HK1142888B - Macrolactone derivatives - Google Patents

Description

Macrolide derivatives

The present invention relates to novel macrolides and to processes for their preparation and use. It has now been found that the microbial strain ST 201196(DSM18870) is capable of forming new macrocycles having strong antifungal activity against the fungus Candida albicans. The compounds are therefore suitable for the treatment of local and/or systemic fungal diseases.

Many anti-infective agents are used therapeutically to treat infectious diseases. However, pathogenic microorganisms are becoming increasingly resistant to the drugs used, and even a huge danger is imminent due to "multidrug-resistant microorganisms" which are not only resistant to a single but also to several anti-infective groups at the same time. Even pathogenic microorganisms have become resistant to all commercially available anti-infective drugs. Infectious diseases caused by this type of microorganism are no longer curable. Therefore, there is a great need for new substances that can be used against drug-resistant microorganisms. Although thousands of anti-infective drugs have been described in the literature, most drugs are too toxic to be used as drugs.

The invention relates to compounds of formula (I) or physiologically tolerated salts of compounds of formula (I),

wherein:

x and Y are independently of each other OH, O- (C)₁-C₆) -alkyl, NH₂Or NH- (C)₁-C₆) -alkyl, or X and Y together form an-O-group;

r1 and R2 are independently Cl or H;

r3 is H, (C)₁-C₆) -alkyl, C (═ O) - (C)₁-C₆) -alkyl or (C)₁-C₆) alkylene-NH- (C)₁-C₆) -alkyl, and

r4 is H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) -an alkyl group.

Preferably the present invention relates to compounds of formula (I) wherein X and Y together form an-O-group, whereby X and Y in the corresponding preferred compounds together with the carbon atom to which they are bonded form an epoxy group.

More preferably the invention relates to compounds of formula (I) wherein R1 and R2 are Cl.

More preferably the invention relates to compounds of formula (I) wherein R1 equals Cl and R2 equals H.

R3 and R4 are preferably, independently of one another, H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) Alkyl, particularly preferably both R3 and R4 are equal to H.

Particularly preferred the present invention relates to compounds of formula (I) as defined below, wherein: x and Y together form an-O-group, R1 and R2 independently of one another are Cl or H, and R3 and R4 independently of one another are H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) -an alkyl group;

and compounds of formula (I) as defined below, wherein: x and Y together form an-O-group, R1 and R2 equal Cl, and R3 and R4 independently of one another are H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) -an alkyl group;

and compounds of formula (I) as defined below, wherein: x and Y together form an-O-group, R1 is Cl, R2 is equal to H, and R3 and R4 are independently of each other H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) -an alkyl group;

and compounds of formula (I) as defined below, wherein: x and Y together form an-O-group, R3 and R4 both equal H, and

wherein R1 and R2 independently of one another equal Cl or H, preferably wherein R1 and R2 equal Cl (also referred to as compounds of formula (II) below):

or wherein more preferably R1 is equal to Cl and R2 is equal to H (also referred to as compounds of formula (III) below):

or wherein more preferably R1 and R2 are equal to H.

(C₁-C₆) Alkyl is a straight-chain or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, butyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl.

If not otherwise indicated, the chiral centers in the compounds of formula (I) may be present in the R or S configuration. The present invention relates to optically pure compounds and stereoisomeric mixtures, such as enantiomeric mixtures and diastereomeric mixtures.

Physiologically tolerated salts of the compounds of the formula (I) are to be understood as meaning their organic and inorganic salts, as described in Remington's Pharmaceutical Sciences, 17 th edition, p.1418, 1985. In view of physical and chemical stability and solubility, sodium, potassium, calcium and ammonium salts are preferable for the acid group; for the basic groups, salts of hydrochloride, sulfate, phosphate or carboxylic acid and sulfonic acids such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid and p-toluenesulfonic acid are preferred.

Furthermore, the present invention relates to all obvious chemical equivalents of the compounds of formula (I) according to the invention. Equivalents of this type are those which exhibit slight chemical differences and thus have the same effect or are converted under mild conditions into the compounds of the invention. The equivalents mentioned include, for example, salts, reduction products, oxidation products, esters, ethers, acetals or amides of the compounds of the formula (I) and equivalents which can be prepared by standard methods by those skilled in the art.

Furthermore, the invention relates to a process for the preparation of a compound of the formula (I) or a physiologically tolerable salt of a compound of the formula (I),

wherein:

x and Y are independently of each other OH, O- (C)₁-C₆) -alkyl, NH₂Or NH- (C)₁-C₆) -alkyl, or X and Y together form an-O-group;

r1 and R2 are independently Cl or H;

r3 is H, (C)₁-C₆) -alkyl, C (═ O) - (C)₁-C₆) -alkyl or (C)₁-C₆) alkylene-NH- (C)₁-C₆) -an alkyl group; and is

R4 is H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) -an alkyl group,

the method comprises the following steps:

1. fermenting the microorganism strain ST 201196(DSM18870) or one of its variants and/or mutants in a medium containing a Cl source under suitable conditions until one or more compounds of formula (I) accumulate in the medium; and

2. isolating the compound of formula (I) from the culture medium; and

3. the compounds of formula (I) are optionally derivatized and/or converted to physiologically tolerable salts.

