NO330438B1 - Cyclipostins, their preparation and use as well as pharmaceutical preparation and microorganism - Google Patents

Description

The present invention relates to new compounds, called cyclipostins, which can be obtained by cultivating Streptomyces specie HAG 004107 (DSM 13381) and their physiologically acceptable salts. The invention further relates to a method for the production of cyclipostins, the microorganism HAG 004107 (DSM 13381), cyclipostins and their physiologically acceptable salts used as pharmaceuticals, in particular as inhibitors of lipases, as well as pharmaceutical preparations with a content of cyclipostin or a physiologically acceptable salt thereof.

JP6056859 and JP4145089 describe cyclipostine derivatives with short-chain side groups. Said compounds are said to exhibit antibiotic and insecticidal properties.

A disease that can be particularly advantageously treated with lipase inhibitors is diabetes mellitus. Diabetes mellitus is a disease characterized by elevated blood sugar concentration due to chronic metabolic disturbances. The metabolic disturbances are due to a lack of insulin or a reduced insulin effect. The missing msuUneffect leads to a weakened utilization of the glucose taken up in the blood by the body's cells. Because of this and because of a new formation of glucose from proteins (gluconeogenesis), there is an increase in the blood glucose level. In addition to this, with reduced insulin action in the fat tissue, the insulin-antagonistic hormones such as glucagon lead to increased lipolysis and thereby to an increased fatty acid concentration in the blood. This leads to ketoacidosis, i.e. an increased formation of ketone bodies (acetoacetic acid, p-hydroxybutyric acid, acetone). Under acute conditions, the degree of biochemical misregulation is life-threatening and leads, without treatment, to diabetic coma and ultimately to rapid death. Diabetes is one of the most frequent chronic metabolic diseases in humans and it is estimated that up to over 3% of the population has a diabetic or pre-diabetic disposition and is thereby acutely threatened.

Therefore, there is a great need for the treatment or cure of diabetes mellitus.

Diabetes is treated with insulin administration and age-related diabetes, so-called non-insulin-dependent (NIDDM) or type H diabetes, is initially administered with sulphonylureas. The principle of action for the sulphonylureas is an increase in insulin secretion from the β-cell in the pancreas in order to compensate for the hormone deficiency or insulin resistance. In case of continued illness, however, insulin must also be used, the insulin effect can be summarized as follows. This peptide hormone lowers the concentration of glucose in the blood and leads to an increase in anabolics and at the same time to an inhibition of catabolic processes:

• Glucose transport in the body cells is increased,

• glycogen production in the liver and muscles is increased,

• lipolysis is inhibited,

• the absorption of fatty acids in the adipose tissue is increased, and

• the uptake of amino acids in the body cells and protein synthesis is increased.

One of the strongest effects of insulin is the inhibition of lipolysis. In type II diabetics, this regulation of lipolysis no longer works 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 the utilization of glucose in the skeletal muscles. Lipolysis, i.e. the release of fatty acids, is controlled by the so-called hormone-sensitive lipase (HSL), which is located in the fat cells and which is inhibited by insulin via a phosphorylation cascade. It would therefore be desirable to have inhibitors, i.e. easier for HSL, which simulate the action of insulin and which are able to reduce the blood fat content. Such agents are suitable for the treatment of type II diabetics for control of fat metabolism, but their use will also be possible for other storage diseases. For all these reasons, new inhibitors of HSL and other lipases are needed and therefore sought.

It has now surprisingly been found that the microorganism strain Streptomyces species HAG 004107, DSM 13381, is able to form very effective new lipase inhibitors that inhibit the hormone-sensitive lipase even in very low concentrations. The new natural compounds are organophosphates consisting of a double ring system, a bicycle, and a substituted hydrocarbon chain, and which specifically inhibit lipases. The ring skeleton is described for the first time with only a methyl group instead of the hydrocarbon chain as an acetylcholinesterase inhibitor, CGA134 736, by R. Neumann & H.H. Peter in Experientia, vol. 43, pp. 1235-1237, 1987 and later the same compound, designated cyclophostin, is described by T. Kurokawa et al. in J. Antibiotics, 46, 1315-1318, 1993. This structurally related compound exhibits no selective lipase inhibitory properties. The substances known up to now exhibit deficiencies that result in insufficient levels of effectiveness, high toxicity and/or unwanted side effects.

A first aspect of the present invention relates to a compound which is characterized in that it is given by formula I

there

R1 means

1. a carbon chain of 10 to 18 carbon atoms that is straight, branched, saturated or

unsaturated, and where the carbon chain is possibly mono- or di-substituted with:

1.1 -OH,

1.2 =0,

1.3 -O-Ci-Ce-alkyl, where alkyl is straight or branched,

R<2>means

1.0 Ci-Ce alkyl, or

2.0 C2-C6-alkenyl.

E is a phosphorus (P),

Xi, X2 and X3 independently of each other means

1.0 -O-,

in all stereochemical forms and mixtures thereof in any ratio, as well as their physiologically acceptable salts.

R<1> has a chain length of 10 to 18 C atoms. The chain may be saturated, e.g. alkyl where alkyl may be straight or branched, or unsaturated, e.g. alkenyl or alkynyl wherein alkenyl or alkynyl is straight or branched. R 1 may be unsubstituted or substituted once or twice, identically or differently, with the groups 1.1 to 1.3 as described above. Preferably, the substituents are in positions 10' to 14'. The substituents 1.1 to 1.3 can also be substituted themselves with one or more groups selected from: alcohol, aldehyde, acetal, ketal, ether, carboxyl, ester, amino, nitrile, nitro, oxime, oximeter and halogen.

Halogen means chloride, bromide, fluoride or pseudohalide, such as cyanide (nitrile). -Ci-C6-alkyl means straight or branched alkyl with 1,2,3,4, 5 or 6 C-atoms such as e.g. methyl, ethyl, i-propyl, tert-butyl and hexyl. -C2-C6-alkenyl means straight or branched alkenyl with 2, 3,4, 5 or 6 C atoms, such as e.g. allyl, crotyl and pentenyl.

