DE10121313A1 - Interconnections for the production of spinosyns - Google Patents

Abstract

The invention relates to intermediates for producing spinosyns, to various methods of producing them, and to the use of said intermediates for producing spinosyn derivatives.

Description

The present invention relates to interconnections for the production of Spinosyns, various processes for their production and their use of these intermediates for the production of spinosyn derivatives.

The spinosyns are known compounds. Are spinosyns Fermentation products from cultures of the Actinomycetes Saccharopolyspora spinosa can be produced. Natural spinosyns consist of a tetracyclic Polyketide backbone (aglycon) with a 12-membered macrolide ring and one 5,6,5-cis-anti-trans-tricyclic, as well as a D-forosamine and a 2,3,4-tri-O- Methyl L-rhamnose sugar content (Kirst et al. (1991), Tetrahedron Letters, 32: 4839). More than 20 different natural spinosyns, the so-called A83543 complex, have been described so far (cf. WO 97/00265, WO 94/20518 and WO 93/09126). These compounds vary in substitution of one or a few Methyl groups on the tetracyclic backbone, on the forosamine or on the tri-methyl Rhamnose sugar moiety. A 17-pseudoaglycon, which contains the forosamine sugar is also isolated from culture broths from S. spinosa.

The main components of formed of S. spinosa A83543 complex are the variants spinosyn A and spinosyn D represent, which are the essential components of the product Spinosad (see FIG. Pesticide Manual, British Crop Protection Council, 11 ^th Ed., 1997, page 1272 and Dow Elanco trade maganzine Down to Earth, Vol. 52, NO :. 1, 1997 and the literature cited therein).

If the amino sugar is absent, the compounds are considered Designated Spinosyn A, D, etc.-17 pseudo aglycone; if the neutral sugar is not is present, the compounds are spinosyn A, D, etc.-9 pseudoaglycone designated. Spinosyns without the two sugar residues are called spinosyn aglycones designated.  

Spinosyns are suitable for controlling arachnids, nematodes and insects, especially Lepidoptera and Diptera. You can expect plants Pests that are currently being controlled with spinosyns become resistant to them can produce active ingredients on the market. Therefore, it is important to find new ones to produce biologically active derivatives of spinosyns, currently used to combat can replace spinosyns used by pests. Synthetic approaches to Preparation of spinosyn derivatives were described by Martynow, J.G. and Kirst, H.A. in J. Org. Chem. 1994, 59, 1548. In this publication the 9,17-Di ketone of the spinosyn aglycon and the 17-keto derivative of the spinosyn aglycon imagines. The spinosyn-17 pseudoaglycone and spinosyn-9 pseudoaglycone are shown in WO 97/00265 and US-A 6 001 981. In it, the manufacture is white teren derivatives described by semisynthetic processes starting from natural products are available. The spinosyn-9 pseudo-aglycon, which at the Position C9 is known to be oxidized by chemical insecticides Derivatization was generated. The 9-keto-spinosyn-aglycone is new in view to the state of the art.

It is the object of the present invention to prepare new interconnections to deliver, which are suitable for the production of Spinosyn derivatives.

The object was achieved by providing compounds of the formula (I)

in which
R ^{1 represents} methyl or ethyl, and
AB represents one of the following groups: -HC = CH-, -HC = C (CH ₃ ) -, -H ₂ C-CH ₂ -,
-H ₂ C-CH (CH ₃ ) -.
R ¹ preferably represents ethyl.
AB preferably represents the group -HC = CH-.

The compounds of formula (I) according to the invention can in stereoisomers Shapes that are either like image and mirror image (enantiomers), or that are do not behave like image and mirror image (diastereomers) exist. The invention concerns both the enantiomers or diastereomers or their respective Mixtures. Like the diastereomers, the racemic forms can be described in be known to separate into the stereoisomerically uniform components. Possibly the isomers can be converted into one another by methods known per se.

The present invention also relates to chemical and biochemical / microbiological process for producing the above-mentioned compounds of general formula (I).

The compounds of the formula (I) are obtained if compounds of the formula (II)

wherein R ¹ and AB have the meanings given above,
with an oxidizing agent, optionally in the presence of a diluent.