The medium is a nutrient solution or a solid medium containing at least one carbon and nitrogen source and conventional inorganic salts.

The Cl source used may be, for example, NaCl or CaCl₂. In the present application, the microbial strain ST 201196(DSM18870) preferably produces compounds of formula (I) in which the radicals R1 and R2 are both equal to Cl or in which R1 is equal to Cl and R2 is equal to H. Preferably, the present invention relates to a process for the preparation of compounds of formula (I) wherein R1 and R2 are both equal to Cl. Furthermore, the present invention preferably relates to a process for the preparation of compounds of formula (I) wherein R1 equals H and R2 equals Cl.

Preferably the present invention relates to a process for the preparation of a compound of formula (I) wherein X and Y form an-O-group, and further R3 and R4 are as hereinbefore described or preferably H.

Particularly preferred the present invention relates to a process for the preparation of compounds of formula (II). More particularly preferred the present invention relates to a process for the preparation of compounds of formula (III). Moreover, the present invention particularly preferably relates to a process for the preparation of compounds of formula (I) wherein R1, R2, R3 and R4 are equal to H.

The process of the invention can be used for both laboratory scale (milliliter to liter scale) fermentations and for industrial scale (cubic meter scale).

Suitable carbon sources for the fermentation are assimilable carbohydrates and sugar alcohols such as glucose, lactose, sucrose or D-mannitol, and carbohydrate-containing natural products such as malt extract or yeast extract. Suitable nitrogen-containing nutrients are: an amino acid; peptides and proteins and their degradation products, such as ProbionF (Applied Microbiology and Biotechnology 1984, 19(1), 23-28), casein, peptone or tryptone; a meat extract; a yeast extract; gluten; ground seeds, such as ground corn, wheat, legumes, soybean, or cotton seeds; distillation residue from ethanol production; a meat food; a yeast extract; an ammonium salt; a nitrate salt. The nitrogen source is preferably one or more synthetically or biosynthetically derived peptides. Inorganic salts are, for example, chlorides, carbonates, sulfates or phosphates of alkali metals or alkaline earth metals, iron, zinc, cobalt and manganese. Trace elements are for example cobalt and manganese.

Conditions suitable for forming the materials of the present invention are as follows: the formation of the substances according to the invention is preferably carried out in a medium which contains 0.05 to 5%, preferably 0.1 to 2.5%, Probion F, 0.02 to 1.0%, preferably 0.05 to 0.5% CaCl₂×2H₂O, 0.02-1.5%, preferably 0.05-0.7% MgSO₄×7H₂O, cyanocobalamine 0.00001-0.001%, and adsorption resin XAD-16 1-5%; or 0.05-5%, preferably 0.1-2.5% oatmeal, 0.2-5.0%, preferably 0.1-2% glycerol, 0.02-1.0%, preferably 0.05-0.5% CaCl₂0.02-1.5%, preferably 0.05-0.7% MgSO₄×7H₂O, and 0.00001% to 0.001% cyanocobalamin. The data given in percentages are each based on the weight of the entire nutrient solution.

The cultivation of the microorganisms is carried out under aerobic conditions, i.e.for example in submerged form and with shaking or stirring in shaker flasks or fermentation flasks or in a solid medium, optionally with introduction of air or oxygen. It can be carried out at a temperature in the range from about 18 to 35 deg.C, preferably from about 20 to 32 deg.C, in particular from 27 to 30 deg.C. The pH range should be between 4 and 10, preferably between 6.5 and 9. Generally, the microorganisms are cultured under these conditions for 3 to 18 days, preferably 144 to 216 hours. The cultivation is advantageously carried out in a plurality of stages, i.e.one or more precultures are first prepared in a liquid nutrient medium and then inoculated into the actual production medium, the main medium, in a volume ratio of from 1: 10 to 1: 100. The preculture is obtained, for example, as follows: the microorganism strain in the form of vegetative cells or fruiting bodies is inoculated into a nutrient solution and allowed to grow for about 3 to 13 days, preferably 96 to 240 hours. Vegetative cells and/or fruit bodies can be obtained, for example, by growing a microorganism strain in a solid or liquid nutrient medium such as yeast agar for about 3 to 15 days, preferably 7 to 10 days.

The substance of formula (I) is isolated or purified from the culture medium according to known methods and taking into account the chemical, physical and biological properties of the natural substance. HPLC is used to determine the concentration of the respective derivative in the medium or in the individual separation stages.

For separation, the culture broth may be centrifuged and/or filtered through a suction filter. The mycelium is freeze-dried together with XAD, and then the natural substance is extracted from the freeze-dried product with an organic solvent such as methanol or 2-propanol. The organic solvent phase contains the natural substances of the invention; it is optionally concentrated in vacuo and further purified.