R<1> means preferably

1. -(CH2)15CH3,

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

3. -(CH2)„CH(OH)(CH2)3CH3,

4. -(CH2)nCH(OH)CH2CH(CH3)2,

5. -(CH2)i2CH(OH)(CH2)2CH3,

6. -(CH2)i3CH(OH)CH2CH3,

7. -(CH2)i4CH(OH)CH3,

8. -CCH2)15CH2(OH),

9. -(CH2)i6CH3, or

10.0 -(CH2)i3C=OCH2CH3,

11.0 -(CH2)i2C=OCH2CH2CH3,

12.0 -(CH2)nC=OCH2CH2CH2CH3,

13.0 -(CH2)i3CH3,

14.0 -CCH2)nCH(CH3)2,

15.0 -(CH2)i4CH3, or

16.0 -(CH 2 ) 1 2 CH(CH 3 ) 2 .

R<2> preferably means C1-C6 alkyl and especially methyl, ethyl or propyl.

Preferred compounds according to the invention are indicated below:

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

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

The ring system alone contains two asymmetric substituted atoms, C atom 3 and the phosphorus atom. Both atoms can be in the R or S configuration. It has surprisingly been found that the strain Streptomyces species HAG 004107, DSM 13381, is able in each case to form several stereoisomers of the compounds of the general formula I, i.e. that the strain synthesizes compounds in which the atoms C3 and P can independently occupy R - or the S configuration. Isomers with the spatial form of the carbon atom (3) in the R and of phosphorus in the S configuration often occur in cultures of Streptomyces species HAG 004107, DSM 13381.

Formula IA:

In addition, however, cyclopostins with other configurations, such as (R,R), (S,S) or (S,R), are also formed, which surprisingly also have a significant lipase-inhibiting effect.

The compounds of formula I or a physiologically acceptable salt thereof can be prepared by fermenting the microorganism Streptomyces species HAG 004107, DSM 13381, or a variant or mutant thereof, under suitable conditions in a culture medium until one or more compounds of the general formula I are present in large quantity in the culture medium and then isolated from this and possibly transferred to physiologically acceptable salts.

The cyclipostins of the invention can preferably be produced from Actinomycetales species, preferably from Streptomyces species HAG 004107, DSM 13381. Streptomyces species HAG 004107, DSM 13381 has an ivory-colored mycelium (RAL 1014) and is characterized by the conidiophores characteristic of streptomycetes.

An isolate has been deposited at the German Collection for Microorganisms and Cell Cultures, "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH", Mascheroder Weg IB, D-38124 Braunschweig, Germany, according to the rule of the Budapest Agreement, March 16, 2000, under the accession no. : Streptomyces species HAG 004107, DSM 13381.

Instead of the strain Streptomyces species HAG 004107, DSM 13381, one can also use its mutants and variants which synthesize one or more compounds of the cyclipostins of the invention. Such mutants can be obtained in a manner known per se by physical means such as irradiation, e.g. with ultraviolet or X-rays, or chemical mutagens such as ethyl methanesulfonate (EMS), 2-hydroxy-4-methoxy-benzophenone (MOB) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

The invention thereby also relates to a method for producing a compound of formula I or a physiologically acceptable salt thereof and which is characterized in that the microorganism Streptomyces species HAG 004107, DSM 13381, or a variant or mutant thereof, is fermented under suitable conditions in a culture medium until a or more compounds of the general formula I are present in the culture medium in increased quantity and that it is then isolated from the culture medium and possibly transferred to physiologically acceptable salts.

Preferably, the strain Streptomyces species HAG 004107, DSM 13381, their mutants and/or variants are fermented in a nutrient solution (also called culture medium) with a carbon and nitrogen source as well as the usual inorganic salts, until the new cyclipostins are present in increased amounts in the culture medium and then the cyclipostins are isolated from this and possibly separated into individual, active components.

The fermentation is preferably carried out under aerobic conditions and it proceeds particularly well at a temperature between 18 and 35°C and a pH value between 6 and 8.

The method of the invention can be used for fermentation on a laboratory scale (milliliter to liter range) and for industrial scale (cubic meter scale). All percentages refer to weight, unless otherwise stated. The mixing ratio in the liquid refers to volume unless otherwise stated.

Suitable carbon sources for the aerobic fermentation are assimilated carbohydrates and sugar alcohols such as glucose, lactose, sucrose or D-mannitol, as well as carbohydrate-containing natural products such as oatmeal, soy flour and malt extract. Nitrogen-containing nutrients include amino acids, peptides and proteins as well as their breakdown products such as peptones or tryptones, further meat extracts, yeast extracts, ground seeds, e.g. of corn, wheat, beans, soya or cotton, distillation liquids from alcohol production, meat milk and yeast extract, but also ammonium salts and nitrates. As inorganic salts, the nutrient solution can e.g. contain chlorides, carbonates, sulphates or phosphates of alkali or alkaline earth metals, iron, zinc, cobalt and manganese.

The formation of the invention's cyclipostins of formula II to XVA proceeds particularly well in a culture medium containing around 0.1 to around 5% and preferably around 0.3 to 3% of oatmeal and trace elements. The percentages always apply to the weight of the total culture medium.

The preferred formation of the cyclisporins of formula VIII to XVA can be particularly well carried out in nutrient solutions containing about 0.1 to 5% and preferably about 0.3 to 2% glycerol and 0.3 to 5%, preferably 0.5 to 3% soy flour and 0.05 to 1.0 g/l, preferably 0.1 to 1.0 g/l sodium chloride.

In the culture or culture medium, Streptomyces species HAG 004107, DSM 13381, a mixture of cyclipostins is formed. Depending on the composition of the culture medium, the quantitative proportions of one or more of the cyclipostins of the invention may vary. In addition, the synthesis of the individual cyclipostins can be controlled by varying the composition of the medium so that one or more of the cyclipostins are not produced at all or produced in an amount that is below the detection limit for the microorganism.

Preferably, the culture medium contains a detectable cyclipostin. Preferably, the cyclipostine A or P or P2 is formed.