That which can be used as a starting compound for the process according to the invention Spinosyn aglycone is known and can be prepared according to that described in WO 01/16303 Process are made. In an analogous manner, those for the fiction Starting compounds usable according to the method based on the corresponding natural spinosyns can be obtained.

A large number of different oxidizing agents are known for the oxidation of alcoholic groups (cf. e.g. oxidation agents in: Organic Synthesis by Oxidation with Metal Compounds; Mijs, de Jonge; Plenum Verlag: New York, 1986;
Manganese Compounds as Oxidizing Agents in Organic Chemistry; Arndt, Open Court Publishing Company: La Salle, IL, 1981; The Oxidation of Organic Compounds by Permanganate Ion and Hexavalent Chromium; Lee, Open Court Publishing Company: La Salle, IL, 1980). Then an oxidation, for example in the presence of permanganates, such as potassium permanganate, halogens, such as chlorine or bromine, metal oxides, such as manganese dioxide or rutenium tetroxide, etc., he follow.

Numerous different oxidizing agents are also special only for the oxidation secondary alcohols described in the literature, such as the Verwen acidic dichromates (see Chromium Oxidations in Organic Chemistry; Cainelli, Cardillo, Springer: Published by New York, 1984; Reagents for Organic Synthesis; Fieser, Vol. 1, Wiley: New York, 1967, pp. 142-147, 1059-1064 and other vols Line). A solution of chromic acid and sulfuric acid in water is called Jones Reagent (Bowden et al. (1946), J. Chem. Soc .: 39; Bowers et al. (1953), J. Chem. Soc .: 2548). Three other chromium (VI) reagents (see message about one comparative study of Jones's, Collins's and Corey's reagent in Warrener et al.  (1978) Aust. J. Chem., 31: 1113) are also known to be used; at for example dipyridine-chromium (VI) oxide (Collins's reagent) (see, for example, Collins et al. (1968), Tetrahedron Lett .: 3363), pyridinium chlorochromate (Corey's reagent) (cf. Review: Luzzio and Guziec (1988), Org. Prep. Proced. Int., 20: 533-584) and Pyridi nium dichromate (see Coates (1969), Corrigan Chem. Ind. (London): 1594; Corey, Schmidt (1979), Tetrahedron Lett .: 399). For acid sensitive substrates for example, the use of chromium (VI) oxide in hexamethyl-phosphoric acid tri amide (HMPA) (see Cardillo et al. (1976), Synthesis: 394), a chromium (VI) - oxide-pyridine complex (cf. Poos et al. (1953), J. Am. Chem. Soc., 75: 422) or Trimethylsilyl chromates (Moiseenkov et al. (1987) J. Org. Chem. USSR, 23: 1646) are also described. Sodium hypochlorite in acetic acid is for the oxidation of a secondary alcohol mentioned in large quantities (cf. Stevens et al. (1980), J. Org. Chem., 45: 2030; Schneider et al. (1982), J. Org. Chem., 47: 364). The oxi dation reagents can also be polymer-bound (see review: McKillop, Young (1979), Synthesis: 401-422). Both chromic acids and per Manganese were used as an oxidizing agent in this way. Are also numerous phase transfer reactions with permanganates (see review: Lee, in Trahanovsky, Ref. 2, pt. D, pp. 147-206), chromic acids (Hutchins et al. (1977), Tetra hedron Lett .: 4167; Landini et al. (1979), Synthesis: 134) and rutenium tetroxide (Morris, Kiely J. Org. Chem. (1987), 52: 1149). Even ultrasound-induced Oxidation reactions are conceivable - this is the use of potassium permanganate mentioned (Yamawaki et al. (1983), Chem. Lett .: 379).

