyl is straight or branched,

1.6 -aryl,

1.7 -C 1 -C 8 -alkylbenzene,

1.8-diphenyl,

1.9 -NH-C ₁ -C ₆ -alkyl, wherein the alkyl is straight or branched,

1.10 -NH-C2-C6-alkenyl, where alkenyl is straight or branched,

1.11 -NH ₂ ,

1.12 = With group

1.13 -S-C 1 -C 6 -alkyl, wherein the alkyl is straight or branched,

1.14 -SC ₂ -C ₆ -alkenyl, wherein the alkenyl is straight or branched,

1.15 halogen, wherein substituents 1.1 to 1.15 may also be substituted,

2. - [-aryl- (CH ₂ ) _n ] m group, wherein the - [-aryl- (CH ₂ ) _n ] m group is mono- or di-substituted as described in 1.1 to 1.15 and are independently integers 0, 1, 2 or 3;

R ² is

C 1 -C 8 -alkyl, wherein alkyl is unsubstituted or substituted one or two times with the substituents described in 1.1 to 1.15,

2. C ₂ -C 8 -alkenyl, wherein alkenyl is unsubstituted or substituted one or two times with the substituents described in 1.1 to 1.15;

3. C 1 -C ₆ -alkynyl wherein alkynyl is unsubstituted or substituted one or two times with the substituents described in 1.1 to 1.15;

E is phosphorus (P) or sulfur (S),

X ¹ , X ² and X ³ are independently of each other

1. -0-,

2. -NH-,

3. -Ν =,

4. -S-, or

5 -CH ₂ - and -CHR ² -, in all their stereochemical forms and mixtures of these forms in any ratio, and their physiologically 'acceptable salts and chemical equivalents.

R ¹ preferably has a chain of 6 to 24 carbon atoms, particularly preferably 10 to 18 carbon atoms. The chain may be saturated, for example -alkyl, where the alkyl moiety may be straight or branched, or may be unsaturated, e.g. -alkenyl or -alkynyl, wherein the alkenyl or alkynyl radical may be straight or branched. R ¹ may be unsubstituted or mono- or polysubstituted or differently substituted with the substituents described in groups 1.1 to 1.15. Preferred are substituents on carbon atoms of number 8 'to 16', particularly preferred are positions 10 'to 14'. The substituents 1.1 to 1.15 may also be substituted by one or more groups selected from the group consisting of: alcohol-, aldehyde-, acetal-, ketal-, ether-, carboxyl-, ester-, amino-, nitrile-, nitro-, oxime-, oxime ether and halogen.

A carbocyclic hydrocarbon chain having 2 to 30 carbon atoms refers to a chain of 2 to 30 carbon atoms with one or more, preferably one, two or three ring systems, preferably containing 4, 5, 6 or 7 carbon atoms. The rings may be mono-, di- or tricyclic, preferably monocyclic, and may be located at the beginning, middle, or end of the hydrocarbon chain. The carbocycles may be aliphatic or aromatic in nature. Examples are diphenyl or alkylbenzene.

A heterocyclic hydrocarbon chain having 2 to 30 carbon atoms refers to a chain of 2 to 30 carbon atoms with one or more, preferably one to three, ring systems in which at least one carbon atom is replaced by a heteroatom such as 0, S or N. The rings may be mono-, dialbo, or tricyclic, preferably monocyclic, and may be located at the beginning, middle, or end of the hydrocarbon chain. The rings may preferably be 4-, 5-, 6- or 7-membered, aliphatic or aromatic in nature. Examples are alkylpiperidine, substituted or unsubstituted.

Aryl refers to an aromatic ring or ring system having 6 to 14 carbon atoms, preferably 6 to 10 carbon atoms, such as optionally substituted alkylphenol or alkylnaphthol. Halogen refers to a chloride, bromide, fluoride or pseudohalide such as cyanide (nitrile).

-C 1 -C ₆ -alkyl refers to straight or branched alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms, such as methyl, ethyl, isopropyl, tert-butyl and hexyl.

-C ₂ -C 8 -alkenyl denotes straight or branched alkyl having 2, 3, 4, 5 or 6 carbon atoms such as allyl, crotyl and pentenyl.

-C 6 -C 6 -alkynyl denotes straight or branched alkyl having 2, 3, 4, 5 or 6 carbon atoms such as propynyl, butynyl and pentynyl.

R ¹ preferably means:

1. - (CH ₂ ) ₁₅ CH ₃ ,

2. - (CH ₂ ) ₁₃ CH (CH ₃ ) ₂ ,

3. - (CH ₂ ) h CH (OH) (CH ₂ ) ₃ CH ₃ ,

4. - (CH ₂ ) _X 2 CH (OH) CH ₂ CH (CH ₃ ) ₂ ,

5. - (CH ₂ ) ₁₂ CH (OH) (CH ₂ ) ₂ CH ₃ ,

6. - (CH ₂ ) ₁₃ CH (OH) CH ₂ CH ₃ ,

7. - (CH ₂ ) ₁₄ CH (OH) CH ₃ ,

8. - (CH ₂ ) ₁₅ CH ₂ (OH),

9. - (CH ₂ ) ₁₆ CH ₃ , or

10.0 - (CH ₂ ) i ₃ C = OCH ₂ CH ₃ ,

11.0 - (CH ₂ ) i ₂ C = OCH ₂ CH ₂ CH ₃ ,

12.0 - (CH ₂ ) ₁₁ C = OCH ₂ CH ₂ CH ₂ CH 3,

13.0 - (CH ₂ ) ₁₃ CH ₃ ,

14.0 - (CH ₂ ) ₁₁ CH (CH ₃ ) ₂ ,

15.0 - (CH ₂ ) ₁₄ CH ₃ , or

16.0 - (CH ₂ ) i ₂ CH (CH ₃ ) 2,

R ² denotes preferably C -C alkyl, in particular methyl, ethyl or pollen.

Preferred compounds of the invention are listed below:

Cyclipostin A2 of formula IIA:

Cyclipostin D of formula V:

OH

ABOUT

Cyclipostin G of formula VIII:

Cyclipostin N of formula X:

Cyclipostin P of formula XI:

Cyclipostin R of Formula XIII:

Cyclipostine R2 of formula XIIIA:

Cyclipostin S of formula XIV:

Cyclipostin T of formula XV:

all their stereochemical forms and mixtures of these forms in any proportion as well as their physiologically acceptable salts and chemical equivalents.

The method of counting carbon atoms for the NMR spectra of the above formulas is as follows:

The ring system itself contains two asymmetrically substituted atoms, carbon atom 3 and phosphorus atom. Both atoms can have the R- or S- configuration. Surprisingly, it has been found that a strain of the genus Streptomyces HAG 004107, DSM 13381 is capable of forming multiple stereoisomers of the compounds of formula I, i.e. the strain is able to synthesize compounds in which the C3 and P atoms independently of each other have the R- or S- configuration. The isomer with the spatial configuration on the carbon atom R- and the S- configuration on the phosphorus atom of the general formula IA is widespread in the culture of the genus Streptomyces HAG 004107, DSM 13381.

In addition, cyclipostins of other configurations are also formed, such as (R, R), (S, S) or (S, R), which, surprisingly, also exhibit a marked bioaccumulation effect on lipases.

A compound of formula I, or a physiologically acceptable salt thereof, or a chemical equivalent thereof, can be prepared by a microorganism of the genus Streptomyces HAG 004107, DSM 13381 or a variant or mutation thereof under appropriate conditions by fermentation in a culture medium until propagation of one or more compounds of the formula I in a culture medium. wherein they can be obtained by final isolation from the culture medium and optionally converted into a chemical equivalent and a physiologically acceptable salt.