Further purification of one or more compounds of the invention is carried out by chromatography on suitable materials, preferably, for example, on molecular sieves, silica gel, alumina, ion exchangers or adsorption resins or Reverse Phase (RP). By means of this chromatography, the natural substance derivatives are isolated. Chromatography of the compounds of the invention is carried out using buffered aqueous solutions or mixtures of aqueous and organic solutions.

Aqueous or mixture of organic solutions is understood to mean all water-miscible organic solvents, preferably methanol, 2-propanol and acetonitrile, or all aqueous buffered solutions which are miscible with organic solvents, in concentrations of from 0 to 100% of the solvent. The buffers used were the same as indicated above.

By means of reverse phase chromatography, e.g. in(adsorption resin, Mitsubishi corporation, Japan) or Amberlite(TOSOHAAS) or on other hydrophobic materials such as RP-8 or RP-18 phases, based on the different polarity of the compounds of the invention. Further, the separation can be carried out by means of normal phase chromatography such as silica gel, alumina, etc.

Chromatography of the derivative of the natural substance is carried out according to methods known to the person skilled in the art, preferably using buffered basic or acidified aqueous solutions or mixtures of aqueous solutions with alcohols or other water-miscible organic solvents. Acetonitrile and/or methanol are preferably used as organic solvent.

Buffered alkaline or acidified aqueous solutions are understood to mean, for example, water, phosphate buffer, ammonium acetate, ammonium formate, citric acid buffer (concentrations of up to 0.5M) and formic acid, acetic acid, trifluoroacetic acid, ammonia, triethylamine or all commercially available acids and bases known to the person skilled in the art, preferably concentrations of up to 1%. With respect to the buffered aqueous solution, 0.1% ammonium acetate is particularly preferred.

Chromatography is for example performed with a gradient starting with 100% water and ending with 100% solvent; preferably, a linear gradient of 5-95% acetonitrile is used.

Alternatively, gel chromatography or hydrophobic phase chromatography may be performed. Gel chromatography on polyacrylamide or mixed polymer gels such as Biogel-P(Biorad) or FractogelTSK HW(Merck, Germany). The sequence of chromatography described above is reversible.

If the compounds of the formula (I) are present as a mixture of stereoisomers, the stereoisomers can be isolated by known methods, for example by chiral column separation.

According to methods known per se (J.March, Advanced Organic Chemistry, John Wiley)&Sons, 4 th edition, 1992), for example by reacting an OH group [ R3 equal to H ] on the 3, 5-dichlorotyrosine amino acid of the compound of formula (I) with an acid chloride or with an acid anhydride in the presence of a base]Derivatisation to acyl [ R3 equal C (═ O) - (C)₁-C₆) -alkyl groups)]And/or the OH group [ R4 equals H ] on the 3-hydroxyvaline amino acid of the compound of formula (I)]Derivatisation to acyl [ R4 equal C (═ O) - (C)₁-C₆) -alkyl groups)]。

According to methods known to the person skilled in the art (J.March, Advanced organic chemistry, John Wiley&Sons, 4 th edition, 1992), for example by reacting with (C) in the presence of a base₁-C₆) Alkyl bromides or, in the case of methylation, methyl iodide or Me₂SO₄Reacting the OH group [ R3 equal to H ] on the 3, 5-dichlorotyrosine amino acid of the compound of formula (I)]Alkylated by alkyl [ R3 equals (C)₁-C₆) -alkyl radical]And/or the OH group [ R4 equals H ] on the 3-hydroxyvaline amino acid of the compound of formula (I)]Alkylated by alkyl [ R4 equals (C)₁-C₆) -alkyl radical]。

Selective differentiation of phenolic hydroxyl Groups (R3 ═ H) and aliphatic hydroxyl Groups (R4 ═ H) for introduction of protecting Groups (t.w.greene, p.g.m.wuts, Protective Groups in Organic Synthesis, John Wiley, was carried out by methods known per se to the person skilled in the art&Sons, 3 rd edition, 1999). For example, Pettus et al (J.Am.chem.Soc.2000, 122, 6160-₁-C₆) Alkyl bromides at K₂CO₃In acetone or with (C)₁-C₆) -alkyl-OH in (CF)₃CO)₂O and CuCl₂In the presence of hydrates, in DBU, to carry out selective alkylation of phenolic hydroxyl groups. Other possibilities for the discrimination between phenolic and aliphatic hydroxyl groups, for the double alkylation [ R3 equal to R4 equal to (C), are carried out by methods known per se to the person skilled in the art₁-C₆) -alkyl radical]Or diacylation [ R3 equal to R4 equal to C (═ O) - (C)₁-C₆) -alkyl radical]Selective deprotection of a compound of formula (I) (T.W.Greene, P.G.M.Wuts, Protective Groups in organic Synthesis, John Wiley&Sons, 3 rd edition, 1999). For example, Jones et al (J.org.chem.2001, 66, 3688-. The phenolic hydroxyl group (R3 ═ H) can also be determined by reaction with H₂N-(C₁-C₆) Alkyl in Cl-[(C₁-C₆) -alkyl radical]-Cl or Br- [ (C)₁-C₆) -alkyl radical]Reaction in the presence of-Br being derivatised as a radical- (C)₁-C₆) alkylene-NH- (C)₁-C₆) -an alkyl group.