In addition to the cyclipostins A to T2 (compounds of formulas II to XVA), further related compounds are also formed in the culture medium of Streptomyces species HAG 004107, DSM 13381, which differ from the compounds of formulas II to XVA by changed residues R 1 and R *). In small amounts, cyclipostins exhibit shortened or further branched residues R<1>. Oxidation (hydroxylation) products of secondary components can also be detected in cultures of Streptomyces HaG 004107, DSM 13381.

The cultivation of the microorganism takes place aerobically, i.e. e.g. submerged under stirring or stirring in shaking flasks or fermenting, possibly during the introduction of air or oxygen.

It can be carried out in temperature ranges around 18 to 35°C, preferably at around 25 to 32°C and especially at 26 to 30°C. The pH range should be between 6 and 8 and preferably between 6.5 and 7.8. The microorganisms are cultivated under these conditions generally for a period of 24 to 300 hours, and in particular 30 to 90 hours.

Preferably, cultivation is done in several stages, i.e. one or more pre-cultures are first added to a liquid culture medium which is then impaled into the actual production medium, the main culture, e.g. in the volume ratio 1:10. The preliminary culture is obtained, for example, by soaking a mycelium in a nutrient solution and then allowing this to grow at approx. 36 to 120 hours, preferably 48 to 96 hours. The mycelium can e.g. is achieved by allowing the stem to grow for around 3 to 40 days and preferably 4 to 10 days on a solid or liquid nutrient medium, e.g. malt-yeast-agar or oatmeal-agar.

The fermentation process can be monitored using the pH values in the culture or the mycelium volume as well as by chromatographic methods, e.g. thin layer chromatography or

HPLC or testing of the biological activity. The cyclipostins of the invention are contained in the mycelium and to a lesser extent also in the culture filtrate. The isolation method described below is used for working up the cyclipostins of the invention, preferably for the purification of cyclipostin A and P.

The isolation or purification of the cyclipostins of the invention from the culture medium takes place according to known methods, taking into account the chemical, physical and biological properties of the natural substances. Thin-layer chromatography can be used for testing the cyclipostin concentration in a culture medium or in the individual isolation steps, e.g. on silica gel with methylene chloride:ethyl acetate or chloroform:methanol, e.g. in a ratio of 98:1, as an eluent, or HPLC. The detection by thin-layer chromatographic separation occurs e.g. by means of dyes such as molybdatophosphoric acid or ^-vapour, whereby the amount of substance formed is suitably compared with a calibration solution.

To isolate the cyclipostins of the invention, the mycelium is first separated from the culture medium using methods known per se and then the cyclipostins are extracted from the cell mass with an optional water-miscible organic solvent. The organic solvent phase contains the cyclipostins of the invention and these are optionally concentrated under vacuum and then purified further as described below.

The culture filtrate is optionally combined with the concentrate from the mycelium extract and extracted with a suitable water-immiscible organic solvent, e.g. with n-butanol or ethyl acetate. The then separated organic phase is optionally concentrated under vacuum and dissolved in 1/30 of the original water:methanol volume.

The further purification of one or more of the cyclipostins of the invention takes place by chromatography on suitable materials, preferably e.g. on molecular sieves, on normal-phase carriers such as e.g. silica gel, aluminum oxide, on ion exchanges or on adsorber resins or on reversed phases. By means of these chromatography methods, the cyclipostins are separated. Chromatography of the cyclipostins takes place with organic solvents or with mixtures of aqueous and organic solutions.

By mixtures of aqueous or organic solutions is meant all water-miscible solvents, preferably methanol, propanol and acetonitrile, in a concentration of 10 to 100% solvent, preferably 60 to 90% solvent, or also all buffered aqueous solutions that are miscible with organic solvents . The buffers used are the same as stated above.

Separation of the cyclipostins due to their different polarity occurs by means of reverse phase chromatography, e.g. on MCI ® (adsorber resin from Mitsubishi, Japan) or Amberlite XAD ® (TOSOHAAS), on other hydrophobic materials, such as e.g. RP-8 or RP-18 phases. In addition, the separation can take place using normal phase chromatography, e.g. on silica gel, aluminum oxide or the like.

The chromatography of the cyclipostins takes place with buffered or acidified aqueous solutions or mixtures of aqueous solutions with alcohols or other water-miscible organic solvents. Propanol and acetonitrile are preferably used as organic solvents.

By buffered or acidified aqueous solutions is meant e.g. water, phosphate buffer, ammonium acetate, citrate buffer in a concentration of 1 mM to 0.5 M as well as formic acid, acetic acid, trifluoroacetic acid or other commercial acids known to the person skilled in the art, preferably in a concentration of from 0.01 to 3%, especially 0.1% .

One chromatographs with a gradient starting with 100% aqueous buffer and ending with 100% solvent, preferably one works according to a linear gradient 50 to 100% 2-propanol or acetonitrile.

Alternatively, a gel chromatography or chromatography on hydrophobic phases can also be carried out.

Gel chromatography is carried out on a polyacrylamide or mixed polymer gel, e.g. Biogel-P 2 ® (Fa. Biorad), Fractogel TSK HW 40 ® (Fa. Merck, Germany or Toso Haas, USA) or a Sephadex ® (Fa. Pharmacia, Uppsala, Sweden).

The order of the aforementioned chromatographies can be reversed.

A further and very effective purification step for cyclipostins is crystallization. CykUpostin crystallizes from solutions in organic solvents and from mixtures of water with organic solvents. The crystallization is carried out in a manner known per se, e.g. by concentrating or cooling saturated cyclipostine solutions.

The cyclipostins of the invention are stable in solid or liquid state and in solutions in the pH range between 4 and 8 and especially between 5 and 7, and can thereby be incorporated into normal galenic preparations.

The present invention includes all stereoisomeric forms of the compounds of formula I to XVA. In the compounds of formula I to XVA, the asymmetric centers contained can all, independently of one another, exhibit S-configuration or R-configuration. The invention also includes all possible enantiomers and diisostereomers, as well as mixtures of two or more stereoisomeric forms, e.g. mixtures of the enantiomers and/or diastereomers, in all proportions. Within the scope of the invention are also enantiomers 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 ratios. When there is a cis/trans isomerism, both the cis form and the trans form and mixtures of these forms in all proportions are within the scope of the invention.