Cyclipostins according to the invention may be produced by Actinomycetales species, preferably by Streptomyces HAG 004107, DSM 13381 species. Streptomyces HAG 004107, DSM 13381 species exhibits an ivory coloration of the mycelium (RAL 1014) and is characterized by conidiophores characteristic of Streptomyces.

The isolate was deposited at the Deutschen Sammlung von Microorganismen und Zellkulturen GmbH, Mascheroder Weg IB, D 38124 Braunschweig, Germany under the Budapest Agreement of 16 March 2000 under the following number: Streptomyces species HAG 004107, DSM 13381.

Instead of the strain Streptomyces species HAG 004107, DSM 13381, mutations and variants that synthesize one or more cyclipostin compounds of the invention may also be used. Such mutants may ^be: prepared by methods known per se by physical means, for example irradiation such as ultraviolet or X-rays, or chemical mutagenesis, for example with ethyl methane sulfonate (EMS), 2-hydroxy-4-methoxybenzophenone (MOB) or N-methyl-N'-nitro -N-nitrosoguanidine (MNNG).

The invention relates to a process for the production of compounds of the formula I or their physiologically acceptable salts, characterized in that the microorganism Streptomyces species HAG 004107, DSM 13381 or its variants or mutations is fermented in the culture medium under suitable conditions until one or more compounds of the formula Also in the culture medium, which are then isolated from the culture medium and optionally converted to their chemical equivalent and physiologically acceptable salts.

Preferably, the strain of Streptomyces species HAG 004107, DSM 13381, mutation and / or variant thereof is fermented in a nutrient solution (also referred to as culture medium) with a carbon and nitrogen source with conventional inorganic soles until cyclipostine accumulates in the culture medium and then cyclipostine is isolated. from the culture medium and, if appropriate, it is divided into individual active components.

The fermentation is preferably carried out under aerobic conditions, preferably at temperatures from 18 to 35 ° C at pH 6-8.

The production method according to the invention can be used for fermentation on a laboratory scale (milliliters to liters) and on an industrial scale (cubic meters). All percentages are by weight unless otherwise stated. The mixture ratio for liquids refers to volume unless otherwise stated.

Preferred carbon sources for aerobic fermentation are assimilatable carbohydrates and sugar alcohols such as glucose, lactose, sucrose or D-mannitol, as well as natural carbohydrate-containing products such as oatmeal, soybean meal and malt extract. Suitable nitrogenous substances are amino acids, peptides and proteins, as well as their degradation products such as peptone or tryptone, as well as meat extract, yeast extract, ground seeds such as corn, wheat, beans, soy or cotton, distillation residue alcohol production, meat meal or yeast extract, as well as ammonium salts and nitrates. The stock solution may contain inorganic salts such as alkali or alkaline earth metal chlorides, carbonates, sulfates or phosphates, iron, zinc, cobalt and manganese.

The formation of cyclipostin of formulas II to XVA according to the invention takes place particularly well in a culture medium containing 0.1 to 5%, preferably 0.3 to 3% of oat flakes and trace elements. The percentages are given in weight percent based on the entire culture medium.

The formation of cyclipostin of formulas VII to XVA according to the invention is particularly preferably carried out in a culture medium comprising 0.1 to 5%, preferably 0.3 to 2% glycerin and 0.2 to 5%, preferably 0.5 to 3% soybean meal, and 0.05 to 1.0 g / l, preferably 0.1 to 1.0 g / l of sodium chloride.

In the culture medium, Streptomyces species HAG 004107, DSM 13381 produces a mixture of cyclipostins. Depending on the composition of the culture medium, the proportions of one or more cyclipostins according to the invention may vary. In addition, it is possible by the composition of the medium to achieve the synthesis of a single cyclipostin by the microorganism so that one or more cyclipostins are not produced at all, or are produced in amounts smaller than the limit of observation.

Preferably, the culture comprises detectable cyclipostin.

Preferably cyclipostin A or P or P2 are formed.

In addition to cyclipostinu and T2 (of II to XVA) forms a Streptomyces species HAG 004107, DSM 13381 in a culture medium other related compounds which are the aforementioned compounds differ different radicals R ¹ and R ² in formulas II to XVA. The lower amounts are shown cyclipostiny to be truncated or more branched radical R ^first In the culture of Streptomyces HAG 004107, DMS 13381 oxidation products (hydroxylation products) are also shown as by-products.

The cultivation of the microorganisms is also carried out aerobically, for example by submerged method, with shaking or stirring in a flask or fermenter, optionally by introducing air or oxygen. It can be carried out in a temperature range from 18 to 35 ° C, preferably from 25 to 32 ° C, especially at temperatures from 26 to 30 ° C. The pH values should be in the range of 6 to 8, preferably in the range of 6.5 to 7.8. The microorganism is generally cultured under these conditions for 24 to 300 hours, preferably 30 to 90 hours.

Preferably, it is cultivated in several stages, i. from. that one or more precultures are first prepared in a liquid culture medium which is then inoculated into its own production medium, the main culture, e.g. in a 1:10 volume ratio. The preculture is obtained e.g. by inoculating the mycelium into the nutrient solution and allowing it to grow for about 36 to 120 hours, preferably 48 to 96 hours. The mycelium may be maintained in the strain for about 3 to 40 days, preferably 4 to 10 days, on a solid or liquid nutrient medium, for example malt-yeast or oatmeal agar.

The course of the fermentation can be monitored by the pH of the culture or mycelium, as well as by chromatographic methods such as thin layer chromatography or high pressure liquid chromatography, or by monitoring biological activity. Cyclipostins according to the invention are also to a small extent also contained in the culture filtrate. The opi15 purification method described below serves to purify cyclipostin according to the invention, preferably to purify cyclipostin A and P.

The isolation or purification of the cyclipostins according to the invention from the culture medium is carried out by known methods with regard to the chemical, physical and biological properties of the natural substances. Thin-layer chromatography, for example on silica gel, using dichloromethane-ethyl acetate or chloroform-methanol (e.g. 98: 1) as eluent or by HPLC can be used to test the concentration of cyclipostine in the culture medium or in individual isolated fractions. Detection by thin-layer chromatography can be carried out, for example, by means of coloring agents such as molybdophosphoric acid or by using pairs of ₁₂ , the amount of the substance being measured by comparison with a standard solution.

In the isolation of cyclipostin according to the invention, the mycelium is first separated from the culture medium in a conventional manner and then cyclipostine is extracted from the cell mass by treatment with a water-miscible solvent. The cyclipostin-containing organic solvent phase of the present invention is subsequently concentrated in vacuo and purified further as described above.

Optionally, the culture filtrate is combined with a mycelium extract concentrate and extracted with a suitable water-immiscible solvent such as n-butanol or ethyl acetate. Finally, the separated organic phase is concentrated in vacuo and dissolved in an appropriate volume of 1/30 water / methanol mixture.

Further purification of one or more cyclipostins according to the invention is carried out by chromatography on suitable materials, preferably, for example, molecular sieves, normal phase carriers such as silica gel, alumina, ion exchange resins or adsorbent resins such as reverse phase RP. Using these chromatographies, cyclipostins are separated. Chromatography of the cycli16 posthins is performed with an organic solvent or a mixture of aqueous and organic solvents.

By the term aqueous or organic solvent mixture is meant all solvents miscible with water, preferably methanol, propanol and acetonitrile, in solvent concentrations of 10 to 100%, preferably 60 to 90% of solvent, or else buffered aqueous solutions which are miscible with organic solvents. The buffers used are the same as described above.