Derivatizing a compound of formula (I) wherein X and Y form an-O-group, into a compound of formula (I) wherein X and Y are independently of each other OH, O- (C)₁-C₆) -alkyl, NH₂Or NH- (C)₁-C₆) -alkyl of the compound of formula (I) (j. march, Advanced Organic Chemistry, John Wiley&Sons, 4 th edition, 1992), for example by reacting epoxy groups with (C)₁-C₆) Alcoholate [ if Y is equal to O- (C)₁-C₆) -alkyl, then X is equal to OH; or if X is equal to O- (C)₁-C₆) Alkyl, then Y is equal to OH]、NH₃[ if Y is equal to NH₂X is then equal to OH; or if X equals NH₂Y is then equal to OH]Or H₂N-(C₁-C₆) Alkyl [ if Y is equal to NH- (C)₁-C₆) -alkyl, then X is equal to OH; or if X is equal to NH- (C)₁-C₆) Alkyl, then Y is equal to OH]And (4) reacting.

Isolates of the microorganism strain ST 201196 were deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (Mascheroder Weg 1B, 38124 Brunswick, Germany) under the provisions of the Budapest convention at 09.11.2003. The following numbers are designated as accession numbers: DSM 18870.

The vegetative cells of the strain ST 201196(DSM18870) have a characteristic rod shape. On solid nutrient media, the microorganism strain ST 201196(DSM18870) forms orange-yellow fruiting bodies which contain round myxospores. Thus, the taxonomy of the microbial strain ST 201196 can be described as myxobacteria sp.

It is also possible to replace the microorganism strain ST 201196(DSM18870) with mutants and/or variants thereof which synthesize one or more compounds of the invention.

Mutants are microorganisms in which one or more genes of the genome have been altered, wherein the one or more genes responsible for the organism's ability to produce the compounds of the invention remain functional and heritable.

Mutants of this type can be generated in a manner known per se by physical means, such as radiation, for example ultraviolet or X-ray beam radiation, or by chemical mutagens, such as Ethyl Methanesulfonate (EMS), 2-hydroxy-4-Methoxybenzophenone (MOB) or N-methyl-N' -nitro-N-nitrosoguanidine (MNNG), or as described by Brock et al in microbiology ("Biology of Microorganisms", Prentice Hall, pp.238-247, 1984).

The variant is a phenotype of the microorganism. Microorganisms have the ability to adapt to their environment and thus exhibit significant physiological flexibility. With respect to phenotypic adaptation, all cells of the microorganism are involved, the nature of the alteration not being genetically determined and being reversible under the conditions of the alteration (H.Stolp, microbiological technology: organnismus, halotats, activites. Cambridge university Press, Cambridge, UK, page 180, 1988).

Screening for synthesis of mutants and/or variants of one or more compounds of the invention was performed according to the following protocol:

-freeze drying the fermentation medium;

-extracting the lyophilisate with an organic solvent;

-extracting the compound from the culture filtrate using a solid phase;

analysis by HPLC, TLC or by detection of biological activity.

The fermentation conditions apply to ST 201196(DSM18870) and to mutants and/or variants thereof.

For the determination of the antifungal activity of the rapidly growing aerobic variants, the broth dilution method (microdilution) according to the procedure of CLSI (clinical laboratory standards institute, M7-A7, Vol.26, No. 2) was used. Measure IC₅₀The value is obtained. IC (integrated circuit)₅₀The value is the concentration of active substance necessary to inhibit the growth of candida albicans in the test organism by 50%.

IC of the Compound of formula (II) against Candida albicans₅₀It was 0.06. mu.g/ml. IC of the Compound of formula (IV) against Candida albicans₅₀It was 0.41. mu.g/ml.

The invention furthermore relates to the use of the compounds of the formula (I) or their physiologically tolerable salts as medicaments in human or veterinary medicine, in particular for the treatment and/or prophylaxis of fungal diseases. Preferably the present invention relates to the use of a compound of formula (I) or a physiologically tolerable salt thereof for the treatment of local and/or systemic fungal diseases.

The invention furthermore relates to medicaments containing at least one compound of the formula (I), wherein one or more compounds of the formula (I) can be administered as such or, preferably, as a mixture with one or more pharmacologically suitable conventional carriers or excipients.

The compounds of the invention are stable in the solid state and in solutions at pH 2-9, in particular at pH5-7, and can therefore be incorporated into conventional galenic preparations.

The medicaments of the invention can be administered orally or parenterally, but rectal administration is also possible in principle. Suitable solid or liquid galenic forms are, for example, granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, aerosols, injectable solutions in the form of drops or ampoules and preparations for the prolonged release of active substances, in which pharmacologically suitable carriers or excipients such as disintegrants, binders, coatings, bulking agents, glidants or lubricants, flavor additives, sweeteners or solubilizers, for example magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk proteins, gelatin, starches, vitamins, cellulose and its derivatives, animal or vegetable oils, polyethylene glycols and solvents such as sterile water, alcohols, glycerol and polyols are customarily used.