In addition, most oxidizing agents that can oxidize primary alcohols in aldehydes are also suitable for the corresponding oxidation of secondary alcohols. Such oxidizing agents for primary alcohols are, for example, pyridinium dichromate, tetrapropylammonium perruthenate (Pr ₄ N ⁺ RuO ₄ ^- ), cerium ammonium nitrate (CAN), silver carbonate on Celite (Fetizon et al. (1968), Acad. Sci., Ser. C , 267: 900), Na ₂ Cr ₂ O ₇ in water (Lee et al. (1970), J. Org. Chem., 35: 3589), lead tetraacetate pyridine, benzoyl peroxide-nickel dibromide or dimethyl sulfoxide in the presence of oxalyl chloride (Swern oxidation), copper (II) sulfate pentahydrate in pyridine, copper (II) acetate in 70% acetic acid, iron chloride in water, chromium (VI) oxide in glacial acetic acid or dichromium trioxide in pyridine. Reagents which can specifically oxidize a secondary hydroxyl group even in the presence of a primary hydroxyl group include, for example, hydrogen peroxide-ammonium molybdate (Trost et al. (1984), Isr. J. Chem., 24: 134), sodium borate (NaBrO ₃ ) - CAN (Tomioka et al., Tetrahedron Lett. 23: 539). N-halosuccinimides (halogen = chlorine, bromine, iodine) can be used as oxidizing agents for hydroxyl groups, even in the presence of other oxidizable groups (cf. Review: Filler Chem. Rev. 1963, 63, 21-43, pp. 22- 28). For example, the combination of N-iodo-succinimide-tetrabutylammonium iodide is suitable for the oxidation of secondary alcohols in high yields (Hanessian et al. (1981), Synthesis: 394).

Other known oxidation methods also include oxidative dehydrogenation, for example in the presence of catalysts such as silver or copper catalyst ren (M. Muhler in: Handbook of Heterogenous Catalysis, VCH, Weinheim, 1997). Other mild catalytic oxidative processes using platinum or Palladium-carbon catalysts are known which also make them sensitive classes of substances, for example carbohydrates (M. Besson et al. (1995), J. Catal. 152: 116-122) or steroids (T. Akihisa et al. (1986), Bull. Chem. Soc. Jpn. 59: 680-685). As an efficient commercial catalyst for the oxida tion is, for example, the inorganic TS-1 catalyst (oxide titanium silicalite), which in aqueous hydrogen peroxide (30% w / w) catalytic oxidation of primary and secondary alcohols (R. Murugawel et al. (1997), Angew. Chem. Int. Ed. Engl., 36: 477-479).

Pyridinium dichromate is preferably used as the oxidizing agent.

The inventive method for producing the new compounds is before preferably carried out using diluents. dilution agents are preferably used in such an amount that the reaction mixture remains easily stirrable throughout the process.  

Depending on the previously mentioned oxidizing agent come as a diluent medium in addition to water or aqueous hydrogen peroxide also acidic dilution agents such as concentrated or partially diluted acetic acid, and basic diluents, such as pyridine.

Practically all inert organic solvents are also suitable. For this include in particular aliphatic, alicyclic or aromatic, optionally halogenated hydrocarbons, such as gasoline, benzene, toluene, xylene, Anisole, chlorobenzene, dichlorobenzene, petroleum ether, hexane, cyclohexane, dichloro methane, 1,2-dichloroethane, chloroform, carbon tetrachloride, ethers such as diethyl ether, dioxane, tetrahydrofuran or ethylene glycol dimethyl or diethyl ether, Ketones such as acetone or butanone, nitriles such as acetonitrile or propionitrile, amides, such as dimethylformamide, dimethylacetamide, N-methylformanilide, N-methylpyrroli don or hexamethylphosphoric triamide, esters such as ethyl acetate, sulf oxides, such as dimethyl sulfoxide or sulfolane.

Of course, the method according to the invention can also be used in mixtures perform the solvents mentioned.

Dichloromethane is particularly preferably used as the diluent.

Furthermore, the reaction according to the inventive method is preferably ren carried out under inert gas.

The reaction temperatures can be in one in the process according to the invention larger range can be varied. Generally one works at temperatures between - 100 ° C and + 150 ° C, preferably at temperatures between - 20 ° C and + 50 ° C.  

The process according to the invention is generally carried out under normal pressure guided. However, it is also possible to increase or decrease pressure work.

To carry out the method according to the invention, the respectively required ones are used Starting compounds in general in approximately equimolar amounts puts. However, it is also possible to use the oxidizing agent in a deficit the.