The distribution of cyclipostins is based on their different polarity and is carried out by reverse phase chromatography, for example MCI® (adsorption resin from Mitsubishi, Japan) or Amberlite® (Tosohaas), on other hydrophobic materials such as RP-8- or RP- 18-stages. In addition, the separation can be carried out by normal phase chromatography, for example silica gel, alumina and the like.

Cyclipostin chromatography is performed with buffered or saturated aqueous solutions or a mixture of aqueous solutions with alcohols or other water-miscible solvents. Propanol and acetonitrile are used as organic solvents. Buffered or saturated aqueous solutions means e.g. water, phosphate buffer, ammonium acetate, citrate buffer in concentrations from 1 mM to 0.5 M as well as formic acid, acetic acid, trifluoroacetic acid or other commonly used acids known to those skilled in the art, preferably in concentrations from 0.01 to 3% %, preferably 0.1%.

Gradient chromatography starts with 100% aqueous buffer and ends with 100% solvent, preferably a linear gradient from 50 to 100% 2-propanol or acetone is used.

Alternatively, gel or hydrophobic phase chromatography may be used.

Gel chromatography is carried out on polyacrylamide or mixed polymer gels such as Biogel-P2® (Biograd), Fractogel TSK HW 40® (Merck, Germany or Toso Haas, OSA) or Sephadex® (Pharmacia, Uppsala, Sweden) ).

The order of chromatography is interchangeable.

I

Another very important method of purifying cyclipostin is crystallization. Cyclipostins crystallize from solutions of organic solvents and mixtures of water with organic solvents. Crystallization is accomplished by known procedures, such as by concentrating or cooling the saturated cyclipostin solution.

The cyclicostostins of the present invention are stable in solid or liquid form and in solution at pH values of 4 to 8, in particular in the range of 5 to 7, and allow processing into galenic forms.

The invention includes other conventional chemical equivalents of compounds of formula I which exhibit slight chemical differences and which have the same effects or are converted under mild conditions to the compounds of the invention. Known equivalents include, for example, ester and ether, as well as oxidation products, reduction products, and hydrogenation products of the compounds of this invention.

The ester and ether derivatives, oxidation products, reduction products and hydrogenation products are prepared by methods described in the literature, for example, in Advanced Organic Synthesis, 4th edition, J. March, John Wiley & Sons., 1992.

The present invention encompasses all stereoisomeric forms of the compounds of Formulas I-XVA. The compounds of formulas I to XVA contain asymmetric centers which may have independently the S- or R- configuration. The invention includes all possible enantiomers and diastereoisomers as well as mixtures of two or more stereoisomeric forms, for example mixtures of enantiomers and / or diastereoisomers in all ratios. Enantiomers are also in enantiomeric forms, as well as left-handed and right-handed antipodes, R- and S-configurations, in the form of racemates and in the form of mixtures of both enantiomers in all proportions subject to the present invention. The invention also relates to cis / trans isomers as well as cis-form or trans-form or mixtures of these forms in all ratios.

Because of their excellent pharmacological properties, the compounds of the present invention are suitable for use as medicaments in human and / or veterinary medicine. They block lipases and are useful in the treatment of diseases caused by a metabolic-related disorder that have causes in lipid-related metabolic disorders. The compounds of the formula I according to the invention show a surprising blocking effect on hormone-sensitive lipase, HSL, an allosteric enzyme in adipocytes, which is blocked by insulin and is responsible for the removal of fats in fat cells and thus for the circulation of fat in the blood stream. Inhibition of this enzyme also corresponds to the insulin-like action of the compounds of the present invention, which ultimately leads to a reduction in the free fatty acid content in the blood and blood sugar. They can also be used in metabolic disorders such as insulin-independent diabetes mellitus, diabetic syndrome and direct pancreatic damage.

The invention also encompasses pharmaceutical compositions comprising one or more cyclipostins of the invention and / or chemical equivalents thereof. They are preferably used in admixture with suitable excipients or carriers. All pharmacologically acceptable carriers and / or excipients are used as the carrier material.

The invention further provides a process for the manufacture of medicaments according to the invention, characterized in that at least one compound according to the invention is admixed with a suitable pharmaceutically acceptable carrier and optionally another suitable active ingredient, excipient or excipient in a form suitable for administration.

The pharmaceutical compositions of this invention are generally administered in the form of oral, topical or parenteral, but are also principally possible as rectal compositions. Suitable solid or liquid galenic forms are, for example, granules, powders, tablets, dragees, (micro) capsules, syrups, emulsions, suspensions, aerosols, drops or injectable solutions in the form of ampoules, as well as retarded release formulations using conventional a carrier and an additional or auxiliary agent, such as disintegrants, binders, coating agents, swelling agents, talc flours or lubricants, flavoring agents, sweeteners or solvents. As a frequently used carrier or excipient, e.g. magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, gelatin, starches, vitamins, cellulose and derivatives thereof, animal or vegetable oils, polyethylene glycols and solvents such as sterile water, alcohol, glycerin and polyhydric alcohols.

Alternatively, the dosage form for oral administration can be formulated as microcapsules, in order to retard release or prolong it for a prolonged period of time, such as by coating or incorporating the active ingredient into a particle of a suitable polymer, wax or the like.

Preferably, the pharmaceutical formulations are prepared in dosage forms wherein each unit contains as an active ingredient a known dose of one or more cyclipostin compounds of the invention and / or a chemical derivative thereof. For solid dosage forms such as tablets, capsules and suppositories, such doses may contain up to 200 mg, preferably about 0.1 to 100 mg, and for injectable solutions in the form of ampoules to 200 mg, preferably about 1.0 to 100 mg per day.

The daily doses administered depend on the body weight, age, severity and condition of the patients. Depending on the conditions, lower or higher doses may be administered. Administration of the daily dose may be by a single dosage form, or by multiple smaller dosage forms, or by multiple-dose dosing at certain intervals. The invention also includes pharmaceutical compositions comprising one or more cyclipostins and / or chemical equivalents thereof according to the invention. It is preferred to use it in admixture with a suitable excipient or carrier. Any pharmacologically acceptable carrier and / or excipient may be used as the carrier.

The action of the compounds of the formula I according to the invention is tested by the following enzyme system:

Preparation of the enzyme

Preparation of partially purified HSL

Isolated rat adipose cells were obtained from adipose tissue in male sex glands of untreated human rats (wistar, 220-250 g) by the collagenase treatment procedure published e.g. in: B.S. Nillson et al., Anal. Biochem. 158, 1986, 399407; G. Frederikson et al., J. Biol. Chem. 256, 1981, 6311-6320; H. Tornquist et al., J. Biol. Chem., 251, 1976, 813-819). Fat cells from 10 rats were washed three times by flotation, once each with 50 ml homogenization buffer (25 ml Tris / HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 10 μς / πιΐ leupeptin, 10 μς (ιηΐ antipatin, 20 μg / ml pepstatin) and dissolved in 10 ml homogenization buffer. Fat cells were placed in a Teflon glass homogenizer (Braun-Melsungen) and homogenized at 1500 rpm at 15 ° C. The homogenate was centrifuged (Sorwall SM24-Rörchen, 5000 rpm, 4 ° C). The phase between the upper layer of fat and the solid pellet was collected and centrifuged again. The phase so obtained was centrifuged again (Sorwall SM24-Rörchen, 20,000 rpm, 45 minutes, 4 ° C). The interphase was collected and treated with 1 g heparin on sepharose (Pharmacia-Biotech, CL-6B, washed 5 times with 25 mM Tris / HCl, pH 7.4, 150 mM NaCl). After incubation for 60 min at 4 ° C (shaking at 10 min intervals), the reaction mixture was centrifuged. The liquid was adjusted to pH 5.2 by addition of acetic acid and incubated for 30 minutes at 4 ° C. The precipitate was centrifuged (Sorwall SM34, 12000 rpm, 10 minutes, 4 ° C) and the residue was suspended in