Due to the valuable pharmacological properties, the compounds of the invention are suitable for use as pharmaceuticals in human and/or animal medicine. They inhibit lipases and have beneficial properties for the treatment of metabolic diseases that cause disturbances in lipid metabolism. The compounds of the invention with the general formula I have the surprising inhibitory effect on the hormone-sensitive lipase, HSL, an allosteric enzyme in adipocytes, which is inhibited by insulin and which is responsible for the breakdown of fat in fat cells and thus for the transfer of fat components into the bloodstream. An inhibition of these enzymes thus corresponds to an insulin-like effect for the compounds of the invention which ultimately leads to a reduction of free fatty acids in the blood and of blood sugar. They can also be used when derailing the metabolism, such as e.g. in non-insulin-dependent diabetes mellitus, in diabetic syndrome and in direct damage to the pancreas.

The invention thus relates to pharmaceutical preparations containing one or more of the cyclipostins of the invention. It is preferred to use it in admixture with suitable excipients or carriers. All pharmacologically acceptable carriers and/or excipients can be used as carriers in humans.

The invention further relates to a method for the production of a medicinal product according to the invention which is characterized by bringing at least one of the compounds of the invention together with a pharmaceutically suitable and physiologically acceptable carrier and possibly further suitable active ingredients, additives or auxiliaries into a suitable administration form .

The pharmaceuticals of the invention are generally administered orally, locally or parenterally, however, in principle rectal application is also possible. Suitable solid or liquid galenic preparation forms are e.g. granules, powders, tablets, dragees (micro-)capsules, drops, syrups, emulsions, suspensions, aerosols, drops or injectable solutions in ampoule form as well as preparations with prolonged active ingredient addition in the production of which usually carriers and additives and/or auxiliaries such as explosives , binders, coating agents, swelling agents, lubricants, flavourings, sweeteners and dissolving agents, are used. Frequently used carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk egg white, gelatin, starch, vitamins, cellulose and derivatives thereof, animal or vegetable oils, polyethylene glycols and solvents such as sterile water, alcohols, glycerol and polyhydric alcohols.

Optionally, dosage units can be prepared in microencapsulated form for oral administration to delay release or to extend it over a longer period of time, e.g. by coating or embedding the active ingredient in pellet form in suitable polymers, waxes and the like.

Preferably, the pharmaceutical preparations are prepared in unit doses and administered as such, whereby each unit contains as active ingredient one or more compounds of the invention's cyclipostins and/or chemical derivatives thereof. In the case of fixed unit doses such as tablets, capsules and suppositories, this dose can be up to 200 mg, preferably around 0.1 to 100 mg, and in the case of injection solutions in ampoule form up to around 200 mg, preferably around 0.1 to 100 mg per day.

The daily dose that is administered depends on the body weight, age, gender and condition of the person receiving treatment. However, higher or lower daily doses may also be appropriate. Administration of the daily dose can take place both by giving everything at once in the form of a single dosage unit, or it can be carried out in several smaller dosage units or also by multiple administrations in divided doses at special intervals. The invention also relates to pharmaceutical preparations containing one or more of the cyclipostins of the invention. It is preferred to use it in admixture with suitable excipients or carriers. As a carrier, in the case of humans, all pharmacologically acceptable carrier materials and/or excipients can be used.

The effect of the invention's compounds of formula I has been examined by the following enzyme test system.

Enzyme preparation:

Preparation of the partially purified HSL:

Isolated rat fat cells are obtained from epididymal adipose tissue of untreated natural rats (Wistar, 220-250 g) by collagenase treatment according to published methods (see, e.g., S. Nilsson et al., Anal. Biochem. 158, 1986, 399-407; g. Fredikson et al., J. Biol. Chem. 256, 1981, 6311-6320; H. Tornquist et al., J. Biol. Chem. 251, 1976, 813-819). The fat cells from 10 rats are washed three times by flotation with 50 ml each time of homogenization buffer (25 ml Tris/HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 10 µg/ml leupeptin, 10 | u.g/ml antipain, 20 jxg/ml pepstatin) and finished in 10 ml homogenisation buffer. The fat cells are homogenized in a Teflon-in-glass homogenizer (Braun-Melsungen) at 10 elevations at 1,500 rpm. minute and 15°C. The homogenate is centrifuged (Sorvall SM24-R6hrchen, 5000 revolutions per minute, 10 min, 4°C). The residue between the overlying fat layer and the pellet is removed and the centrifugation is repeated. The resulting underlying mass is centrifuged once more (Sorvall SM24-Rohrchen, 20,000 revolutions per min., 45 min., 4°C). The underlying is taken out and 1 g of heparin-Sepharose (Pharmacia-Biotech, CL-6B, washed 5 times with 25 mM Tris/HCl, pH 7.4, 150 mM NaCl) is added. After incubation for 60 minutes at 4°C (shaking at intervals of 15 minutes), the whole is centrifuged (Sorvall SM24-Rohrchen, 3000 revolutions, 10 min., 4°C). The supernatant is brought to pH 5.2 by adding glacial acetic acid and incubated for 30 min. at 4°C. The precipitate is collected by centrifugation (Sorvall SS34, 12000 rpm, 10 min, 4°C) and suspended in 2.5 ml of 20 mM Tris/HCl, pH 7.0, 1 mM EDTA, 65 mM NaCl, 13 % sucrose, 1 mMDTT, 10 µg/ml leupeptin/pepsMin/antipain. The suspension is dialysed overnight at 4°C against 25 mM Tris/HCl, pH 7.4, 50% glycerol, 1 mM DTT, 10 µg/ml leupeptin, pepstatin, antipain and then brought onto a hydroxyapatite column (0.1 g per 1 ml suspension, equilibrated with 10 mM potassium phosphate, pH 7.0, 30% glycerol, 1 mM DTT). The column is washed with 4 volumes of equilibration buffer at an elution rate of 20 to 30 ml/hour. HSL is eluted with 1 volume of equilibration buffer containing 0.5 M potassium phosphate and then dialyzed (see above) and concentrated 5- to 10-fold by ultrafiltration (Amicon Diaflo PM 10 Filter) at 4°C. The partially purified HSL can be stored for 4 to 6 weeks at -70°C.

