HK1061655B - Caloporoside derivatives, methods for the production thereof and their use - Google Patents

Description

Calcosporin derivative, preparation method and application thereof

The invention relates to novel active substances (carosporin derivatives) which are formed during the fermentation of the microorganisms gloeophorus dichlorus bres.st001714, DSM13784, to a process for their preparation, to their use as medicaments, to medicaments containing them and to the microorganisms gloeophorus dichlorus bres.st001714, DSM 13784.

In 1994, calosporins were first described as inhibitors of phospholipase C (w. weber et al, j. antibiotics,47,1188-1194). In the same year, two other similar secondary metabolites were isolated (r.shann et al, nat. prod, lett.,4,171-178). The compounds of the invention of formula I (below) and the materials described therein differ structurally.

Cancer is a disease of humans and animals, which is often fatal and is caused by uncontrolled growth of cells of the body itself. Cancer is the name given to the formation of malignant growth of tumors (tumors or carcinomas) or malignant degeneration and maturation disorders of leukocytes (leukemias). Cancer cells or tumor cells are formed by transformation of the body's own cells. Malignancy of a cancer cell manifests itself by growth autonomy, that is, it can grow uninhibited and infiltrative without adapting to the structural arrangement of organs and destroying tissues. One definitive hallmark of malignancy is the formation of metastases at sites distant from the tumor after the tumor cells have spread with blood or lymph. Cancer is one of the most common causes of death in humans, and methods and means to cure or treat malignant degeneration are therefore highly desirable.

Possible treatments for malignant tumors in addition to surgical removal of the tumor, possible underlying approaches have been radiation therapy with X-ray, alpha, beta, gamma radiation, immunotherapy and chemotherapy. Immunotherapy is used to a limited extent today. Chemotherapy of tumors means the administration of cytotoxic (cytostatic) therapy to tumors and residual tumor cells after local surgical or radiation therapy. These substances specifically interfere with the specific processes of cell division, and thus tissue reactions with a high proportion of dividing cells (e.g., rapidly growing tumor tissue) are more sensitive. What is needed isAlkylated compounds are used, for example cyclophosphamide (The Merk Index, 12)^thPage 463), metabolic antagonists such as methotrexate (the merk Index, 12)^thPage 1025), alkaloids such as vincristine (The Merk Index, 12)^thPage 1704) and antibiotics such as daunomycin (The Merk Index, 12)^thPage479) and doxorubicin (The Merk Index, 12)^thPage 581-. However, all these drugs have such a number of disadvantages that the death of the patient is only delayed and cannot be avoided due to the various side effects. In addition, drug resistance occurs in degenerating (cancerous) cells. Thus, the current drugs have no cytostatic effect, but are toxic due to side effects. Furthermore, the fact that cytostatics are used in combination or in succession to exceed the efficacy of individual cytostatics (monotherapy) has been shown, so that it is possible that multiple chemotherapies do not add significant side effects. For all these reasons, new chemotherapeutic drugs are highly desirable and are sought worldwide.

Cyclin-dependent kinases (═ CDKs) play a central role in regulating the cell cycle. They catalyze phosphorylation reactions to initiate a cascade of reactions that lead to the transition from the G1 phase (growth phase 1) to the S phase (synthesis phase) of the cell cycle. Cyclin-dependent kinases therefore represent a good therapeutic target for the treatment of cancer and other diseases of patho-pathological disorders of cell proliferation. Low molecular weight inhibitors that regulate cell cycle and prevent uncontrolled cell division would be useful drugs for treating cancer patients.

Applicants have surprisingly found that DSM13784 is capable of efficiently producing novel cytostatics that inhibit cyclin-dependent kinases at very low concentrations in the microbial strain gloeophorus dichlorus (Fr.: Fr.) bres. st001714.

The invention therefore relates to the active substances (carosporin derivatives) produced by the strain gloeophorus dichlorus (Fr.: Fr.) bres. st001714, DSM13784 and their physiologically tolerated salts, esters and obvious chemical equivalents.

The invention thus relates to compounds of the formula I:

wherein R is₁、R₂And R₃Are each, independently of one another, H or an acyl radical having 2 to 10 carbon atoms, preferably 2 to 6 atoms, particularly preferably 2 atoms; and R is₄Is H or-C (O) (CH)₂)_nCOOH, wherein n is 1 to 7, preferably 1 to 3, particularly preferably 1 or 2; but R is₁、R₂、R₃And R₄Not all are H at the same time.

The acyl group of the compounds of formula I may be straight-chain or branched, saturated or mono-or di-unsaturated.

Acyl having 2 carbon atoms means, for example, acetyl.