2.5 ml 20 mM Tris / HCl, pH 7.0, 1 mM EDTA, 65 mM NaCl, 13% sucrose, 1 mM DTT, 10 µg / ml leupeptin / pepstatin / antipatin. The suspension was dialyzed overnight against a 25 mM Tris / HCl solution, pH 7.4, 50% glycerin, 1 mM DTT, 10 µg / ml leupeptin, pepsatin, antipatin, and then placed on a column of hydroxyapatite (0.1 g per ml suspension) , equilibrated with 10 mM potassium phosphate, pH 7.0, 30% glycerin, 1 mM DTT). The column was washed with four volumes of equilibration buffer with a flow rate of 20 to 30 ml / h. HSL was eluted with one volume of equilibration buffer containing 0.5 M potassium phosphate and then dialyzed (see above) and concentrated 5 to 10 times by ultrafiltration (Amicon Diaflo PM 10 filter) at 4 ° C. The partially purified HSL may be stored at -70 ° C for 4-6 weeks.

determination

To prepare the substrate, 25-50 pCi of [ ³ H] trioleoylglycerine (in toluene), 6.8 μπιοί of unlabeled trioleoylglycerine and 0.6 mg of phospholipid (phosphatidylcholine / phosphatidylinositol) were mixed

3: 1 w / v), the mixture was dried with N ₂ and then dissolved in 2 ml of 0.1 M KPi (pH 7.0) with sonication (Branson 250, microsyringe, setting 1-2, 2x1 min. V) 1 minute intervals). After addition of 1 ml KPi and renewed ultrasound (4 x 30 seconds, on ice for 30 second intervals), 1 ml of 20% BSA (bovine serum albumin) (in KPi) (final trioleoylglycerin concentration 1.7 mM) was added. For the reaction, 100 μΐ of substrate solution was pipetted to 100 μΐ HSL solution (HSL prepared as above, diluted in 20 mM KPi, pH 7.0, mM EDTA, 1 mM DTT, 0.02% BSA, 20 μρ / πιΐ pepstatin (10 µg / ml leupeptin) and incubated for 30 minutes at 37 ° C. After addition of 3.25 ml methanol / chloroform / heptane (10: 9: 7) and 1.05 ml 0.1 MK ₂ CC> 3,

0.1 M boric acid (pH 10.5) was mixed well and finally centrifuged (800 x g, 20 min). After phase separation, one equivalent of the upper phase (1 ml) was collected and radioactivity was measured using a liquid scintillation counter.

evaluation

The substances were tested as described above in four independent measurements. Blocking of HSL enzymatic activity with a test compound was determined by comparison with an unprotected control reaction. The IC ₅₀ was calculated using a curve with at least 10 concentrations of the test compound. The Graphit software package, Elsevier-Biosoft, was used for data analysis. In this test, the compounds of the invention exhibited the following effects:

Cyclipostin A, P, P2 and R block lipolysis in rat adipocytes with IC50 - 0.2 μ 0,2, inhibit human hormone-sensitive lipase (HSL) with trioleoylglycerin as substrate: IC50 = ~ 0.07 to 0.5 μΜ. HSL from rats was blocked with NBD (4-chloro-7-nitrobenzo-2-oxa-1,3-diazole) as substrate, at concentrations from 4 nM to 10 nM.

Cyclipostine protects the hormone-sensitive lipase (HSL) as well as the mono-acyl-glycerin-lipase of the rat extract in submicromolar concentrations.

The invention is further explained in the following examples. The percentages given are percentages by weight. Mixtures for liquids are given in percent by volume unless otherwise stated.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of a glycerin culture of Streptomyces species HAG 004107, DSM 13381

100 ml of nutrient solution (malt extract 2.0%, yeast ex23 tract 0.2%, glucose 1.0%, (NH4) ₂ HPO4 0.05%, pH 6.0) in a sterile 300 ml Erlenmeyer flask were inoculated with strain Streptomyces species HAG 004107, DSM 13381 and incubated for 7 days at 28 ° C and 180 rpm on a rotary shaker. Finally, 1.5 ml of this culture was diluted with 1.5 ml of 99% glycerin and stored at 20 ° C.

Example 2

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

A sterile Erlenmeyer flask was filled with 100 ml of the following nutrient solution: 15 g / l glucose, 15 g / l soybean meal, 5 g / l grain, 2 g / l CaCO3 and 5 g / l NaCl and inoculated with one tube (same nutrient). solution with 2% agar) on a slant agar or 1 ml glycerol culture (see Example 1) and the shaker was operated at 180 rpm at 28 ° C. A 48-96 hour old submerged culture (inoculation amount of about 10%) from the same nutrient solution is sufficient to inoculate a 10 and 200 L fermenter.

Example 3

Preparation of culture of Streptomyces species HAG 004107, DSM 13381 in an Erlenmeyer flask

A sterile 300 ml Erlenmeyer flask was filled with 100 ml of the following nutrient solution: 20 g / 1 dog oatmeal,

2.5 ml of trace element solution, and seeded with a 10% seed amount of preculture (Example 2) and incubated on a shaker at 180 rpm at 28 ° C. The culture could be inoculated into a cyclipostin fermenter after two days. The trace element solution had the following composition:

g / 1 CaCl ₂ x 2H ₂ O, g / 1 Fe (III) citrate,

0,2 g / l MnSO ₄ x H ₂ O,

0.1 g / 1 of _ZnCl2,

02 025 g / 1 CuSO ₄ x 5H ₂ O

0.02 g / l Na-tetraborate,

0.004 g / l CoCl ₂ x 6H ₂ O,

0.01 g / l Na-molybdate.

Example 4 ^!

Production of cyclipostins of formulas II to IX

A 200 L fermenter was charged with 90 L nutrient solution under the following conditions:

Nutrient medium:

g / 1 oatmeal in water,

2.5 ml / l trace elements, pH 7.8 (before sterilization).

The nutrient solution was heat-sterilized for 30 minutes and, after cooling, inoculated with 5% by volume of the seeding material obtained according to Example 3.

Trace element solution:

g / 1 CaCl ₂ x 2H ₂ O, g / 1 Fe (III) citrate,

0,2 g / l MnSO ₄ x H ₂ O,

0, 1 g / 1 of _ZnCl2,

0.025 g / l CuSO ₄ x 5H ₂ O,

0.02 g / l Na-tetraborate,

0.004 g / l CoCl ₂ x 6H ₂ O,

0.01 g / l Na-molybdate.

Duration of the process: 72 hours.

Incubation temperature: 27 ° C.

Stirring speed: 65 rpm.

Aeration: 6 m ^{3 of} air per hour.

The fermentation was carried out without the addition of suds suppressors. The production maximum was reached after 48 hours.

Example 5

Production of cyclipostins of formulas X to XVA

A 200 liter fermenter processes 100 liter contents under the following conditions:

Nutrient medium:

5g / L glucose;

20g / L glycerin;

20g / l soya meal;

5g / l yeast extract

3g / l sodium chloride;

2.5 ml / l trace element solution pH 7.0 (before sterilization)

Duration of the process: 72 hours.

Incubation temperature: 27 ° C.