Analysis:

To prepare the substrate, mix 25 - 50 u.Ci[<3>H]trioleoylglycerol (in toluene), 6.8 umol unlabelled trioleoylglycerol and 0.6 mg phospholipids

(phosphatidylcholine/phosphatidylinositol 3:1 weight/volume), after which the whole is dried over N2 and taken up in 2 ml of 0.1 M KPi (pH 7.0) by ultrasonic treatment (Branson 250, Mikrospiss, setting 1 - 2.2 x 1 min with 1 min interval). After adding 1 ml of KP, and new ultrasound treatment (4 x 30 sec. on ice in 30 second intervals), 1 ml of 20% BSA (Bovine serum albumin) (in KPi) is added (final concentration trioleoylglycerol 1.7 mM). For the reaction, 100 ul substrate solution is pipetted into 100 ul HSL solution (HSL prepared as above, diluted in 20 mM KPi, pH 7.0, 1 mM EDTA, 1 mM DTT, 0.02% BSA, 20 µg/ml pepstatin, 10 ug/ml leupeptin) and the whole is incubated for 30 min. at 37°C. After adding 3.25 ml of methanol/ldoroform/heptane (10:9:7) and 1.05 ml of 0.1 M K2CO3, 0.1 M boric acid (pH 10.5), the whole is mixed well and centrifuged (800 x g , 20 min.). After phase separation, an equivalent of the upper phase (1 ml) is taken out and the radioactivity is determined by liquid scintillation counting.

Rating:

The substances are tested in the usual way in four independent approaches. The inhibition of enzymatic activity of HSL due to a test substance is determined by comparison with an uninhibited control reaction. The ICso values are calculated via an inhibition curve with at least 10 concentrations of the test substance. For analysis of the data obtained, the program package GRAPHIT, Elsevier-BIOSOFT, is used. In this test, the compounds showed the following effect: The cyclipostins A, P, P2 and R inhibit lipolysis in rat adipocytes with IC50=~0.2 uM, they inhibit the human hormone-sensitive lipase (HSL) with trioleylglycerol as substrate: IC50=~0 .07 to 0.5 µM. HSL from rats is inhibited, with NBD (4-chloro-7-nitrobenzo-2-oxa-1,3-diazole) as substrate, in concentrations of from 4 nM to 10 riM.

The cyclipostins inhibit both the hormone-sensitive lipase (HSL) and also the mono-acyl-glycerol lipase in the rat extracts at submicromolar concentrations.

The invention is explained in more detail in the following examples. Percentages refer to the weight. The mixing ratios of the liquids refer to volumes unless otherwise stated.

Examples

Example 1

Preparation of a glycerol culture of Streptomyces species HAG 004107, DSM 13381.

100 ml nutrient solution (malt extract 2.0%, yeast extract 0.2%, glucose 1.0%

(NH4)2HP040.05%, pH 6.0) in a sterile 300 ml Erlenmeyer flask was inoculated with the strain Streptomyces species HAG 004107, DSM 13381 and incubated for 7 days at 28°C and 180 rpm. minute on a rotary shaker. 1.5 ml of this culture was then diluted with 1.5 ml of 99% glycerol and stored at -20°C.

Example 2

Preparation of a pre-culture in the Erlenmeyer flask of Streptomyces species HAG 004107, DSM 13381.

A sterile 300 ml Erlenmeyer flask with 100 ml of the following nutrient solution: 15 g/l glucose, 15 g/l soy flour, 5 g/l corn starch, 2 g/l CaCO3 and 5 g/l NaCl is impaled with a culture grown on a slant tube (same nutrient solution, however with 2% agar) or with 1 ml of a glycerol culture (see example 1) and incubated on a shaking machine at 180 revolutions per minute. minute and 28°C. For impinging 10 or 2001 fermenters, a 48- to 96-hour-old submerged culture (inoculation amount approx. 10%) of the same nutrient solution is sufficient.

Example 3

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

Sterile 300 ml Erlenmeyer flask with 100 ml of the following nutrient solution:

20 g/l dog oats

2.5 ml trace element solution

inoculate with 10% inoculation amount of the pre-culture from example 2 and incubate on a shaking machine at 180 revolutions per minute and 28°C. The culture can be used after 2 days to obtain the cyclipostins or to inoculate the ferments. The trace element solution had the following composition:

3 g/l CaCl2x 2 H20

1 g/l Fe(III) citrate;

0.2 g/l MnSO 4 x H 2 O;

0.1 g/lZnCk;

0.025g/lCuSO4x5H2Os

0.02 g/l Na tetraborate

0.004 g/l CoCl 2 x 6 H 2 O

0.01 g/l Na molybdate.

Example 4

Preparation of cyclipostins of formulas II to IX.

A 2001 fermenter is operated with 90 liters of nutrient solution under the following conditions: Nutrient medium: 20 g/l oatmeal in water;

2.5 ml/l trace element.

pH 7.8 (before sterilization)

The nutrient solution is heat sterilized for 30 minutes and after cooling it is inoculated with 5% of the volume with impe material, obtained according to example 3.

Trace element solution: 3 g/l CaCl2x 2 H20

1 g/l Fe(in) citrate;

0.2g/lMnSO4xH2O;

0.1 g/l ZnCl2;

0.025 g/l CuS04x 5 H20,

0.02 g/l Na tetraborate

0.004 g/l CoCl 2 x 6 H 2 O

0.01 g/l Na molybdate.

Process duration: 72 hours

Incubation temperature: 28°C

Stirring speed: 90 stirrings per minute

Ventilation: 6 m<5> air per hour.

The fermentation was carried out without the addition of an antifoam agent. The production maximum was reached after approx. 40 to 76 hours.

Example 5

Preparation of cyclipostins X to XVA.

A 2001 fermenter with 100 liter filling is operated under the following conditions: Nutrient medium: 5 g/l glucose; 20 g/l glycerol; 20 g/l soy flour; 5 g/1 yeast extract; 3 g/l NaCl;

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

Process duration: 72 hours

Incubation temperature: 27°C

Stirring speed: 65 stirrings per minute

Ventilation: 6 m3 air per hour.