Examples of saturated, unbranched acyl groups are acetic acid residues (C ═ 2), propionic acid residues (C ═ 3), butyric acid residues (C ═ 4), valeric acid residues (C ═ 5), caproic acid residues (C ═ 6), heptanoic acid residues (C ═ 7), octanoic acid residues (C ═ 8), nonanoic acid residues (C ═ 9), decanoic acid residues (C ═ 10).

Examples of monounsaturated unbranched acyl groups are acrylic acid residues (C ═ 3), crotonic acid residues (C ═ 4) or vinylacetic acid residues (C ═ 4).

An example of a diunsaturated unbranched acyl group is a sorbic acid residue (C ═ 6).

Calorosporin is a very poorly active antibiotic consisting of one salicylic acid and one disaccharide. The two structural units are connected by an alkyl chain. The glycosyl units in the compounds of formula I may be disaccharides, which in each case consist of an aldohexose of the D-pyranose type (e.g. D-glucopyranose or D-galactopyranose) and an aldonic acid (e.g. gluconic acid) of the aldohexose. The sugar moiety is preferably D-mannopyranosyl-D-mannonic acid, which is not or is not defined by R above₂、R₃And/or R₄Instead of that.

The invention also relates to

a) A compound of formula I (wherein R₁Acetyl group; r₂＝R₃＝R₄H (═ carosporin B: molecular formula C)₃₈H₆₂O₁₆MW 774.9)) and physiologically tolerable salts thereof;

b) a compound of formula I (wherein R₁＝R₃Acetyl group; r₂＝H；R₄(II) malonic acid monoacyl (═ Caroroside C: molecular formula: C)₄₃H₆₆O₂₀MW 902.99)) and physiologically tolerable salts thereof;

c) a compound of formula I (wherein R₁＝R₂＝H；R₃Acetyl group; r₄(II) malonic acid monoacyl (═ Caroroside D: molecular formula: C)₄₁H₆₄O₁₉MW 806.96)) and physiologically tolerable salts thereof;

d) a compound of formula I (wherein R₁＝R₃＝H；R₂Acetyl group; r₄(II) malonic acid monoacyl (═ Caroroside E: molecular formula: C)₄₁H₆₄O₁₉MW 806.96)) and physiologically tolerable salts thereof;

e) a compound of formula I (wherein R₁＝R₂＝R₄＝H；R₃Acetyl group; (═ Carorosporin F: molecular formula C₃₈H₆₂O₁₆MW 774.9)) and physiologically tolerable salts thereof.

Unless otherwise indicated, the chiral centers of the compounds of formula I may be in the R or S configuration. The present invention relates to optically pure compounds and also to stereoisomeric mixtures, such as mixtures of enantiomers and mixtures of diastereomers.

According to the invention, the compound of formula I can be obtained by fermenting Gloeoporus dichlorus (Fr.: Fr.) Bres. ST001714, DSM13784 or a variant or mutant thereof in a culture medium under suitable conditions until one or more of the carbasporine derivatives of formula I accumulate in the culture medium. The calosporine derivatives are obtained by subsequent isolation of the compounds and, where appropriate, conversion into chemical equivalents and their physiologically tolerable salts.

The invention therefore also relates to a process for the preparation of a compound of the formula I, which comprises fermenting the microorganism Gloeoporus dichlorus (Fr.: Fr.) Bres.ST001714, DSM13784 or a variant or mutant thereof in a medium under suitable conditions until one or more compounds of interest have accumulated in the medium, and subsequently isolating the compounds from the medium and, where appropriate, converting them into chemical equivalents and/or their physiologically tolerable salts.

The strains ST001714, DSM13784, mutants and/or variants thereof are preferably fermented in nutrient solutions or in solid medium (also referred to as medium) with carbon and nitrogen sources and customary inorganic salts until the compounds according to the invention accumulate in the medium, and these compounds are then isolated from the medium and, where appropriate, divided into the individual active components.

The process of the invention can be used for fermentation on a laboratory scale (in the milliliter to liter range) and on an industrial scale (in the cubic meter range).

The strain Gloeoporus dichlorus (Fr.: Fr.) Bres. ST001714 was grown in pre-culture medium. Isolates were deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascherder Weg 1B, 3300Brunswick, Germany, on 13.10.2000 under the provisions of the Budapest treaty, with the deposition number: DSM 13784.

Gloeophorus dichlorus (Fr.: Fr.) bres. st001714, DSM13784 had white mycelium and purple spores. It occurs preferentially in birch, but can also infect other hosts, such as alder, salix, populus, elm, prunus.