The processing takes place according to usual methods.

The new compounds of the general formula (I) can also be obtained by bioconverters sion are prepared starting from compounds of general formula (II).

Selective and / or stereospecific oxidations of hydroxyl groups of natural products and synthetic compounds by bioconversion using microorganisms or their enzymes are described in the literature. In particular, cells and / or enzymes from Actinomycetes (Nocardia, Streptomy ces) and other Gram-positive (Bacillus, Clostridium) or Gram-negative bacteria were used for the specific oxidation of steroid compounds, the chemical accessibility of which is limited. Examples that relate in particular to enzyme classes such as hydroxysteroid dehydrogenases or cholesterol oxidases are listed in the following table:

According to the invention, compounds of the formula (II)

wherein R ¹ and AB have the meanings given above,
contact with a microorganism in an aqueous nutrient medium under aerobic conditions and then isolate compounds of the formula (I).

Instead of the microorganisms, enzyme extracts and purified ones can also be used Enzymes, if necessary after adding or regenerating the required Co factors, are used, which are based on conventional methods based on these Microorganisms are available.

A microorganism is preferably produced for the process according to the invention the genus Bacillus or from the group of the Actinomycetes, in particular from the Genus Streptomyces, or a mushroom, in particular from the class of the Zygomycetes, and here preferred the genus Zygorhynchus or Mucor - or starting there of enzyme extracts produced or purified enzymes - used.

A strain from which is particularly preferred for the process according to the invention Species Bacillus simplex, Streptomyces argillaceus, Streptomyces griseus, Zygorhynchus moelleri or Mucor circinelloides used.

A strain with the characteristic features of the following strains is very particularly preferably used for the method according to the invention:

The strains listed in the table are from the German collection of micro organismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, in accordance with the provisions of the Budapest contract has been deposited.

The strains are described in more detail in Example 9. It can't just be the one behind put strains as such, but mutants of it can be used as long as these mutants have the characteristic features of the deposited strains. This means that the mutants still have the ability to invent to be able to carry out bioconversion in accordance with the invention.

The aqueous nutrient medium preferably contains an assimilable carbon source and an assimilable nitrogen source.

The compounds of formula (I) are produced, for example, when a strain from the species Streptomyces argillaceus, Streptomyces griseus, Bacillus megaterium, Bacillus simplex, Zygorhynchus moelleri or Mucor circinelloides in a water gene nutrient medium under aerobic conditions in the presence of compounds  of the formula (II) are fermented. Typically, the microorganisms in a nutrient medium containing a carbon source and possibly a protein contains like material, fermented. Preferred carbon sources include Glu cose, brown sugar, sucrose, glycerin, starch, corn starch, lactose, dextrin, Molasses etc. Preferred nitrogen sources include cottonseed flour, yeast, autolyzed baker's yeast, solid milk components, soybean meal, corn meal, pancreatic or papainic digested casein breakdown products, solid distilla ingredients, animal peptone broths, pieces of meat and bone, etc. Combinations of these carbon and nitrogen sources are preferred used. Trace elements, such as zinc, magnesium, manganese, cobalt, Iron etc. do not have to be added to the fermentation medium as long as Lei tation water and unpurified components used as medium components become.

The compounds of the general formula (I) can be prepared at any temperature rature induced that sufficient growth of the microorganisms ge guaranteed. The temperature is preferably between 21 ° C. and 32 ° C., particularly preferably about 28 ° C.

In general, optimal production of the compounds of formula (I) within 1 to 10 days after adding the compounds of formula (II) to the Culture obtained, preferably in about 2 to 6 days. The fermentation broth usually remains weakly basic during the fermentation (pH 7.4 to 8.0). The final pH is partly dependent on the buffer that may be used and in part from the initial pH of the culture medium. Preferably the pH set to about 6.5 to 7.5 before sterilization, particularly preferably to pH 7.2.