The fermentation was carried out without the addition of agents to suppress the formation of foam. The production maximum was reached after approx. 48 hours.

Example 6

Isolation of cyclipostin mixtures from the culture solution of Streptomyces species HAG 004107, DSM 13381.

After completion of fermentation of Streptomyces species HAG 004107, DSM 13381, 100 liters of culture broth from the fermenter, obtained according to example 4, are filtered while adding approx. 2% filtration aid (e.g. Celite ® ) and the cell mass (10 litres) is extracted with 40 1 of methanol. The active ingredient-containing methanolic solution is freed from mycelium by filtration and concentrated under vacuum. The concentrate is taken up on a prepared, 7 liter ®MCI GEL, CHP20P column. It is eluted with a gradient of water after propanol-2. The column flow-through (20 liters per hour) is collected from fractions of 10 liters and the cyclipostin-containing fractions (19 to 21) are each concentrated in vacuo. Using HPLC, the fractions are examined (see example 7). Fraction 19 contains cyclipostin A to E and their isomers, fraction 20 cyclipostin F and isomers thereof, fraction 21 the inhibitors cyclipostin N, P, P2, Q, R, S and T and their isomers.

Example 7

HPLC analysis of the cyclipostins.

HPLC analysis of the cyclipostins is carried out in an HP 1100 © facility with YMC-Pack Pro C18 © columns [AS-303,250 x 4.6 mm, S-5 um, 120 A°]. The flow rate is 1 nil/minute, the column temperature 40°C. A gradient of 0.05% trifluoroacetic acid to acetonitrile is used. 100% acetonitrile is reached as eluent after 11 minutes and then it is further eluted with this solvent unchanged (isocratic). The detection takes place by means of ultraviolet absorption at 210 nm. Proceeding in this way, the cyclipostins have the following retention time:

Cyclipostin A 12.7 minutes,

Cyclipostin A 2 12.6 minutes,

Cyclipostin F 13.2 minutes,

Cyklipostin 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,

Cyklipostin S 18.5 minutes,

Cyklipostin T 19.1 minutes, and

Cyclipostin T 2 18.7 minutes,

Example 8

Preparation of pure cyclipostin A and A 2.

Fraction 19, obtained according to Example 6, is concentrated under vacuum and 1 g of the concentrate, dissolved in water:methanol (1:1), is applied to a Nucleoprep 100-5 CisAB ® column (21 x 250 mm). It is eluted with a gradient from 50% acetonitrile in 0.01% trifluoroacetic acid to 100% acetonitrile. The flow rate is 50 ml/min. The column throughput was checked by measuring the light absorption at 210 nm and by testing the lipase inhibitory properties. Fractions of 60 ml are extracted. In fractions 34 and 35 there is cyclipostin A, in fractions 41 to 44 cyclipostin A 2. These fractions are combined, evaporated under vacuum and successively separated on a SP 250/10 Nucleosil 100-5 C18 HD © column. As a gradient, 50% to 66% acetonitrile in 0.01% trifluoroacetic acid was chosen, the pH value of the solution was adjusted with a drop of ammonium hydroxide solution to 4.0. The fractions containing the pure compounds were pooled and freeze-dried. They yielded 5.4 mg of pure cyclipostin A as a wax-like substance and 3 mg of cyclipostin A 2 as an oil.

Example 9

Characterization of cyclipostin A.

Appearance: soluble in oxygen-containing organic solvents, but only sparingly soluble in water and petroleum ether, neutral, colourless, wax-like substance.

UV maximum: 228 nm in methanol.

IR band: 1752 and 1671 cm"<1>.

By high-resolution FAB mass spectrometry with a nitrobenzyl alcohol:LiCl matrix, the following molecular weights were obtained: 467.2757 amu, corresponding to the total formula for cykhpostinin A-Li on C23H41O7PL1. This gives a total formula for cyclipostin A of C23H41O7P, molecular weight: 460. In electrospray mass spectrometry, there is a peak at 461 amu, corresponding to (M + H)<+>; beyond this the characteristic peak at 221 amu, corresponding to C7H10O6P. In ESI-negative mode one finds 459 amu (M - H)\ 337 amu (C16H34O5P) and 219 amu (CyHgOeP). To determine the position of the alcohol group, it was derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide and the probe examined by electron ionization mass spectrometry. The trimethylsilyl derivative was obtained:

The mass 554 amu. The position of the silylated hydroxy group is indicated by the intensive ions at 497 amu (α-cleavage) and 159 amu (α-cleavage). NMR signals: see Table 1.

Example 10

Characterization of cyclipostin B.

Cyclipostin B is described as in example 8 for cyclipostin A, isolated by repeating the chromatographic steps several times and characterized as in example 9.

Appearance: soluble in oxygen-containing, organic solvents, however in water and petroleum ethers slightly soluble, neutral, colourless, wax-like substance.

UV maximum: 228 nm in methanol.

In electron spray mass spectrometry, in positive ionization mode (ESI, positive) there is a peak at 461 amu, corresponding to (M + H)<+>; beyond this the characteristic peak at 221 amu, corresponding to C^HioOgP. In ESI-negative mode, one finds 459 amu (M - H)"; 337 amu (C16H34O5P) and 219 amu (CvHgOftP). To determine the position of the alcohol group, derivatization is done with N-methyl-N-trimethylsilyl-trifluoroacetamide and the sample is examined with electron ionization mass spectrometry The trimethylsilyl derivative with a mass of 554 amu is obtained:

The position of the silylated hydroxy group is indicated by intensive ions at 511 amu (α-cleavage) and 145 amu (α-cleavage).

General formula for cyclipostin B: C23H41O7P, molecular weight: 460.

Example 11

Characterization of cyclipostin C.

Cyclipostin C is described as in example 8 for cyclipostin A, isolated by repeating the chromatographic steps several times and characterized as in example 9.

Appearance: soluble in oxygen-containing organic solvents, but only slightly soluble in water and petroleum ether, neutral, colourless, wax-like substance.