Mutants and variants of the strain Gloeoporus dichlorus (Fr.: Fr.) Bres. ST001714, DSM13784, which synthesize one or more compounds of the invention, may also be used instead. Such mutants can be generated by physical means, for example by irradiation (e.g.with UV or X-rays) or by chemical mutagens such as Ethyl Methanesulfonate (EMS), 2-hydroxy-4-Methoxybenzophenone (MOB) or N-methyl-N' -nitro-N-nitrosoguanidine (MNNG), in a manner known per se.

Screening for mutants and variants that synthesize one or more compounds of the invention is performed according to the following protocol:

-freeze-drying the plate culture;

-extracting the lyophilisate with an organic solvent;

-extracting the compound from the culture filtrate with a solid phase; and

analysis by HPLC, TLC or analytical bioactivity.

The fermentation conditions described below were applied to Gloeoporus dichlorus (Fr.: Fr.) Bres. ST001714, deposited isolate DSM13784 and mutants and variants thereof.

Gloeoporus dichlorus (Fr.: Fr.) Bres. ST001714, DSM13784 produces a calosporine derivative in a nutrient solution containing a carbon and nitrogen source and usually inorganic salts.

Suitable and preferred sources of carbon for fermentation are assimilable sugars and sugar alcohols such as glucose, lactose, sucrose or D-mannitol, and sugar-containing natural products, for example, malt extract. Suitable nitrogen-containing nutrients are: amino acids, peptides and proteins and their degradation products such as casein, peptone or tryptone, but also meat extract, yeast extract, ground seeds such as seeds of cereals, wheat, beans, soybeans or cotton trees, distillers' grains, meat meal or yeast extract, but also ammonium salts and nitrates, but especially also synthetic or biosynthetic peptides. The nutrient solution may contain inorganic salts such as chlorides, carbonates, sulphates or phosphates of alkali or alkaline earth metals, iron, zinc, cobalt and manganese.

The compounds of the invention are particularly preferred in a nutrient solution containing about 0.05% to 5%, preferably 1% to 2%, of malt extract, 0.05% to 3%, preferably 0.05% to 1%, of yeast extract and 0.2% to 5%, preferably 0.5% to 2%, of glucose, 0.5% to 3%, preferably 0.5% to 3%, of cellulose powder and trace amounts of ammonium sulfate. The percentages in each case are based on the weight of the entire nutrient solution.

In this nutrient solution, Gloeoporus dichlorus (Fr.: Fr.) Bres. ST001714, DSM13784 produced a mixture of carbasporin derivatives. The amount of one or more of the calosporine derivatives of the invention may vary depending on the composition of the nutrient solution. Furthermore, it is possible to control the synthesis of various calosporine derivatives by varying the composition of the medium so that certain calosporine derivatives are not produced at all or are produced in amounts below the detectable limit by the microorganism.

The microorganisms are cultured aerobically, that is to say, for example, by shaking or agitation submerged culture in shake flasks or fermenters, where appropriate with introduction of air or oxygen or in solid media. The fermentation can be carried out at a temperature in the range from about 18 to 35 deg.C, preferably from about 20 to 30 deg.C, in particular from 25 to 30 deg.C. The pH should be in the range of 5 to 8, preferably between 5.5 and 6.5. The microorganism is generally cultured under these conditions for 24 to 720 hours, preferably 288 to 576 hours.

The cultivation is advantageously carried out in a plurality of stages, i.e.one or more precultures are first produced in a liquid medium and then transferred to the actual production medium, for example a 1: 10 volume ratio main medium. The preculture is obtained, for example, by transferring the mycelium to a nutrient solution and allowing it to grow for about 36 to 120 hours, preferably 48 to 72 hours. The mycelium can be obtained, for example, by growing the strain in a solid or liquid nutrient medium such as malt/yeast agar or potato/dextrose agar for about 3 to 40 days, preferably 10 to 30 days.

The progress of the fermentation can be monitored based on the pH of the broth or the volume of mycelium and chromatographic methods such as High Performance Liquid Chromatography (HPLC) or specific biological viability.

The separation method described below is used for purifying the inventive carbasporangin derivative.

The carosporin derivative of the present invention is isolated or purified from the culture broth using a known method in consideration of the chemical, physical and biological properties of natural substances. HPLC can be used to analyze the concentrations of various caloroside derivatives in the culture broth or at various separation stages, and the amounts of the produced substances are conveniently compared using a calibration solution.

For isolation of the compounds of the invention, the culture broth or the culture with the solid medium is lyophilized, and the carosporin derivative is then extracted from the lyophilizate with an organic solvent, optionally miscible with water. The organic solvent phase contains the natural substances according to the invention, which are, where appropriate, concentrated in vacuo and further purified.

Further purification of one or more compounds of the invention is carried out by chromatography on a suitable substance, for example preferably on molecular sieves, silica gel, alumina, on ion exchangers or adsorption resins or on Reverse Phase (RP). The calosporine derivative is isolated by this chromatography. The chromatography of the carbasporangin derivative is carried out with an aqueous buffer solution or a mixture of water and an organic solution.