Stirring speed: 65 rpm.

Aeration: 6 m ^{3 of} air per hour.

The fermentation is carried out without the addition of suds suppressors. Maximum production is achieved after about 48 hours.

Example 6

Isolation of the cyclipostin mixture from Streptomyces species HAG 004107, DSM 13381 culture medium.

After the fermentation of Streptomyces species HAG 004107, DSM 13381, 100 l of the fermentation broth obtained according to Example 4 was added, with the addition of about 2% of the filtering agent (e.g. Celite®), filtered and the cell mass (10 l) was extracted with 40 l of methanol. The methanolic solution of the drug was freed from the mycelium by filtration and concentrated in vacuo. The concentrate was poured onto a prepared column of 7LMCI GEL, CHP20P. Eluted with a gradient of water and propan-2-ol. The effluent from the column (20 liters per hour) was collected in fractions (10 liters each) and the fractions containing cyclipostins (fractions 19-21) were concentrated in vacuo. Fractions were examined by HPLC (see Example 6). Fraction 19 contained cyclipostin A through E as well as them

isomers, fraction 20 contained cyclipostin F a its isomers, fraction 21 contained as their isomers. cyclipostin N, P, P2, Q, R S and T as well Example 7 HPLC analysis of cyclipostins high pressure liquid chromatography HPLC analysis

cyclipostins were performed using an HP 1100® with a YMC-Pack Pro C18® column [AS-303, 250 x 4.6 mm, S-5 µm, 120 ° C]. The flow rate was 1 ml / min, the column temperature was 40 ° C. The column was eluted with a gradient of 0.05% trifluoroacetic acid in acetonitrile. Pure acetonitrile was achieved after 11 minutes and then the column was eluted isocratically with only this solvent. Detection was performed by measuring ultraviolet light absorption at 210 nm. Under these conditions, cyclipostins had the following

retention times: Cyclipostin A 12.7 minutes Cyclipostin A 2 12.6 minutes Cyclipostin F 13.2 minutes Cyclipostin N 15, 9 minutes Cyclipostin P 17.7 minutes Cyclipostin P 2 17, 3 minutes Cyclipostin Q 18.3 minutes Cyclipostin R 16.7 minutes Cyclipostin R 2 16, 4 minutes Cyclipostin WITH 18.5 minutes Cyclipostin T 19.1 minutes and Cyclipostin T 2 18.7 minutes.

Example 8

Obtaining pure cyclipostin A and A2

Fraction 19, obtained according to Example 6, was evaporated in vacuo, 1 g of a contrast agent dissolved in water-methanol (1: 1) was added and the mixture was loaded onto a Nucleoprep 100-5 C18 AB® column (21 x 250 mm). . The column was eluted with a gradient of 50% acetonitrile in 0.01% trifluoroacetic acid to 100% acetonitrile. The flow rate was set at 50 ml per minute. The output from the column was examined by measuring the light absorption at 210 nm to test the blocking action on the lipase. Fractions of 60 ml were collected. Fractions 34 and 35 contained cyclipostin A, and fractions 41-44 contained cyclipostin A2. These fractions were combined, evaporated in vacuo and then separated on a SP 250/10 Nucleosil 100-5 C18 HD ® column (21 x 250 mm). 50 to 66% acetonitrile in 0.01% trifluoroacetic acid was used as a gradient, the pH of the solution was adjusted to 4.0 by dropwise addition of ammonium hydroxide solution. Fractions containing pure compounds were combined and evaporated to dryness. 5.4 mg of pure cyclipostin A were obtained as a waxy substance and 3 mg of cyclipostin A2 as an oil.

Example 9

Characterization of cyclipostin

Appearance: Substance soluble in oxygen-containing organic solvents, while sparingly soluble in water and light petroleum, neutral, colorless, waxy substance.

UV-peak: 228 nm in methanol.

IR bands: 1752 and 1671 cm ^-1 .

The following molecular weight was found by high resolution mass spectrometry in the nitrobenzyl alcohol / LiCl matrix: 467.2757 amu, corresponding to the cyclipostin A-Li C23H41O7PL1 sum formula. This gives a summary formula for cyclipostin A C23H41O7P, molecular weight 460.

By means of electrospray mass spectrometry in a consumable ionization mode (ESI, positive), 461 amu peaks corresponding to the found (M + H) ^{+ were found} , in addition a characteristic peak at 221 amu, corresponding to C7H10O5P. In the ESI negative mode they were gives peaks at 459 amu (M-H) ^- (C16H34O5P) and 219 amu (C 7 H _S O6P) · For the determination of the position of the alcohol group of the compound is derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide and the sample was analyzed by mass spectrometry using electron ionization. A trimethylsilyl derivative is formed:

weighing 554 amu. The position of the silylated hydroxy group was shown by the intense ions 497 (α-cleavage) and 159 amu (α-cleavage).

NMR signals: see Table 1

Table 1:

^! 1 H and ¹³ C NMR chemical shifts of cyclipostin A in methanol-d 6 at 300 K

³ H ¹³ C First 171.08 (1.4 Hz) ^b) 2 - 114.61 (3.4 Hz) ^bl 3 3.87 40.75 4 4.46 / 3.86 66.04 5 4.31 / 4.25 69.39 (6.0 Hz) ^b) 6 - 161.47 (8.0 Hz) ^b) 7 2,4 0 17.89 ( ^4.6 Hz) ^bl I ' 4.25 71.61 (6.6 Hz) ^b) 2 ' 1.73 31.16 (6.6 Hz) ^b> 3 ' 1, 41 26.39 n ' 3.49 72,45 n ± l 1.46 to 1.33 38.44, 38.15 4'-14 '(a) 1.37-1.26 30.85 to 30.58

15 ' 1.34 23.84 16 ' 0.91 14.43

(a) except n and n + 1.

(b) in brackets, the ¹³ C / ³¹ P interaction constants.

Example 10

Characterization of cyclipostin B

Cyclipostin B was prepared as described in Example 8 for cyclipostin A, repeating the chromatography procedure several times and characterized as in Example 8.

Appearance: substance soluble in oxygen-containing organic solvents, while poorly soluble in water and petroleum ether, neutral, colorless, waxy substance.

UV-peak: 228 nm in methanol.

By the means of electrospray mass spectrometry in positive ionization mode (ESI, positive) gives the peaks 461 arou corresponding to the found (M + H) ^+, also a characteristic peak at 221 amu, corresponding C7Hi ₀ O ₆ P. In the ESI negative mode gives peaks 459 amu (MH), 337 amu (C16H34O5P) and 219 amu (C; HgO6P). To determine the position of the alcohol group, the compound was derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide and the sample was examined by electron ionization mass spectrometry. The resulting trimethylsilyl derivative weighs 554 amu:

, OSi (CH ₃ ) ₃ "A ⁰ OSi (CH ₃ ) ₃

The position of the silylated hydroxy group was demonstrated by the intense ions of pti 511 (α-cleavage) and 145 amu (cleavage).

Summary formula of cyclipostin B: C 23 H 41 O 7 P, molecular weight: 460.

Example 11

Characterization of cyclipostin C

Cyclipostin C was prepared as described in Example 8. for cyclipostin A, repeated the chromatography procedure and characterized as in Example 8.

Appearance: substance soluble in oxygen-containing organic solvents, while sparingly soluble in water and petroleum ether, neutral, colorless, waxy substance.

UV-peak: 228 nm in methanol.