Dosage units for oral administration optionally may be microencapsulated to delay release or extend it over a longer period, for example by coating or embedding the active substance in particulate form in a suitable polymer, wax or the like.

The pharmaceutical preparations are preferably prepared and administered in dosage units, each unit containing as active ingredient a dose of one or more compounds of the natural substance derivatives according to the invention. In the case of solid dosage units such as tablets, capsules and suppositories, the dosage may be up to about 500 mg/day, but is preferably from about 0.1 to 200 mg/day; for injection solutions in the form of ampoules, up to about 200 mg/day, but preferably from about 0.5 to 100 mg/day.

The daily dose administered depends on the weight, age, sex and condition of the mammal. However, in some cases higher or lower daily doses may also be appropriate. Administration of the daily dose may be carried out by a single administration of the individual dosage units or of a plurality of smaller dosage units and by a plurality of administration of the subdivided doses at intervals.

The medicaments of the invention are prepared by optionally mixing one or more compounds of formula (I) according to the invention with one or more conventional carriers or excipients and forming a suitable administration form.

The following examples serve to explain the invention in more detail, without limiting the scope of the invention in any way.

The percentages are by weight. The mixing ratio of the liquids is in terms of volume unless otherwise specified.

Example 1: storage at-135 ℃ ST 201196(DSM18870)

Agar plates (1% fresh baker's yeast; 1% CaCl)₂×2H₂O; 0.477% HEPES (20 mM); 0.00005% cyanocobalamin; 1.8% agar; pH7.2) was inoculated with the microorganism strain ST 201196(DSM18870) and incubated at 30 ℃ for about 7 to 10 days. The surface culture was scraped from the surface of the agar with a sterile spatula and suspended in a cold medium1ml of tyrose peptone Medium (1% tyrose peptone (Difco); 0.15% MgSO. sup.; 0.15%) in a tube₄×7H₂O; pH 7.0), and storing at-135 deg.C.

Example 2: storage at-196 ℃ ST 201196(DSM18870)

Agar plates (1% fresh baker's yeast; 1% CaCl)₂×2H₂O; 0.477% HEPES (20 mM); 0.00005% cyanocobalamin; 1.8% agar; pH7.2) was inoculated with the microorganism strain ST 201196(DSM18870) and incubated at 30 ℃ for about 7 to 10 days. The cells of the surface culture were scraped from the agar surface with a sterile spatula and suspended in 1ml of tyrose peptone medium (1% tyrose peptone (Difco); 0.15% MgSO 2) in a low temperature test tube₄×7H₂O; pH 7.0), and stored at-196 ℃.

Example 3: preparation of a preculture of ST 201196(DSM18870) in Erlenmeyer flasks

100ml of nutrient solution (1% fresh baker's yeast; 1% CaCl) in a 300ml sterile conical flask₂×2H₂O; 0.477% HEPES (20 mM); 0.00005% cyanocobalamin; 1.8% agar; pH7.2) was inoculated with the microorganism strain ST 201196(DSM18870) and incubated at 30 ℃ and 180rpm for 7 days on a rotary shaker. Subsequently, 10ml of this preculture were used in each case for the preparation of the main culture.

Example 4: preparation of a liquid Main culture with Medium 1 ST 201196(DSM18870)

100ml of the following nutrient solution (1% Probion F; 0.1% CaCl)₂×2H₂O；0.2％MgSO₄×7H₂O; 0.00005% cyanocobalamin; 2% adsorption resin XAD-16, pH 8.4) was applied to fresh agar plates (1% fresh baker's yeast; 1% of CaCl₂×2H₂O; 0.477% HEPES (20 mM); 0.00005% cyanocobalamin; 1.8% agar; pH7.2) and shaking10ml (10%) of preculture (see example 3) or inoculation of the culture incubated at 180rpm and 30 ℃ on the plates. The maximum yield of the substance according to the invention is reached after 144 to 216 hours. The same nutrient solution as described in example 3 for 144-196 hours of submerged culture (10% inoculation) was sufficient to inoculate a 10 to 200 liter fermentor.

Example 5: preparation of a liquid Main culture with Medium 2 ST 201196(DSM18870)

Mixing 100ml of the following nutrient solution (1% oatmeal, 0.5% glycerol, 0.1% CaCl)₂×2H₂O；0.2％MgSO₄×7H₂O; 0.00005% cyanocobalamin; 2% adsorption resin XAD-16, pH 9.0) was applied to fresh agar plates (1% fresh baker's yeast; 1% of CaCl₂×2H₂O; 0.477% HEPES (20 mM); 0.00005% cyanocobalamin; 1.8% agar; pH7.2) and incubated at 180rpm and 30 ℃ on a shaker, 10ml (10%) of a preculture (see example 3) or inoculation of the culture. The maximum yield of the substance according to the invention is reached after 144 to 216 hours. The same nutrient solution as described in example 3 for 144-196 hours of submerged culture (10% inoculation) was sufficient to inoculate a 10 to 200 liter fermentor.