Mixtures of water or organic solutions refer to all water-miscible organic solvents, preferably methanol, propanol and acetonitrile, in a concentration of 5 to 80%, preferably 20 to 50%, or all buffered aqueous solutions which are miscible with organic solvents. The buffer to be used is the same as described above.

The isolation of the calosporine derivatives is carried out by reverse phase chromatography based on the difference in polarity, for example in MCI^®(adsorption resins from Mitsubishi, Japan) or Amberlite XAD^®(TOSOHAAS), on other hydrophobic substances such as RP-8 or RP-18 phases. Separation can also be carried out by normal phase chromatography such as on silica gel, alumina, etc.

Chromatography of the calosporine derivative is carried out with a buffered or acidified aqueous solution or a mixture of an aqueous solution and ethanol or other water-miscible organic solvent. Propanol and acetonitrile are preferably used as organic solvents.

Buffered or acidified aqueous solutions refer to, for example, water, phosphate buffer, ammonium acetate solution, citrate buffer, at a concentration of 0 to 0.5M, and formic acid, acetic acid, trifluoroacetic acid or all commercially available acids known to the skilled person, preferably at a concentration of 0 to 1%. For aqueous buffer solutions, 0.1% ammonium acetate is preferred.

Chromatography is performed in a gradient starting with 100% water and ending with 100% solvent, preferably with a linear gradient of 20% to 100% propionic acid or acetonitrile.

An alternative is to perform gel chromatography or hydrophobic chromatography.

Gel chromatography on polyacrylamide or copolymer gels, e.g. Biogel-P2^®(Biorad) or Fractogel TSK HW 40^®(Merck, Germany or Toso Haas, USA).

The order of the above chromatography may be reversed.

The compounds of the invention are stable in solid form and in solution at a pH in the range of 3 to 8, preferably 5 to 7, so that they can be incorporated into conventional pharmaceutical preparations.

Due to their valuable pharmacological properties, one or more of the compounds of the present invention are suitable for use in human or veterinary medicine as a medicament.

The invention thus relates to the use of compounds of the formula I or physiologically tolerated salts thereof for producing cytostatics for the treatment of cancer.

The invention also relates to all obvious chemical equivalents of the compounds of the formula I according to the invention. These equivalents are compounds that exhibit slight chemical differences, that is, compounds that have the same efficacy or are convertible under mild conditions into the compounds of the invention. The above equivalents also include, for example, the salts, reduction products, esters, ethers, acetals or amides of the compounds of the invention, and equivalents which can be prepared by the skilled worker by standard methods, and also include all optical antipodes, diastereomers and all stereoisomeric forms.

Physiologically tolerable salts of the compounds of the formula I are organic and inorganic salts thereof, as described in Remington's Pharmaceutical Sciences (17)^thedition, p 1418 (1985)). Sodium, potassium, calcium and ammonium salts of acidic groups are preferred due to physical and chemical stability and solubility; preference is given to salts of basic groups of hydrochloric acid, sulfuric acid, phosphoric acid or carboxylic or sulfonic acids, such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid and p-toluenesulfonic acid.

Esters, ethers and acetals may be synthesized by literature, for example, as in Advanced Organic Synthesis, 4^thEdition，J.March，John Wiley &Sons, 1992 or Protective Groups in organic Synthesis 3^rd Edition，T.W.Greene &P.G.M.Wuts，John Wiley&Sons, 1999.

The carboxyl group can be exemplified by LiAlH₄Reducing to alcohol.

The glycoside moiety of the compound of formula I can be initially removed by basic hydrolysis (w.weber et al, j.antibiotics,47,1188-1194). Any desired sugar residue may then be introduced by glycosylation (e.g., K ö nigs-Knorr reaction). Corresponding methods are described in the literature, for example, in carbohydrate chemistry, J.F. Kennedy, Oxford University Press, 1988.

The mechanism of action of the calosporine derivatives is not known, but their important effects can be detected.

Inhibitors of CDKs are detected by an assay in which the rate of phosphorylation of specific peptide substrates by cyclin-dependent kinases is determined. Cyclin-dependent kinases are activated by binding to specific cyclins. The [ gamma-P ] -phosphate is transferred from [ gamma-P ] -ATP to the peptide substrate by the enzyme. The assay was performed in 96-well microtiter plates: the radioactivity of [ gamma-P ] -phosphate transferred to the substrate was determined.

Carlo sporeIC of glycoside derivatives₅₀The values are shown in Table 1; it is the concentration at which 50% of CDK-4 is inactivated.