By means of electrospray mass spectrometry in positive ionization mode (ESI, positive), 461 amu peaks corresponding to found (M + H) ^{+ were found} , in addition a characteristic peak at 221 amu corresponding to C7H10O6P. Peaks of 459 amu (MH) -, 337 amu (C 16 H 34 O 5 P) and 219 amu (C ₇ H ₈ C> ₆ P) were found in the ESI negative mode. To determine the position of the alcohol group, the compound was derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide and the sample was examined by electron ionization mass spectrometry. The resulting trimethylsilyl derivative weighs 554 amu:

The position of the silylated hydroxy group was shown by intensive ions at 525 amu (α-cleavage) a. 131 amu (cleavage).

Molecular formula cyclipostinu B ₂ C 3 H ₄₁ O7P, molecular weight: 460th

Example 12

Characterization of cyclipostin F

Fraction 20 obtained according to Example 6 was separated as described in Example 8 and cyclipostin F was obtained by repeating the chromatographic procedure several times and characterized as in Example 9.

Retention time: 13.2 min.

Appearance: substance soluble in oxygen-containing organic solvents, while sparingly soluble in water and petroleum ether, neutral, colorless, waxy substance.

UV-peak: 228 nm in methanol.

By the means of electrospray mass spectrometry in positive ionization mode (ESI, positive) gives the peaks 459 amu, corresponding to the found (M + H) ^+, also a characteristic peak at 221 amu, corresponding to C 7 H ₁₀ O6P. In ESI negative mode, peaks of 457.6 amu (MH) -, 336 amu (C ₁₆ H 32 O ₅ P) and 219 amu (C ₇ H ₅ O ₆ P) were found.

Cyclipostin B overall formula: C 23 H 39 O 7 P / molecular weight: 458.

Example 13

Characterization of cyclipostin P

Fraction 21, obtained according to Examples 5 and 6, was separated as described in Example 9 and cyclipostin P was obtained by repeated chromatography (210 mg) and characterized as in Example 9. Cyclipostin P was crystallized by dissolving 210 mg in 3 ml. propan-2-ol and 13 ml acetonitrile and 8 ml water.

Retention time: 17.7 min.

Appearance: substance soluble in oxygen-containing organic solvents, while poorly soluble in water and petroleum ether, neutral, colorless, waxy substance.

UV-peak: 228 nm in methanol.

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

The following molecular mass was found by high resolution mass spectrometry in the nitrobenzyl alcohol matrix: 445.2717 amu, corresponding to the (M + H) ⁺ sum formula of cyclipostin P: C23H42O6P. This gives a summary formula for cyclipostin P C 23 H 41 O 6 P, molecular weight 444. Using electrospray mass spectrometry in positive ionization mode (ESI, positive), found 445 amu peaks corresponding to the found (M + H) ⁺ , in addition a characteristic peak at 221 am, corresponding C7H10O ₆ P. In the ESI negative mode peaks gives 443 amu (M-H), 321 amu (C16H32O4P) and 219 amu (C ₇ H ₈ O _6).

NMR data is shown in Table 2.

Table 2: Chemical shifts of cyclipostin P in MeOD at 300 K.

^Σ Η ¹³ C 1 - 171.08 2 - 114.60 (3.0 Hz) ^al 3 3.87. 40.74 4 4.47 / 3.85. 66, 05 5 4.30 / 4.25 69.40 (6.0 Hz ⁾ 6 - 161.47 (8.0 Hz ⁾ 7 2.40 17.90 (4.6 Hz) ^and> I ' 4.24 71.62 (6.9 Hz) ^a) 2 ' 1.73 31.16 (6.3 Hz ⁾ 3 ' 1.41 26, 38 4'-13 ' 1.34-1.29 30.76 to 30.11 14 ' 1.34-1.29 33.07

15 ' 1.31 23.72 16 ' 0.89 14.42

a) Interaction constants ¹³ C / ³¹ P are shown in brackets.

Example 14

Characterization of cyclipostin P2

Fraction 21, obtained according to Examples 5 and 6, was separated as described in Example 8 and cyclipostin P was obtained by repeated chromatography (130 mg) and characterized as in Example 9.

Retention time: 17.1 min.

Appearance: substance soluble in oxygen-containing organic solvents, while only slightly soluble, neutral, colorless, waxy substance in water and petroleum ether.

UV-peak: 228 nm in methanol.

The following molecular mass was found by high resolution mass spectrometry in the nitrobenzyl alcohol matrix: 445.2721 amu, corresponding to the (M + H) ⁺ sum formula of cyclipostin P: C23H42O6P. This gives a summary formula for cyclipostin P2 C23H41O6P, molecular weight 444. Using electrospray mass spectrometry in positive ionization mode (ESI, positive), 445 amu peaks corresponding to (M + H) ⁺ , in addition a characteristic peak at 221 amu, corresponding to C7H10O6P. Peaks of 443 amu (MH), 321 amu (ΟιθΗ3 ₂ θ4Ρ) and 3 219 amu (Ο ^ ΗθΟςΡ) were found in ESI negative mode.

NMR data is shown in Table 3.

Table 3: Chemical shifts of cyclipostin P2 in MeOD at 300 K.

¹³ C 1 - 171.05 2 - 114.60 (3.2 Hz ⁾ 3 3.87 40.74 4 i 4.46 / 3.85 66.02 5 4.30 / 4.25 69.38 (6.0 Hz) ^al 6 - 161.46 (8.0 Hz ⁾ 7 2.40 17.90 (4.6 Hz ⁾ I ' 4.24 71.60 (6.9 Hz) ^al 2 ' 1.73 31.16 (6.3 Hz) ^a) 3 ' 1.41 26, 39 4'-II 1.34-1.29 31.04 to 30.11 12 ' 1.29 28.53 13 ' 1.17 40.25 14 ' 1.52 29, 15 15 ', 16' 0.87 23, 04

a) Interaction constants ¹³ C / ³¹ P are shown in brackets.

Example 15

Production and characterization of cyclipostin N

Fraction 21 obtained according to Examples 5 and 6 was separated as described in Example 8 and cyclipostin N was obtained by repeated chromatography (2 mg) and characterized as in Example 9.

Retention time: 15.9 min.

Appearance: substance soluble in oxygen-containing organic solvents, while only slightly soluble, neutral, colorless, waxy substance in water and petroleum ether.

UV-peak: 228 nm in methanol.

By means of high resolution mass spectrometry in the matrix of nitrobenzyl alcohol, a quasi-molecular ion (M + H) ⁺ 417.2405 amu was found, corresponding to the general formula of cyclipostin N: C2iH3gOeP (Theory: 417.2406). Characteristic peak in ESI positive mode: 221 amu.

Table 4: Chemical shifts of cyclipostin N in MeOD at 300 K.

³ H ¹³ C 1 - 171.07 2 - 114.60 (3.1 Hz ⁾ 3 3, 87 40.74 4 4.45 / 3.84 66, 03 5 4.30 / 4.25 69.39 (5.9 Hz) ^a) 6 - 161.47 (8.0 Hz ⁾ 7 2.40 ' 1.90 (4.9 Hz) ^a) I ' 4.24 71.60 (6.6 Hz ⁾ 2 ' 1.73 31.16 (6.2 Hz) ^and> 3 ' 1.41 26, 38 4'-II 1.35-1.26 30.76 to 30.11 12 ' 1.35-1.26 33.06 13 ' 1.31 23.72 14 ' 0.89 14.41

The coupling constants ¹³ C / ³¹ P are shown in brackets.

Example 16

Production and characterization of cyclipostin R

Fraction 21 obtained according to Examples 5 and 6 was separated as described in Example 8 and cyclipostin R was obtained by repeated chromatography (8 mg) and characterized as in Example 9.