UV maximum: 228 nm in methanol.

In electron spray mass spectrometry, in positive ionization mode (ESI, positive) there is a peak at 461 amu, corresponding to (M + H)<+>; beyond this the characteristic peak at 221 amu, corresponding to C7H10O6P. In ESI-negative mode, one finds 459 amu (M - H)"; 337 amu (Ci6H3405P) and 219 amu (C7H806P). To determine the position of the alcohol group, derivatization is done with N-methyl-N-trimethylsilyl-trifluoroacetamide and the sample is examined by electron ionization mass spectrometry The trimethylsilyl derivative with a mass of 554 amu is obtained:

The position of the silylated hydroxy group is indicated by intensive ions at 525 amu (0 cleavage) and 131 amu (α cleavage).

General formula for cyclipostin C: C23H41O7P, molecular weight: 460.

Example 12

Characterization of cyclipostin F.

Fraction 20, obtained according to example 6, is described as in example 8, separated and cyclipostin F is isolated by repeating the chromatographic steps several times and characterized as in example 9.

Retention time: 13.2 minutes.

Appearance: soluble in oxygen-containing organic solvents, but only slightly soluble in water and petroleum ether, neutral, colourless, wax-like substance. UV maximum: 228 nm in methanol.

In dextron spray mass spectrometry, in positive ionization mode (ESI, positive) there is a peak at 459 amu, corresponding to (M + H)<+>; beyond this the characteristic peak at 221 amu, corresponding to C7H10O6P. In ESI-negative mode one finds 457.6 amu (M -H)"; 336 amu (C16H32O5P) and 219 amu (C7H806P).

General formula for cyclipostin F: C^HjgC^P, molecular weight: 458.

Example 13

Characterization of cyclipostin P.

Fraction 21, obtained after examples 5 and 6, is described as in example 8, separates and cyclipostin P is isolated (210 mg) by repeating the chromatographic steps several times and is characterized as in example 9. By dissolving the 210 mg in 3 ml of propanol -2 and 13 ml of acetonitrile and the addition of 8 ml of water, cyclipostin P was crystallized. After filtering off and washing with cold acetonitrile, 135 mg of cyclipostin with a melting point of 58 - 59°C were weighed out.

Retention time: 17.7 minutes.

Appearance: soluble in oxygen-containing organic solvents, but only slightly soluble in water and petroleum ether, neutral, colourless, wax-like substance. UV maximum: 228 nm in methanol.

IR signals: 2917,2852, 1753,1671,1471, 1214, 996 and 832 cm"<1>.

By high-resolution FAB mass spectrometry with a nitrobenzyl alcohol matrix, the following molecular weight was obtained: 445.2717 amu, corresponding to (M + H)<+>for cyclipostin P with C23H42O&P. Based on this, a formula for cyclipostin P of C23H42O6P is obtained. Molecular weight: 444.

In electron spray mass spectrometry, in positive ionization mode (ESI, positive) there is a peak at 445 amu, corresponding to (M + H)<+>; beyond this the characteristic peak at 221 amu, corresponding to C7H10O6P. In ESI-negative mode one finds 443 amu (M - H)"; 321 amu (C16H34O4P) and 219 amu (C7H806P).

NMR data are given in Table 2.

Example 14

Characterization of cyclipostin P 2.

Fraction 21, obtained after examples 5 and 6, is described as in example 8, separated and cykpostin P 2 is isolated (130 mg) by repeating the chromatographic steps several times and characterized as in example 9.

Retention time: 17.1 minutes.

Appearance: soluble in oxygen-containing, organic solvents, but only sparingly soluble in water and petroleum ether, neutral, colorless, oily substance.

UV maximum: 228 nm in methanol.

By high-resolution FAB mass spectrometry with a nitrobenzyl alcohol matrix, the following molecular weights are found: 445.27217 amu, corresponding to (M + H)<+>for cyclipostin P with C23H42O6P. Based on this, a formula for cyclipostine P 2 of C23H41O6P is obtained. Molecular weight: 444.

In electron spray mass spectrometry, in positive ionization mode (ESI, positive) there is a peak at 445 amu, corresponding to (M + H)<+>; beyond this the characteristic peak at 221 amu, corresponding to C7H10O6P. In ESI-negative mode one finds 443 amu (M - H)<->; 321 amu (Ci6H34O4P) and 219 amu (C7H8O6P).

NMR data for cyclipostin P 2 are reproduced in Table 3.

Example 15

Extraction and characterization of cyclipostin N.

Fraction 21, obtained after examples 5 and 6, is described as in example 8, separated and cyclipostin N is isolated (2 mg) by repeating the chromatographic steps several times and characterized as in example 9.

Retention time: 15.9 minutes.

Appearance: soluble in oxygen-containing organic solvents, but only slightly soluble in water and petroleum ether, neutral, colourless, oil-like substance. UV maximum: 228 nm in methanol.

By high-resolution mass spectrometry under FAB conditions, a quasimolecular ion (M + H)<+> was observed at 417.2405 corresponding to a formula of C^iHsgOeP (theory: 417.2406). Characteristic fragment in ESr mode: 221 amu.

Example 16

Acquisition and characterization of cyclipostin R.

Fraction 21, obtained according to examples 5 and 6, is described as in example 8, separated and cyclipostin R is isolated (8 mg) by repeating the chromatographic steps several times and characterized as in example 9.

Retention time: 16.7 minutes.

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

UV maximum: 228 nm in methanol.

By high-resolution mass spectrometry with FAB conditions, a quasimolecular ion (M + H) was observed at 431.2561 corresponding to a formula of C22H40OGP. (Theory: 431.2562). Characteristic fragments in ESf^ mode: 221 amu.

Example 17

Acquisition and characterization of cyclipostin R 2.

Fraction 21, obtained according to examples 5 and 6, is described as in example 8, separated and cyclipostin R 2 is isolated (8 mg) by repeating the chromatographic steps several times and characterized as in example 9.

Retention time: 16.4 minutes.

Appearance: soluble in oxygen-containing, organic solvents, but only sparingly soluble in water and petroleum ether, neutral, colorless, oily substance.