The production of the compounds according to the invention takes place both in shakes bottles as well as in stirring fermenters. If the cultivation in shake bottle or large kettles and tanks is carried out, preferably the  vegetative form instead of the spore form of the microorganisms for inoculation applies to a significant lag phase in the production of the metabolites and thus to avoid inefficient use of the equipment. So it is before partial, inoculate a vegetative inoculum in an aqueous nutrient medium this medium with an aliquot of a bottom or slant tube culture to deliver. Having a fresh, active, vegetative inoculum this way has been prepared, this is aseptically into further shaking bottles or more transferred suitable equipment for the fermentation of the microorganisms. The The medium in which the vegetative inoculum is produced can be identical or be different from the medium used for the production of the Invention modern compounds is used as long as there is a sufficient microorganism growth guaranteed.

In general, the preparation of the compounds according to the invention is under Use of the above microorganisms under aerobic conditions in Stirring fermenters accomplished. However, the manufacture is independent of the ver used fermenters and starter cultures. The compounds of the invention can also be obtained from shake cultures. For large volume fennenta a vegetative inoculum is preferably used. The vegetative Ino kulum is obtained by inoculating a small volume of the culture medium with the Spore form, mycelium fragments or a lyophilized pellet of the microorganism mus made. The vegetative inoculum is then placed in a fermentation kettle transferred, after a suitable incubation period in the presence of the verbin of the general formula (II) the compounds of the invention in one optimal yield can be produced. Usually one gets aerobic Submerged fermentation process passed sterile air through the culture medium. For The air volume used is an efficient growth of the microorganisms in the range of about 0.25 to about 0.5 volumes of air per volume of culture medium per minute (vvm). An optimal ratio in a 10 l boiler is not drive 0.3 vvm with motion by a conventional one at around 200-500 rpm, preferably with 300 rev / min rotating propeller. The addition  a small amount, such as 1 ml / l, of an anti-foaming agent, such as silicone to the fermentation medium is necessary if foaming is a Represents problem.

Preferred fermentation conditions and media are described in the examples ben.

The biotransformation of the compounds of formula (II) to those of the invention Connections generally start after about 48 hours and take for min at least 6 days during the fermentation period. The peak production will reached between about 5 to 7 days fermentation time using Bacillus strains after 3-4 days.

The compounds according to the invention as a biotransformation product can be obtained from be isolated from the fermentation medium by conventional methods.

The methods according to the invention are primarily in the biomass of the fermented Microorganisms are present, but can also be found in small quantities in the culture of the fermentation broth. The culture broth can be done easily be separated by filtering through a filter press.

Various methods can be used to convert the ver isolate and clean bonds from the fermentation broth, such as. B. by chromatographic adsorption processes (for example by column chromatography graphie, liquid-liquid distribution chromatography, gel permeation chromatography graphie) followed by elution with a suitable solvent, and crystallization from solvents, as well as combinations thereof. In a preferred cleaning the compounds according to the invention are processed from the biomass from which Mycelia or extracted from extracts of the supernatant. The latter can be found under Ver use of adsorption resins such as XAD, HP20 or Lewapol be put. Column chromatography methods, preferably over silica gel  or modified silica gels are used to do an initial cleaning respectively. The final purification of the compounds according to the invention is before preferably by preparative high performance liquid chromatography (HPLC) enough.

The compounds of the invention can be used to prepare biologically active substances ven, especially insecticidal, spinosyn derivatives can be used.

One uses, for example, for glycosidation of the verbin according to the invention of the formula (Ia) an activated forosamine (D-forosamine: cf. EP-A 0 375 316) as aminosugars of formula (III), wherein LG for a known, suitable departure group (LG = leaving group), such as bromine or 2,2,2-trichloroethanimi dat (= trichloroacetimidate) can be obtained from WO 97/00265 and US-A 6 001 981 known 9-keto-spinosyn A-9 pseudoaglycone of the formula (IVa) (see scheme 1).