Retention time: 16.7 min.

Appearance: substance soluble in oxygen-containing organic solvents, while poorly soluble in water and petroleum ether, neutral, colorless, waxy substance.

UV-peak: 228 nm in methanol.

Quasi-molecular ion (M + H) ⁺ 431.2561 amu was found by high resolution mass spectrometry under FAB-MS conditions, corresponding to the cyclipostin R total formula: C22H40O6P (Theory: 431.2562). Characteristic peak in ESI positive mode: 221 amu.

Table 5: Chemical shifts of cyclipostin R in MeOD at 300 K.

¹³ c 1 - 171.06 2 - 114.58 (3.2 Hz) ^al 3 3.87 40.75 4 4.45 / 3.85 66, 04 5 4.30 / 4.25 69.40 (6.0 Hz ⁾ 6 ^; - 161.48 (8.0 Hz ⁾ 7 2.40 17.90 (5.0 Hz) ^al I ' 4.24 71.61 (7.0 Hz) ^and> 2 ' 1.73 31.16 (6.2 Hz ⁾ 3 ' 1.41 26.38 4'-12 ' 1.37-1.25 30.74 to 30.10 13 ' 1.17 33.06 14 ' 1.30 23.71 15 ' 0.89 14.40

a) Interaction constants ¹³ C / ³¹ P are shown in brackets.

Example 17

Production and characterization of cyclipostin R2

Fraction 21, obtained according to Examples 5 and 6, was separated as described in Example 8 and cyclipostin R2 was obtained by repeated chromatography (8 mg) and characterized as in Example 9.

Retention time: 16.4 min.

Appearance: substance soluble in oxygen-containing organic solvents, while only slightly soluble, neutral, colorless, waxy substance in water and petroleum ether.

UV-peak: 228 nm in methanol.

Quasi-molecular ion (M + H) ⁺ 431.2564 amu was found by high resolution mass spectrometry under FAB-MS conditions, corresponding to the cyclipostin R2 summary formula: C22H40O6P (Theory: 431.2562). Characteristic peak in ESI positive mode: 221 amu.

Table 6: Chemical shifts of cyclipostin R2 in MeOD at 300 K.

¹³ C 1 - 171.06 (1.7 Hz ⁾ 2 - 114.58 (3.1 Hz) ^al 3 3.87 40.75 4 4.46 / 3.85 66,03 5 4.30 / 4.25 69.39 (6.0 Hz) ^and> 6 - 161.47 (8.0 Hz ⁾ 7 2.40 17.90 (4.9 Hz) ^and> I ' 4.24 71.60 (6.9 Hz ⁾ 2 ' 1.73 31.16 (6.6 Hz ⁾ 3 ' 1.41 26, 38 4'-10 ' 1.37-1.25 31.02 to 30.10 11 ' 1.29 28.51 12 ' 1.16 40,24 13 ' 1, 51 29.15 14 ', 15' 0.87 23,02

a) Interaction constants ¹³ C / ³¹ P are shown in brackets.

Example 18

Production and characterization of cyclipostin

Fraction 21 obtained according to Examples 5 and 6 was separated as described in Example 8 and cyclipostin S was obtained by repeated chromatography (0.7 mg) and characterized as in Example 9.

Retention time: 18.5 min.

Appearance: substance soluble in oxygen-containing organic solvents, while poorly soluble in water and petroleum ether, neutral, colorless, solid substance.

UV-peak: 228 nm in methanol.

Quasi-molecular ion (M + H) ⁺ 459.2883 amu was found by high resolution mass spectrometry under FAB-MS conditions, corresponding to the cyclipostin S total formula: C 24 H 44 O 6 P (Theory: 459.2575). Characteristic peak in ESI positive mode: 235 amu.

Table 7: Chemical shifts of cyclipostin S in MeOD at 300 K.

³ H ¹³ C 1 - 170.87 (1.4 Hz ⁾ 2 - 113.66 (3.1 Hz ⁾ 3 3.85 40.77 4 4.45 / 3.85 66, 04 5 4.29 / 4.24 69.17 (6.0 Hz) ^and> 6 - 165.80 (8.3 Hz ⁾ 7 2.98 / 2.82 25.05 (4.6 Hz) ^and> 8 1.16 10.86 I ' 4.25 71.57 (6.9 Hz ⁾ 2 ' 1.74 31.19 (6.3 Hz) ^a) 3 ' 1.42 26.41 4'-13 ' 1.34-1.29 30.76 to 30.11 14 ' 1.34-1.29 33.07 15 ' 1.31 23.73 16 ' 0.89 14.43

a) Interaction constants ¹³ C / ³¹ P are shown in brackets.

Example 19

Production and characterization of cyclipostin T

Fraction 21, obtained according to Examples 5 and 6, was separated as described in Example 8 and cyclipostin T was obtained by repeating the chromatography procedure (5 mg) several times and characterized as in Example 9.

Retention time: 19.1 min.

Appearance: substance soluble in oxygen-containing organic solvents, while only slightly soluble, neutral, colorless, solid substance in water and petroleum ether.

UV-peak: 228 nm in methanol.

By using High Performance hmotriostnej spectrometry under the conditions of the FAB-MS found the quasi-molecular ion (M + H) ⁺ 473.3030 amu, corresponding to the empirical formula cyclipostinu T: ₂ C 5 H ₄ N 6 O ₆ (theory: 473.3032). Characteristic peak in ESI positive mode: 249 amu.

Table 8: Chemical shifts of cyclipostin T in MeOD at 300 K.

^X H ¹³ C 1 - 171.98 (1.7 Hz ⁾ 2 -r 114.39 (3.1 Hz) ^a! 3 3.87 40.78 4 4.46 / 3.85 66, 02 5 4.29 / 4.26 69.23 (5.9 Hz) ^al 6 - 164.69 (8.7 Hz) ^; 7 2.89 / 2.83 33.35 (4.5 Hz ⁾ 8 1.65 20.63 9 0.98 13.84 I ' 4.25 71.57 (6.6 Hz ⁾ 2 ' 1.74 31.18 (6.2 Hz ⁾ 3 ' 1.42 26, 42

4'-13 ' 1.34-1.29 30.78 to 30.11 14 ' 1.34-1.29 33.06 15 ' 1.31 23.72 16 ' 0.89 14.42

a) Interaction constants ¹³ C / ³¹ P are shown in brackets.

Example 20

Production and characterization of cyclipostin T2

Fraction 21 obtained according to Examples 5 and 6 was separated as described in Example 8 and cyclipostin T2 was obtained by repeated chromatography (4 mg) and characterized as in Example 9.

Retention time: 18.7 min.

Appearance: substance soluble in oxygen-containing organic solvents, while poorly soluble, neutral, colorless, solid substance in water and petroleum ether.

UV-peak: 228 nm in methanol.

Quasi-molecular ion (M + H) ^x 473.3035 amu was found by high resolution mass spectrometry under FAB-MS conditions, corresponding to the cyclipostin T total formula: C 25 H 46 O 6 P (Theory: 473, 3032). Characteristic peak in ESI positive mode: 249 amu.

Table 9: Chemical shifts of cyclipostin T2 in MeOD at 300 K.