Example 6: preparation of the substances according to the invention in a fermenter

The 1 liter and 50 liter fermenters were operated under the following conditions:

inoculation: 20 percent of

Nutrient medium: 1% oatmeal; 0.5% glycerol; 0.1% yeast extract; 0.1% CaCl₂×

2H₂O；0.2％MgSO₄×7H₂O; 0.00005% cyanocobalamin; 2% adsorption

Resin XAD-16

Incubation temperature: 30 deg.C

Stirring speed: 200rpm

Ventilating: 0.6m³/h

pH regulator: adjusting pH to 7.6 + -0.3 with KOH before sterilization

pO₂A regulator: is free of

An antifoaming additive: 0.05% Desmophen (Bayer Corp. (Bayer))

Operating time: 155 hours

Using 10% KOH or 10% H₂SO₄The pH adjustment is performed.

Example 7: isolation of the compounds of formulae (II) and (III) from the shake culture of the microorganism strain ST 201196(DSM18870)

After completion of the fermentation of the microorganism strain ST 201196(DSM18870), the culture broth from example 4 (60 l culture broth) was filtered. The XAD-containing biomass was freeze-dried and subsequently extracted with methanol (4 × 5 l). The methanol extract was reduced to 8 liters in vacuum and then applied filled with about 3.0 liters of CHP-20P material (C:)Gel, 75-150 μ, Mitsubishi chemical Corporation) on a preparative column. Elution was performed with a gradient of 10% to 95% methanol. The effluent of the column (120ml/min) was collected in fractions (1 liter per fraction). Fractions 11 to 14 were combined, the solvent was removed on a rotary evaporator and the pool of fractions was freeze dried (yield about 0.9 g).

Example 8: pre-separation of compounds of formulae (II) and (III) by RP-18 chromatography

From 11 th to 14 th of example 7The pool of fractions is dissolved in 100ml of methanol and applied to a laboratory havingPhenomenex of 10 uC 18(2) pre-column (size: 60 mm. times.21.2 mm)mu.C 18(2) column (size: 250 mm. times.50 mm) was eluted over a gradient of 40 minutes with a solution of 5% to 95% acetonitrile in water (0.1% ammonium acetate, pH adjusted to 4.6 with acetic acid). The flow rate was 190ml/min and the fraction size was 190 ml. Fractions 28-29 and 31 were subsequently subjected to further work-up.

Example 9: purification of the Compound of formula (II)

Fraction 31 from example 8 was first freeze-dried (yield about 98mg) and then dissolved in 50ml of methanol, again withPhenomenex of Prep MS C1810 μm Pre-column (Waters, size: 19X 10mm)Axia 5. mu. m C18(2) column (size: 100 mm. times.30 mm) was purified by HPLC. Elution was performed with a gradient of 5% to 95% acetonitrile in water (0.1% ammonium acetate was added, pH was adjusted to 4.6 with acetic acid) over 40 minutes. The effluent of the column was collected according to UV in fractions (50 ml/min). Fractions 4 to 14 containing the compound of formula (II) were freeze dried to yield 38mg (> 95% purity).

Example 10: characterization of the Compound of formula (II)

Colorless solid, crystals crystallized from acetonitrile/water

UV：208、232、286nm

ESI-MS：MW＝815.3312

Experimental formula (II): c₄₂H₅₅Cl₂N₃O₉

Optical rotation (MeOH): -0.19 °, α_D＝-38°

Table 1: NMR chemical shift of macrolide (II); c is 3mg/ml, d₆300K in DMSO

Example 11: purification of the Compound of formula (III)

Fractions 28-29 from example 8 were again run withWaters of Prep MS C1810 μm Pre-column (Waters, size: 19X 10mm)Purification by HPLC was carried out on a 10. mu. m C18 column (size: 100 mm. times.30 mm). Elution was performed with a gradient of 10% to 95% acetonitrile in water (10% formic acid added, pH 2.0) over 40 minutes. The effluent of the column was collected according to UV in fractions (70 ml/min). Fractions 45 to 47 containing the compound of formula (III) were lyophilized to yield about 5.4mg (> 50% purity).

Example 12: characterization of the Compound of formula (III)

Colourless solid

UV：204、232、286nm

ESI-MS：MW＝799.3002

Experimental formula (II): c₄₂H₅₆ClN₃O₉

Table 2: NMR chemical shifts of macrolide (III); c 5mg/ml, in d₆300K in DMSO

Example 13: synthesis of Compound of formula (IV)

The compound of formula (II) (80mg, 0.098mmol) was dissolved in 5ml of acetonitrile, and the solution was treated with potassium carbonate (27mg, 0.196mmol) and methyl iodide (70mg, 0.490mmol) at room temperature. The mixture was then stirred at 50 ℃ for 12 hours. Filtering the solution to obtain a filtrate containingPhenomenex of Prep MS C1810 μm Pre-column (Waters, size: 19X 10mm)Axia 5. mu. m C18(2) column (size: 100 mm. times.30 mm) was purified by HPLC. Elution was performed with a gradient of 5% to 95% acetonitrile in water (0.1% ammonium acetate added, ph4.6, set up with acetic acid) over 40 minutes. The effluent of the column was collected according to UV in fractions (50 ml/min). Fractions 4 and 5 containing the compound of formula (IV) were lyophilized to yield 50mg (61% yield, > 95% purity).