Scheme 1

glycosidation

The representation of an activated forosamine as amino sugar of the formula (III), at for example the α-D-forosaminylbromide hydrobromide or the α-L-N-Fmoc- Forosaminylbromids (LG = bromine), and its glycosidation reaction is over WO 97/00265 and US-A 6 001 981 and D. A. Evans et al. (1993), J. Am. Chem. Soc. 115: 4497-4513. Also shown is an activated aminozu of the formula (III), for example 1- (2,2,2-trichloroethanimidate) -4,6- dideoxy-4- (dimethylamino) -5-C-methyl-β-D-ribo-hexopyranose-2,3-diacetate (LG = 2,2,2-trichloroethane imidate), and its stereoselective glycosidation reaction known (see I. Sato et al. (1999), Chem. Lett. 9: 867-868; see also the Use of trichloroacetimidates in the production of glucospingolipids: G.R. Duffin et al. (2000), J. Chem. Soc., Perkin Trans. 1: 2237-2242).

The biological effectiveness of the 9-keto-spinosyn A-9 pseudoaglycon of the formula (IVa) against Stomoxys calcitrans (stable fly) and Phormia regina (blow fly) is already in WO 97/00265 and US-A 6 001 981.

Furthermore, WO 97/00265 and US-A 6 001 981 are two subsequent reactions (Paths a and b) with the 9-keto-spinosyn A-9 pseudoaglycone of the formula (IVa) knows in which the reactivity of the carbonyl group in the 9-position is used (cf. Scheme 2).  

For example, it is possible to use the 9-keto-spinosyn A-9 pseudoaglycone of the formula (IVa) with the Grignard reagent methyl magnesium chloride (V) to a chromato graphically separable mixture of (9S) - and (9R) -9-methyl-Spinosyn A-9-Pseu to implement the doaglycone of the formulas (VIa) and (VIb) (route a). In another be Known embodiment is the reaction of the 9-keto-spinosyn A-9 pseudo aglycons of formula (IVa) with morpholine (VII) in the presence of sodium cyano borohydride to form the 9-deoxy-9- (N-morpholinyl) spinosyn A-9 pseudoaglycons of formula (VIII) explained (see Scheme 2, path b).

Scheme 2

Carbonyl reactions with the 9-keto-spinosyn A-9 pseudoaglycone

Route a: MeMgCl (V), tetrahydrofuran
Path b: morpholine (VII), methanol, sodium cyanoborohydride

For the (9S) - and (9R) -9-methyl-spinosyn A-9 pseudoaglycone of the formula (VIa) and (VIb) is a biological, in particular insecticidal, activity against aphis gossipii (cotton aphid), Heliothis virescens (tobacco budworm) and Stomoxys calcitrans (stable fly) already described in WO 97/00265 and US-A 6 001 981. Also for the 9-deoxy-9- (N-morpholinyl) -spinosyn A-9 pseudoaglycone of the formula (VIII) is a corresponding biological, especially insecticidal, effectiveness against Stomoxys calcitrans (stable fly) in WO 97/00265 and US-A 6 001 981.

Examples example 1 Production of the Spinosyn aglycon from Tracer®

The compound of the formula (II) in which R ^{1 is} ethyl and AB is the group -HC = CH- (hereinafter called compound (2)) was prepared as described in WO 01/16303.

Example 1a) Production of the 5,6-dihydrospinosyn aglycon from Tracer®

The compound of formula (II), wherein R ^{1 is} ethyl and AB represents the group -H ₂ C-CH ₂ - (5,6-dihydrospinosyn aglycone, hereinafter referred to as compound (4)), was analogous to WO 01/16303 made from 5,6-dihydrospinosyn A. The preparation of 5,6-dihydrospinosyn A from spinosyn A is known from WO 97/00265 and US Pat. No. 6,001,981. Spinosyn A for its part was produced from Tracer® as described in WO 01/16303.

Example 2 Strains used

Table: Strains capable of biotransforming compound (2) to the corresponding 9-keto derivative (hereinafter referred to as compound (1)):

Example 3 Biotransformation with S. argillaceus DSM 14030, S. griseus DSM 14029, Actinomycetales sp. DSM 14077, Actinomycetales sp. DSM 14078, Streptomyces sp. DSM 14079, Zygorhynchus moelleri WP0796 or Mucor circinelloides WP0799

Exemplary protocol of the biotransformation for the production of the connection (I) from compound (2).