Calcosporin B	1.5μM
		Calcosporin C	3.1μM
Calcosporin D	1.8μM
		Calcosporin E	1.8μM
Calcosporin F	1.5μM

The invention also relates to medicaments containing at least one compound according to the invention.

One or more compounds of the inventive carbasporangin derivatives can in principle be administered directly without dilution. The preferred application is in admixture with suitable excipients or carrier materials. Useful carrier substances are those which are pharmacologically suitable and are customary in pharmaceuticals and/or excipients.

The medicaments of the invention are generally administered orally or parenterally, but may in principle also be administered rectally. Suitable solid or liquid pharmaceutical preparations are, for example, granules, powders, (coated) tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, aerosols, drops or ampoules, and products for the sustained release of the active substance, in the production of which usual carriers and additives and/or auxiliaries, such as disintegrants or binders, coatings, swelling agents, glidants or lubricants, flavoring agents, sweeteners or solubilizers, are used. Common carriers or excipients which may be mentioned are, for example, magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyols.

Oral dosage units may be microencapsulated in time for delayed release or extended period of time, for example by coating or embedding the active material particles in a suitable polymer, wax or the like.

The pharmaceutical products are preferably manufactured and administered in dosage units, each containing as its active ingredient a specific dose of one or more compounds of the inventive calosporine derivative. For solid dosage units such as tablets, capsules and suppositories, the daily dose may be up to about 500mg, but preferably from about 0.1 to 200mg, and for the form of ampoules, up to about 200mg, but preferably from about 0.5 to 100mg per day.

The daily dose to be administered depends on the weight, age, sex and physical condition of the mammal. However, in certain cases, a slightly higher or lower daily dosage may also be appropriate. Administration of the daily dose may be effected in a single dose from a single dosage unit or from a plurality of small dosage units, or in divided doses following a particular interval in a plurality of doses.

The medicaments according to the invention are produced by converting one or more compounds according to the invention into suitable dosage forms with the usual carriers, where appropriate additives and/or excipients.

The invention is further illustrated by the following examples. The percentage values are based on weight. Unless otherwise indicated, the mixing ratio of the liquids is based on volume.

Examples

Example 1: preparation of Glycerol cultures of Gloeoporus dichlorus (Fr.: Fr.) Bres. ST001714, DSM13784

Placing in 300ml for sterilization100ml of nutrient solution (malt extract 2.0%, yeast extract 0.2%, glucose 1.0%, (NH) in Erlenmeyer flask₄)₂HPO₄0.05%, pH6.0) was mixed with the strain gloeophorus dichlorus (Fr.: fr.) bres. st001714, DSM13784 were incubated together for 7 days. 1.5ml of this culture was taken, then diluted with 2.5ml of 80% glycerol and stored at-135 ℃.

Example 2: preparation of precultures in Erlenmeyer flasks containing Gloeoporus dichlorus (Fr.: Fr.) Bres. ST001714, DSM13784

30ml of nutrient solution (2.0% malt extract, 0.2% yeast extract, 1.0% glucose), (NH) in a 100ml sterile conical flask₄)₂HPO₄0.05%, ph6.0) was mixed with the strain gloeophorus dichlorus (Fr.: fr.) bres. st001714, DSM13784 were incubated together for 4 days. Then, 2ml of this preculture was used to inoculate the plate to prepare the main culture.

Example 3: preparation of a Primary culture of Gloeoporus dichlorus (Fr.: Fr.) Bres. ST001714, DSM13784 on solid culture plates

Sterile 25X 25cm plates (from Nunc) were poured into 200ml of the following nutrient solution: 20g/l malt extract, 2g/l yeast extract, 10g/l glucose and 0.5g/l (NH)₄)₂HPO₄pH 6.0. These plates were inoculated with 2ml of preculture each. The maximum yield of one or more compounds of the invention is reached after about 480 hours.

Example 4: production of a Calorosporin derivative

50 plates of 25X 25cm were prepared and inoculated with preculture:

nutrient medium:

malt extract 20g/l

2g/l yeast extract

10g/l glucose

0.5g/l(NH₄)₂HPO4

pH6 (before sterilization)

Incubation time: 480 hours

Incubation temperature: 25 deg.C

Example 5: isolation of a mixture of Carcosporins from a plate culture of Gloeoporus dichlorus (Fr.: Fr.) Bres. ST001714, DSM13784

After completion of the fermentation of Gloeoporus dichlorus (Fr.: Fr.) Bres. ST001714, DSM13784, the plate culture obtained according to example 3 was lyophilized and the lyophilizate was extracted with 5 l of methanol. The methanol solution containing the active substance was filtered to remove the residue and concentrated in vacuo. The concentrate was diluted with water and applied to a previously prepared 1.0 liter MCI GEL, CHP20P column. Elution was performed with a gradient from water to 100% acetonitrile. The column effluent (25ml/min) was fractionated (25ml per fraction) and fractions containing the carbasporangin derivative (fractions eluting from 40% acetonitrile to 100% acetonitrile) were combined. Concentration in vacuo and freeze drying gave 8.5g of a tan powder.