Example 14: characterization of the Compound of formula (IV)

Colorless solid, crystals crystallized from acetonitrile/water

UV：235、286nm

MW＝830.85

Experimental formula (II): c₄₃H₅₇Cl₂N₃O₉

Table 3: NMR chemical shifts of the compound of formula (IV); c is 3mg/ml, d₆300K in DMSO

Example 15: synthesis of Compounds of formula (V) and formula (VI)

The compound of formula (II) (30mg, 0.037mmol) was dissolved in 10ml of 1, 2-dichloroethane and the solution was treated with isobutylamine (500. mu.l, 5.03mmol) at room temperature. Stirring the mixture at 70 deg.C for 48 hr, and filtering with Hu PiPrep MS C18 10μmPhenomenex of preplumn (Waters, size: 19X 10mm)Axia 5. mu. m C18(2) column (size: 100 mm. times.30 mm) was purified by HPLC. Elution was performed with a gradient of 5% to 95% acetonitrile in water (0.1% ammonium acetate was added, pH was adjusted to 4.6 with acetic acid) over 40 minutes. The effluent of the column was collected according to UV in fractions (50 ml/min). The 70 th fraction containing the compounds of formulae (V) and (VI) was freeze-dried to give 3mg of the two compounds in a ratio of 55: 45.

Example 16: characterization of the Compound of formula (V)

MW＝889.97

Experimental formula (II): c₄₆H₆₆Cl₂N₄O₉

Table 4: NMR chemical shifts of the compound of formula (V); c is 3mg/ml, d₆300K in DMSO

Example 17: characterization of the Compound of formula (VI)

MW＝916.00

Experimental formula (II): c₄₈H₆₈Cl₂N₄O₉

Table 5: NMR chemical shifts of the compound of formula (VI); c is 3mg/ml, d₆300K in DMSO

Example 18: determination of antifungal Activity against Candida albicans

Preparation of the active substance [ e.g. a compound of formula (II) or a compound of formula (IV) ]]1000. mu.g/ml stock solution in methanol. The test microorganism strain (Candida albicans FH 2173) was stored at-80 ℃. The inoculum was prepared from a fresh liquid preculture. A preculture was prepared from one drop of material stored at-80 ℃ and 30ml of nutrient medium (Sabouraud dextrose broth, Difco) and incubated at 37 ℃ and 180rpm for 24 hours. The inoculum was adjusted so as to obtain the necessary number of colony forming units after inoculation of the experimental vessel. For this purpose, the inoculum is adjusted by means of a photometer at a wavelength of 590nm10⁷CFU/ml (CFU: colony forming unit). After conditioning the inoculum, the suspension was diluted 1: 100 with nutrient broth (Mueller Hinton broth, Difco). Microtiter plates were inoculated within 15 minutes of inoculum preparation. The exact colony count was determined by surface culture means. Dilution series were prepared beforehand on microtiter plates using stock solutions of the active substance and nutrient medium (Mueller Hinton broth, Difco). The active substance was present in a volume of 20. mu.l and treated with 20. mu.l of inoculum in order to obtain a total test volume of 40. mu.l. The inoculated microtiter plates were then capped and incubated at 37 ℃ in 5% CO₂And incubation at 95% atmospheric humidity for 20 hours. For each test, the control without active substance, the sterile control and ciprofloxacin and nystatin as reference substances were tested jointly on 384-well microtiter plates. The microtiter plates were read by measuring the absorbance at a wavelength of 590nm by means of a photometer. IC was then calculated from the values of the dilution series according to standard methods₅₀The value, i.e. the concentration of active substance necessary for 50% inhibition of the growth of the test organism candida albicans.

Sai Nuofei-An Wante

Science of natural products

IndustrieparkH811 deposit receipt at original deposit

65926 Frankfurt/Main is deposited by the International depositary as shown below this page

Submit according to 7.1

¹In the case of clause 6.4(d), the date is the date on which the identity of the international depository has been obtained

Table DSMZ-BP/4 (one page only) 08/2006

Sai Nuofei-An Wante

Science of natural products

IndustrieparkH811

65926 Frankfurt (Frankfurt/Main)

Statement of survival

The International depositary shown below this page

Submit according to clause 10.2

¹Indicating the date of the original deposit or, when a new deposit or transfer is made, the latest relevant date (date of the new deposit or date of the transfer)

²When indicated in items 10.2(a), items (ii) and (iii), refer to the most recent survival test

³Marking with X in application boxes

⁴Filling in if the information requires filling in and if the test result is negative