UV maximum: 228 nm in methanol.

By high-resolution mass spectrometry under FAB conditions, a quasimolecular ion (M + H) was observed at 431.2564 corresponding to a formula of C^HtoOeP. (Theory: 431.2562). Characteristic fragments in ESF^ mode: 221 amu.

Example 18

Acquisition and characterization of cykKpostin S.

Fraction 21, obtained according to examples 5 and 6, is described as in example 8, separated and cyclipostin S is isolated (0.7 mg) by repeating the chromatographic steps several times and characterized as in example 9.

Retention time: 18.5 minutes.

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

UV maximum: 228 nm in methanol.

By high-resolution mass spectrometry under FAB conditions, a quasimolecular ion (M + H) was observed at 459.2883 corresponding to a formula of C24H44O6P. (Theory: 459.2575). Characteristic fragments in ESI^ mode: 235 amu.

Example 19

Acquisition and characterization of cyclipostin T.

Fraction 21, obtained according to examples 5 and 6, is separated as described in example 8 and cyclipostin T is isolated (5 mg) by repeating the chromatographic steps several times and characterized as in example 9.

Retention time: 19.1 minutes.

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

UV maximum: 228 nm in methanol.

By high-resolution mass spectrometry under FAB conditions, a quasimolecular ion (M + H) was observed at 473.3030 corresponding to a formula of C25H46O6P. (Theory: 473.3032). Characteristic fragments in EST^ mode: 249 amu.

Example 20

Acquisition and characterization of cyclipostin T 2.

Fraction 21, obtained according to examples 5 and 6, is described as in example 8, separated and cyclipostin R is isolated (8 mg) by repeating the chromatographic steps several times and characterized as in example 9.

Retention time: 18.7 minutes.

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

UV maximum: 228 nm in methanol.

By high-resolution mass spectrometry with FAB conditions, a quasimolecular ion (M + H) was observed at 473.3035 corresponding to a formula of C25H446O6P.

(Theory: 473.3032). Characteristic fragments in ESI^ mode: 249 amu.

Example 21

Inhibition of the hormone-sensitive lipase (HSL).

The hormone-sensitive lipase from rats was inhibited with trioleyl-glycerol as substrate in the following concentrations (IC50):

Cyclipostin A: 20 nM,

Cyclipostin N: 450 nM,

Cyclipostin P: 30 nM,

Cyclipostin P 2: 40 nM,

Cyclipostin R: 10 nM,

Cyclipostin R 2: 220 nM,

Cyclipostin S: 20 nM,

Cyclipostin T: 200 nM,

Cyclipostin T 2: 60 nM,

Claims (12)

1. Compound, characterized in that it is given by formula I there R<1>means 1. a carbon chain with 10 to 18 C atoms which is straight, branched, saturated or unsaturated, and where the carbon chain is optionally mono- or disubstituted with: 1.1 -OH, 1.2 =0, 1.3 -O-Ci -Ce alkyl, where alkyl is straight or branched, R<2> means 1.0 C 1 -C 6 alkyl, or 2.0 C 2 -C 6 alkenyl. E is a phosphorus (P), Xi, X2 and X3 independently of each other mean 1.0 -O-, in all stereochemical forms and mixtures thereof in any ratio, as well as their physiologically acceptable salts. 2. Compound with formula I or a physiologically acceptable salt thereof according to claim 1, characterized by R<1>means I. 0 -(CH2)isCH3, 2.0 -(CH2),3CH(CH3)2, 3.0 -(CH2)iiCH(OH)(CH2)3CH3)4.0 -(CH2)iiCH(OH)CH2CH (CH3)2, 5.0 -(CH2)i2CH(OH)(CH2)2CH3, 6.0 -(CH2)i3CH(OH)CH2CH3> 7.0~<CH2)i4CH(OH)CH3, 8.0 -CCH2)15CH2(OH), 9.0 -CCH2)i6CH3>or 10.0 -(CH2)13C=OCH2CH3, II. 0 -(CH2)i2C=OCH2CH2CH3j12.0 -(CH2)uC=OCH2CH2CH2CH3j13.0 -fCH2)i3CH3, 14.0 ^CH2)nCH(CH3)2, 15.0 -{CH2)i4CH3, or 16.0 -(CH2)i2CH(CH3 )2. 3. Compound with formula I or a physiologically acceptable salt thereof according to claim 1 or 2, characterized in that R 2 means C 1 -C 6 alkyl. 4. Compound of formula I or a physiologically acceptable salt thereof according to claim 3, characterized in that R<2> means -CH3, -CH2CH3 or -CH2CH2CH3. 5. Method for producing a compound of formula I or a physiologically acceptable salt thereof according to one or more of claims 1 to 4, characterized in that the microorganism Streptomyces species HAG 004107, DSM 13381, or a variant or mutant thereof, is fermented under suitable conditions in a culture medium until amounts of one or more of the compounds of the general formula I occur in the culture medium, and then to isolate the compounds from this and optionally transfer the compounds to physiologically acceptable salts. 6. Method according to claim 5, characterized in that the fermentation is carried out under aerobic conditions at a temperature of between 18 and 35°C and at a pH value of between 6 and 8. 7. Compound with formula I or a physiologically acceptable salt thereof according to one or more of claims 1 to 4 for use as a medicine. 8. Compound of formula I or a physiologically acceptable salt thereof according to one or more of claims 1 to 4 for the treatment of diabetes mellitus, such as non-insulin dependent diabetes mellitus, and diabetic syndrome. 9. Compound of formula I or a physiologically acceptable salt thereof according to claim 8 for use as an anti-diabetic agent. 10. Pharmaceutical preparation, characterized in that it contains at least one compound of formula I or a physiologically acceptable salt thereof according to one or more of claims 1 to 4 in combination with a suitable pharmaceutical carrier and/or excipient. 11. Process for the production of pharmaceutical preparations as defined in claim 10, characterized in that at least one compound of formula I or an acceptable salt thereof according to one or more of claims 1 to 4 is brought into a suitable administration form together with suitable excipients and/or carriers. 12. Microorganism, characterized in that it is Streptomyces species HAG 004107, DSM 13381.