¹³ c. 1 - 170, 98 (1.7 Hz ⁾ 2 - 114.40 (3.1 Hz ⁾ 3 3.87 40.78 4 4.46 / 3.85 66, 02 5 4.29 / 4.25 69.23 (5.9 Hz) ^al 6 - 164.69 (8.7 Hz) ^and> 7 2.90 / 2.83 33, 35 (4.5 Hz ⁾

8 1.65 20, 63 9 0.98 13.84 I ' 4.24 71.57 (6.9 Hz ⁾ 2 ' 1.74 31.18 (6.2 Hz) ^and> 3 ' 1.42 26, 42 4'-II 1.37-1.25 31,03-30,11 ' 12 ' 1.29 28.52 13 ' 1.17 40.25 14 ' 1.52 29, 15 15 ', 16' 0.87 23.03

a) Interaction constants ¹³ C / ³¹ P are shown in brackets.

Example 21

Hormone-sensitive lipase (HSL) blocking

I

IC 50 values for blocking hormone-sensitive lipase in rats with trioleylglycerin as substrate:

Cyclipostin A: 20 nM, Cyclipostin N: 450 nM, Cyclipostin P: 30 nM, Cyclipostin P2: 40 nM, Cyclipostin R: 10 nM, Cyclipostin R2: 220 nM, Cyclipostin WITH: 20 nM, Cyclipostin T: 200 nM, Cyclipostin T2: 60 nM.

Claims (14)

PATENT CLAIMS A compound of formula I R ' O o (I) FROM? where R ¹ is 1. a hydrocarbon chain of 2 to 30 carbon atoms, which may be straight, branched, saturated or unsaturated, carbocyclic or heterocyclic, the hydrocarbon chain optionally substituted once or twice with: 1.1 -OH group, 1.2 = 0 group, 1.3 -O-C 1 -C 8 -alkyl, wherein the alkyl is straight or branched, 1.4 -0-C ₂ -C 6 -alkenyl, wherein alkenyl is straight or branched, 1.5 -C 1 -C 8 -alkyl, wherein the alkyl is straight or branched, 1.6-aryl group, 1.7 -C 1 -C ₆ -alkylbenzene, 1.8-diphenyl, 1.9 -NH-C 1 -C 8 -alkyl, wherein the alkyl is straight or branched, 1.10 -NH-C ₂ -C ₆ -alkenyl, wherein alkenyl is straight or branched, 1.11 — NH ₂ , 1.12 = With group 1.13 -S-C 1 -C 8 -alkyl, wherein the alkyl is straight or branched, 1.14 -SC ₂ -C 6 -alkenyl, wherein alkenyl is straight or branched; 1.15 halogen, wherein substituents 1.1 to 1.15 may also be substituted, 2.0 - [-aryl- _{(CH2) n] m} group, in which - [-aryl- _{(CH2) n]} m is unsubstituted or substituted one or two substituents described in 1.1 to 1.15, anam are independently integers 0 , 1, 2 or 3; R ² is 1.0 C 1 -C ₆ -alkyl wherein alkyl is unsubstituted or substituted one or two times with the substituents described in 1.1 to 1.15, 2.0 C ₂ -C 6 -alkenyl, wherein alkenyl is unsubstituted or substituted one or two times with the substituents described in 1.1 to 1.15, 3.0 C 1 -C ₆ -alkynyl wherein alkynyl is unsubstituted or substituted one or two times with the substituents described in 1.1 to 1.15, E is phosphorus (P) or sulfur (S), X ¹ , X ² and X ³ are independently of each other 1.0 -0-, 2.0 -NH-, 3.0 -N =, 4.0 -S-, or 5.0 -CH ₂ - and -CHR ² -, in all their stereochemical forms and mixtures thereof in any ratio, as well as their physiologically acceptable salts and chemical equivalents. A compound of formula (I) or a physiologically acceptable salt thereof according to claim 1, wherein R ¹ represents a hydrocarbon chain of 10 to 18 carbon atoms which may be straight, branched, saturated or unsaturated, carbocyclic or heterocyclic or aromatic, wherein the hydrocarbon chain is unsubstituted. or substituted once or twice with the substituents described in 1.1 to 1.15. A compound of formula I, or a physiologically acceptable salt thereof, according to claim 1 or 2, wherein: R ¹ is I. 0 - (CH ₂ ) ₁₅ CH ₃ , 2.0 - (CH ₂ ) i ₃ CH (CH ₃ ) ₂ , 3.0 - (CH ₂ ) _u CH (OH) (CH ₂ ) ₃ CH ₃ , 4.0 - (CH ₂ ) n CH (OH) CH ₂ CH (CH ₃ ) ₂ , 5.0 - (CH ₂ ) ₁₂ CH (OH) (CH ₂ ) ₂ CH ₃ , 6.0 - (CH ₂ ) ₁₃ CH (OH) CH ₂ CH ₃ , 7.0 - (CH ₂ ) i ₄ CH (OH) CH ₃ , 8.0 - (CH ₂ ) and ₅ CH ₂ (OH), 9.0 - (CH ₂ ) and ₆ CH ₃ , 10.0 - (CH ₂ ) i ₃ C = OCH ₂ CH ₃ , II. 0 - (CH ₂ ) ₁₂ C = OCH ₂ CH ₂ CH ₃ , 12.0 - (CH ₂ ) ₁₁ c = and ₂ ch ₂ ch ₂ ch ₃ , 13.0 - (CH ₂ ) ₁₃ CH ₃ , 14.0 - (CH ₂ ) _{X 1} CH (CH ₃ ) ₂ , 15.0 - (CH ₂ ) i ₄ CH ₃ , or 16.0 - (CH ₂ ) i ₂ CH (CH ₃ ) ₂ . A compound of formula I or a physiologically acceptable salt thereof, according to claims 1, 2 or 3, wherein R ² is alkyl having 1 to 6 carbon atoms. A compound of formula I, or a physiologically acceptable salt thereof, according to claim 4, wherein R ² is -CH ₃ , -CH ₂ CH _3, or -CH ₂ CH ₂ CH ₃ . A compound of the formula I or a physiologically acceptable salt thereof according to one or more of claims 1 to 5, which can be prepared by the microorganism Streptomyces species HAG 004107, DSM 13381 or one of its variants or mutations under appropriate conditions by fermentation in the culture medium until accumulation. % of one or more compounds of formula (I) in a culture medium, wherein the compound is subsequently isolated from the culture medium, and optionally converted into a chemical equivalent and a physiologically acceptable salt. Process for the preparation of a compound of the formula I or a physiologically acceptable salt thereof according to one or more of claims 1 to 6, characterized in that the microorganism Streptomyces species HAG 004107, DSM 13381 or one of its variants or mutations is fermented in the culture medium under suitable conditions. until one or more compounds of formula (I) have accumulated in the culture medium, wherein the compound is subsequently isolated from the culture medium, and optionally converted to a chemical equivalent and a physiologically acceptable sqIi. Process for the preparation of a compound of the formula I according to claim 7, characterized in that the fermentation is carried out under aerobic conditions at temperatures from 18 to 35 ° C, at pH values from 6 to 8. A compound of formula I or a physiologically acceptable salt thereof according to one or more of claims 1 to 6 for use as a medicament. A compound of formula I or a physiologically acceptable salt thereof according to one or more of claims 1 to 6 for use as a medicament for inhibiting lipases. A compound of formula I or a physiologically acceptable salt thereof according to one or more of claims 1 to 6 for use as an antidiabetic agent. Pharmaceutical preparation, characterized in that it contains at least one compound of the formula I or a physiologically acceptable salt thereof according to one or more of claims 1 to 6. A process for the preparation of a pharmaceutical composition according to claim 12, characterized in that at least one compound of the formula I or a physiologically acceptable salt thereof according to one or more of claims 1 to 6 is brought into a suitable dosage form together with a suitable excipient and / or carrier. . 14. Streptomyces species HAG 004107, DSM 13381.