Example 6: preliminary isolation of the Calcosporin derivatives by RP18 chromatography

0.5g of the product obtained in example 4 was applied to Nucleosil^®100-7C18HD column (column: 40 mm. times.250 mm). Elution was performed with a gradient of 20% acetonitrile (+ water and 0.1% ammonium acetate) to 100% acetonitrile at a flow rate of 35 ml/min. The column effluent was collected in fractions (35 ml). The calosporine derivatives are mainly found in the 39 th to 68 th parts. They were combined, the solvent removed in vacuo and lyophilized. The purities of the thus obtained calosporins B (22.4mg) and F (10.5mg) were already more than 95%. The resulting calosporin C (part 41; 43.4mg), D (parts 43-45; 76.2mg) and E (parts 43-45; 76.2mg) were about 70% pure and were therefore further purified by chromatography.

Example 7: purification of Calcosporin C, D and E

20mg of the carosporin C fraction concentrated in example 5 was added to the LUNA^®A5 μ C18(2) column (cartridge: 10 mm. times.250 mm) and chromatography with a gradient of 25 to 35% acetonitrile in 0.1% ammonium acetate/water. EluentThe flow rate was 6.5ml/min, 6.5ml per fraction. Calorosporin C is in sections 35 to 42. The above fractions were lyophilized to give karosporin C (7.5mg) with a purity of greater than 95%.

25mg of the mixture of caloroside D and caloroside E obtained in example 5 was applied to the LUNA^®A5 μ C18(2) column (cartridge: 10 mm. times.250 mm) and chromatography with a 30 to 40% acetonitrile gradient in 0.1% ammonium acetate/water. The flow rate of the eluate was 6.5ml/min, 6.5ml per fraction. Calcosporin D in sections 18 and 19, and Calcosporin E in sections 20 to 21. The above fractions were lyophilized to give a mixture of carosporin D (7.0mg) and carosporin E (6.0mg) with a purity of greater than 95%.

The physicochemical and spectral properties of the substances of the invention are summarized below:

carosporin B:

the molecular formula is as follows: c₃₈H₆₂O₁₆

Molecular weight: 774.9

UV_{Maximum of}：208、244、310

¹H-and¹³C-NMR: see Table 2

High resolution FAB mass spectrum showed strong MH at m/z 775.4120Da⁺Signal, and calculated mass (for C)₃₈H₆₃O₁₆Single isotope) 775.4116Da fit well.

C, carosporin C:

the molecular formula is as follows: c₄₃H₆₆O₂₀

Molecular weight: 902.99

UV max: 208, 244, 310

¹H-and¹³C-NMR: see Table 3

High resolution FAB mass spectra showed strong M + H at M/z 903.4264Da⁺Signal, and calculated mass (for C)₃₆H₇₁O₂Single isotope) 903.4284Da fit well.

Carosporin D:

the molecular formula is as follows: c₄₁H₆₄O₁₉

Molecular weight: 860.96

UV_{Maximum of}：208、244、310

¹H-and¹³C-NMR: see Table 4

High resolution FAB mass spectra showed strong M + Na at M/z 883.3942Da⁺Signal, and calculated mass (for C)₄₁H₆₄O₁₉Na, single isotope) 883.3939Da fit well.

Carosporin E:

the molecular formula is as follows: c₄₁H₆₄O₁₉

Molecular weight: 860.96

UV_{Maximum of}：208、244、310

¹H-and¹³C-NMR: see Table 4

High resolution FAB mass spectra showed strong M + Na at M/z 833.3942Da⁺Signal, and calculated mass (for C)₄₁H₆₄O₁₉Na, single isotope) 883.3939Da fit well.

Carosporin F:

the molecular formula is as follows: c₃₈H₆₂O₁₆

Molecular weight: 774.9

UV_{Maximum of}：208、244、310

¹H-and¹³C-NMR: see Table 5

High resolution FAB mass spectra are shown inStrong M + H at M/z 775.4128Da⁺Signal, and calculated mass (for C)₃₈H₆₃O₁₆Single isotope) 775.4116Da fit well.