Table DSMZ-BP/9 (one page only) 8/2006

Sai Nuofei-An Wante

Science of natural products

IndustrieparkH811 deposit receipt at original deposit

65926 Frankfurt/Main is deposited by the International depositary as shown below this page

Submit according to 7.1

¹In the case of clause 6.4(d), the date is the date on which the identity of the international depository has been obtained

Table DSMZ-BP/4 (one page only) 08/2006

Sai Nuofei-An Wante

Science of natural products

IndustrieparkH811

65926 Frankfurt (Frankfurt/Main)

Statement of survival

The International depositary shown below this page

Submit according to clause 10.2

¹Indicating the date of the original deposit or, when a new deposit or transfer is made, the latest relevant date (date of the new deposit or date of the transfer)

²When indicated in items 10.2(a), items (ii) and (iii), refer to the most recent survival test

³Marking with X in application boxes

⁴Filling in if the information requires filling in and if the test result is negative

Table DSMZ-BP/9 (one page only) 8/2006

Claims (20)

1. A compound of formula (I) or a physiologically tolerable salt of a compound of formula (I),

wherein:

x and Y are independently of each other OH, O- (C)₁-C₆) -alkyl, NH₂Or NH- (C)₁-C₆) -alkyl, or X and Y together form an-O-group;

r1 and R2 are independently Cl or H;

r3 is H, (C)₁-C₆) -alkyl, C (═ O) - (C)₁-C₆) -alkyl or (C)₁-C₆) alkylene-NH- (C)₁-C₆) -alkyl, and

r4 is H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) -an alkyl group.

2. A compound of formula (I) as claimed in claim 1, wherein X and Y together form an-O-group.

3. The compounds of the formula (I) as claimed in claim 1, wherein R3 and R4 independently of one another are H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) -an alkyl group.

4. The compounds of the formula (I) as claimed in claim 2, wherein R3 and R4 independently of one another are H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) -an alkyl group.

5. The compounds of the formula (I) as claimed in claim 1, wherein X and Y together form an-O-group, R1 and R2 are each independently of the other Cl or H, and R3 and R4 are each independently of the other H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) -an alkyl group.

6. The compounds of the formula (I) as claimed in claim 2, wherein X and Y together form an-O-group, R1 and R2 are each independently of the other Cl or H, and R3 and R4 are each independently of the other H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) -an alkyl group.

7. A compound of the formula (I) as claimed in claim 3, wherein X and Y together form an-O-group, R1 and R2 are each independently of the other Cl or H, and R3 and R4 are each independently of the other H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) -an alkyl group.

8. The compounds of the formula (I) as claimed in claim 4, wherein X and Y together form an-O-group, R1 and R2 are each independently of the other Cl or H, and R3 and R4 are each independently of the other H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) -an alkyl group.

9. The compound of formula (I) as claimed in any of claims 1 to 8, wherein R1 and R2 equal Cl.

10. The compound of formula (I) as claimed in any of claims 1 to 8, wherein R1 equals Cl and R2 equals H.

11. The compound of formula (I) as claimed in any of claims 1 to 8, wherein R3 and R4 are equal to H.

12. The compound of formula (I) as claimed in claim 9, wherein R3 and R4 equal H.

13. The compound of formula (I) as claimed in claim 10, wherein R3 and R4 are equal to H.

14. The compound of formula (I) as claimed in any of claims 1 to 8, wherein R1, R2, R3 and R4 are equal to H.

15. The compound of formula (I) as claimed in claim 11, wherein R1, R2, R3 and R4 are equal to H.

16. The use of a compound of the formula (I) or a physiologically tolerable salt thereof as claimed in any of claims 1 to 15 for the preparation of a medicament for the treatment and/or prophylaxis of fungal diseases.

17. A medicament containing at least one compound of formula (I) as claimed in any one of claims 1 to 15.

18. A process for the preparation of a compound of formula (I) or a physiologically tolerable salt of a compound of formula (I),

wherein:

x and Y are independently of each other OH, O- (C)₁-C₆) -alkyl, NH₂Or NH- (C)₁-C₆) -alkyl, or X and Y together form an-O-group;

r1 and R2 are independently Cl or H;

r3 is H, (C)₁-C₆) -alkyl, C (═ O) - (C)₁-C₆) -alkyl or (C)₁-C₆) alkylene-NH- (C)₁-C₆) -alkyl, and

r4 is H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) -an alkyl group,

the method comprises the following steps:

1. fermenting the microorganism strain ST 201196 deposited under DSM18870 in a medium containing a Cl source under suitable conditions until one or more compounds of formula (I) accumulate in the medium; and

2. isolating the compound of formula (I) from the culture medium; and

3. the compounds of formula (I) are optionally derivatized and/or converted to physiologically tolerable salts.

19. A process as claimed in claim 18, wherein the reaction is carried out in the reaction of the formula (I)In the compounds, X and Y together form an-O-group, R1 and R2 independently of one another are Cl or H, and R3 and R4 independently of one another are H, (C)₁-C₆) -alkyl or C (═ O) - (C)₁-C₆) -an alkyl group.

20. The microorganism strain ST 201196 with the accession number DSM 18870.