0 Table 2: in methanol-d₄And DMSO-d₆Method for determining Calorosporin B at 300K¹H and¹³chemical shift of C

						DMSO	MeOD	DMSO	MeOD
1	-	-	171.24	a)
					2	-	-	a)	a)
3	-	-	162.22	163.57
					4	6.68	6.77	115.28	116.81
5	7.17	7.25	～131.6	b
					6	6.57	6.67	121.02	～123.0
7	-	-	137.90	139.57
					8	a)	a)	a)	a)
9	a)	a)	a)	a)
					10-20	1.30-1.20	1.32-1.27	29.01-28.69	30.76-30.47
21	1.30	1.38/1.32	24.81	26.52
					22	1.54/1.45	1.65/1.52	35.31	37.00
23	4.81	4.95	70.04	73.09
					24	1.15	1.24	19.69	20.22
1‘	-	-	173.43	175.35
					2‘	3.95	4.13	70.89	72.97
3‘	3.95	4.17	70.04	71.59
					4‘	3.54	3.82	67.96	69.80
5‘	3.65	3.86	78.94	80.12
					6‘	3.74/3.44	3.90/3.69	62.11	63.25
1“	4.53	4.71	100.27	101.34
					2“	3.74	3.95	70.37	72.46
3“	3.22	3.44	73.67	75.30
					4“	3.21	3.49	67.54	69.00
5“	3.04	3.22	77.15	78.23
					6“	3.72/3.31	3.88/3.63	61.80	63.25
Ac-C‘	-	a)	-	a)
					Ac-Me	a)	a)	a)	a)

a) No signal for these protons/carbon atoms was observed in MeOD or DMSO (aggregate).

Table 3: in methanol-d₄Method for determining Calorosporin C at 300K¹H and¹³chemical shift of C

	1H	13C
			1	-	175.90
2	-	116.73
			3	-	163.67
3-Ac-Me	a)	a)
			3-Ac-C‘	-	a)
4	6.73	116.73
			5	7.16	～132.6b)
6	6.55	123.53
			7	-	139.51
8	2.49	43.06
			9	1.53	24.75
10-20	1.36-1.26	30.78-30.62
			21	1.38/1.33	26.54
22	1.65/1.52	37.02
			23	4.95	73.04
24	1.24	20.22
			1‘	-	175.17
2‘	4.17	72.82
			3‘	4.08	71.60
4‘	3.71	69.79
			5‘	4.07	76.38
6‘	4.60/4.14	66.43
			7‘	-	170.55
8‘	3.27(c	～44.8
			9‘	-	173.12
1“	4.93	99.40
			2“	5.33	73.40
3“	3.72	73.40
			4“	3.42	69.34

5“	3.34	78.19
			6“	3.89/3.62	63.11
2“-Ac-Me	2.11	21.17
			2“-Ac-C‘	-	172.56

a) No signal was observed for these nuclei (signal becomes very broad).

b) Signal broadening

c) Proton signals at the 8' position were only observed in freshly prepared solutions (rapid exchange with deuterium).

Table 4: in methanol-d₄Method for determining Calorosporin D and E at 300K¹H and¹³chemical shift of C

	1HCaloporosid D	13CCaloporosid D	1HCaloporosid E	13CCaloporosid E
					1	-	-	176.28	176.28
2	-	-	120.37	120.37
					3	-	-	162.12	162.12
4	6.61	6.61	114.91	114.91
					5	7.06	7.06	131.53	131.53
6	6.58	6.58	122.23	122.23
					7	-	-	147.22	147.22
8	3.04	3.04	36.31	36.31
					9	1.55	1.55	33.35	33.35
10-20	1.36-1.25	1.36-1.25	31.07-30.61	31.07-30.61
					21	1.38/1.32	1.38/1.32	26.53	26.53
22	1.65/1.53	1.65/1.53	37.01	37.01
					23	4.95	4.95	73.13a)	73.07a)
24	1.24	1.24	20.23	20.23
					1‘	-	-	175.18	175.40
2‘	4.17	4.15	72.80	72.80
					3‘	4.08	4.15	71.61	71.53
4‘	3.71	3.85	69.78	69.27

5‘	4.07	4.08	76.40	77.07
					6‘	4.58/4.14	4.59/4.24	66.35	65.43
7‘	-	-	～170.4	～170.4
					8‘	3.28	3.28	～45.0	～45.0
9‘	-	-	～173.0	～173.0
					1“	4.93	4.84	99.40	100.20
2“	5.33	4.03	73.41	70.27
					3“	3.72	4.78	73.39	77.44
4“	3.42	3.71	69.32	66.35
					5“	3.34	3.38	78.35	78.00
6“	3.91/3.62	3.89/3.65	63.09	62.96
					2“/3“-Ac-Me	2.11	2.00	21.18	20.98
2“/3“-Ac-C‘	-	-	172.59	172.36

a) These signals do not match clearly.

Table 5: in methanol-d₄Method for determining Calorosporin F at 300K¹H and¹³chemical shift of C

	1H	13C
			1	-	174.95
2	-	117.32
			3	-	162.18
4	6.68	115.42
			5	7.17	133.17
6	6.67	122.65
			7	-	146.91
8	2.93	36.57
			9	1.56	33.27
10-20	1.36-1.25	30.93-30.62
			21	1.35	26.53

22	1.65/1.53	37.02
			23	4.95	73.05
24	1.24	20.23
			1‘	-	175.15
2‘	4.17	72.98
			3‘	4.09	71.73
4‘	3.71	69.88
			5‘	3.85	79.59
6‘	3.90/3.61	63.57
			1“	4.88	99.76
2“	5.37	73.52
			3“	3.63	73.56
4“	3.46	69.27
			5“	3.28	78.33
6“	3.91/3.63	63.06
			Ac-C‘	-	173.03
Ac-Me	2.13	21.21

Example 8: biological assay for CDK-4 inhibitors

To determine IC₅₀The stock solution of the natural substance of the present invention was prepared at a concentration of 10 mM. A384-well flash plate was coated with 50. mu.l (50. mu.g/well) of biotin-labeled peptide substrate at room temperature for 2 hours and then washed 3 times with PBS buffer. In the reaction, 30. mu.l of the carosporin derivative diluted with buffer and 20. mu.l of a previously mixed ATP/cyclin D1/CDK4 solution (final concentration: 1. mu. Ci 33P-. gamma. -ATP, 2. mu.M ATP and 1. mu.g of the enzyme mixture) were pipetted onto the plate. After 2 hours reaction at 37 ℃, the plates were washed 3 times with 80 μ l of 3% phosphoric acid each time and then counted for 30 seconds using a MicroBeta counter. Percent inhibition was determined using a mathematical equation. To determine IC₅₀Values, 10 concentrations of freshly diluted solutions of the substance of the invention in DMSO were analyzed.

Claims (14)

1. A compound of formula I:

wherein R is₁、R₂And R₃Are each independently of the other H or an acyl radical having 1 to 10 carbon atoms; r₄Is H or-C (O) (CH)₂)_nCOOH, wherein n is 1 to 7; but R is₁、R₂、R₃And R₄Not all are H at the same time.

2. A compound of the formula I as claimed in claim 1, in which R is₁、R₂And R₃Are independently of each other, H or acetyl; and R is₄Is H or malonic acid monoacyl.

3. A compound of the formula I as claimed in claim 1, in which R is₁Is acetyl; and R is₂、R₃And R₄Is H.

4. A compound of the formula I as claimed in claim 1, in which R is₁And R₃Is acetyl; r₂Is H; and R is₄Is malonic acid monoacyl.

5. A compound of the formula I as claimed in claim 1, in which R is₁And R₂Is H; r₃Is acetyl; and R is₄Is malonic acid monoacyl.

6. A compound of the formula I as claimed in claim 1, in which R is₁And R₃Is H; r₂Is acetyl; and R is₄Is malonic acid monoacyl.

7. A compound of the formula I as claimed in claim 1, in which R is₁、R₂And R₄Is H; and R is₃Is acetyl.

8. A compound of the formula I as claimed in claim 1, which is obtainable by fermenting the microorganisms gloeophorus dichlorus (Fr.: Fr.) bres. st001714, DSM13784 or one of its variants or mutants under suitable conditions, isolating one or more charosporin derivatives and, where appropriate, converting them into physiologically tolerable salts, or a physiologically tolerable salt thereof.

9. A process for the preparation of a compound of the formula I as claimed in one of claims 1 to 7 or a physiologically tolerable salt thereof, which comprises fermenting the microorganisms gloeophorus dichlorus (Fr.: Fr.) bres.st001714, DSM13784 or one of its variants or mutants under suitable conditions, isolating one or more carosporin derivatives and, where appropriate, converting them into a physiologically tolerable salt.

10. A process as claimed in claim 9, wherein the fermentation is carried out aerobically at a temperature of from 18 to 35 ℃ and a pH of from 5 to 8.

11. Use of a compound of formula I as claimed in any one of claims 1 to 8 or a physiologically tolerable salt thereof in the preparation of a medicament for use as a CDK inhibitor.

12. The use as claimed in claim 11, wherein the medicament is prepared for the treatment of cancer or other diseases with pathological disorders of cell proliferation.

13. A medicament containing at least one compound of the formula I as claimed in any of claims 1 to 8 or a physiologically tolerable salt thereof.

14. A process for the manufacture of a medicament as claimed in claim 13, which comprises converting at least one compound of the formula I as claimed in any of claims 1 to 8 or a physiologically tolerable salt thereof and suitable excipients and/or carriers into a suitable dosage form.