Example 3a) Preparation of the precultures of S. argillaceus DSM 14030, S. griseus DSM 14029, Actinomycetales sp. DSM 14077, Actinomycetales sp. DSM 14078, Streptomyces sp. DSM 14079, Zygorhynchus moelleri WP0796 or Mucor circinelloides WP0799

The strains S. argillaceus DSM 14030, S. griseus DSM 14029, Actinomycetales sp. DSM 14077, Actinomycetales sp. DSM 14078, Streptomyces sp. DSM 14079, Zygorhynchus moelleri WP0796 or Mucor circinelloides WP0799 grown in the following media: Medium 1: yeast malt Medium: D-glucose 0.4%, yeast extract 0.4%, malt extract 1.0%, ad 1 liter of lei  water (pH with aqueous HCl to 6.5 for the mushrooms or 7.2 for the bacteria discontinued) or TSB Medium (Medium 2): Trypticase Soy Broth (Difco) (30 g / l), ad 1 liter of tap water. The pH of the medium was increased with aqueous NaOH 7.2 set. The media were each at 121 ° C and 1.1 bar overpressure for 20 Minutes sterilized.

2 ml portions of a mycelium suspension in 50% glycerol were used as inoculum for 2 × 150 ml Set up medium 1 in a 1000 ml Erlenmeyer flask and 72 hours at 240 Rotations per minute (rpm) incubated on a rotary shaker. This Cultures were either used to make new glycerin preserves, which -20 ° C, or they served as an inoculum for the production cultures (see 3b).

Example 3b) Production of the production cultures of S. argillaceus DSM 14030) S. griseus DSM 14029, Actinomycetales sp. DSM 14077, Actinomycetales sp. DSM 14078, Streptomyces sp. DSM 14079, Zygorhynchus moelleri WP0796 or Mucor circinelloides WP0799

To produce the production cultures, 2 ml of a preculture as described under 3a) were used as inoculum for 100 × 150 ml of medium GS (glucose (20 g / l), soy flour (20 g / l), starch (20 g / l), NaCl (2.5 g / l), CaCO ₃ (5 g / l), MgSO ₄ × 6 H ₂ O (0.5 g / l), KH ₂ PO ₄ (0.25 g / l) after the inoculation, the pH was adjusted to 6.8 with KOH and sterilization was carried out for 20 minutes at 121 ° C. and 1.1 bar gauge pressure, and the compound (2) was mixed in a methanolic concentration of 50-500 mg / l medium Solution (100 mg / ml methanol) was added either at the beginning of the fermentation or during the fermentation at any time up to a fermentation period of 160 hours. The biotransformation was stopped after 240 hours. Daily samples were used to monitor the biotransformation process of 50 ml, which were removed sterile and examined by means of analytical HPLC, under which conditions, preferably with S. argillac eus DSM 14030, conversion rates up to 100% of connection (2) to connection (1) achieved.

Example 4 Biotransformation with Bacillus simplex DSM 14028, B. megaterium DSM 333 and B. megaterium DSM 339

Exemplary protocol of the biotransformation for the production of the connection (1) from compound (2).

Example 4a) Preparation of the primary precultures of Bacillus simplex DSM 14028, B. megaterium DSM 333 and B. megaterium DSM 339

Bacillus simplex DSM 14028, B. megaterium DSM 333 and B. megaterium DSM 339 in the following medium gene: TSB Medium (Medium 2): Trypticase Soy Broth (Difco) (30 g / l), ad 1 liter Tap water. The medium was at 121 ° C and 1.1 bar pressure for 20 minutes sterilized.

2 ml of a bacterial suspension in 50% glycerol was used as an inoculum for 150 ml Medium 2 is placed in a 1000 ml Erlenmeyer flask and 48 hours at 240 rpm incubated on a rotary shaker. This medium was either used to make new canned glycerol stored at -20 ° C, or it served as an inoculum for the production cultures (see 4b).

Example 4b) Production culture (10 liter scale)

To prepare the production culture, 1 × 150 ml was added as in 4a) written preculture as inoculum for 10 liters of Medium 3 (LB Broth Medium,  Sigma) used. Before inoculation, 1 ml of defoamer was added to the medium SAG 5693 (Union Carbide, USA) added per liter, and for 30 minutes at 1.1 bar sterilized with steam. This production culture was at 90 ° C at 90 ° C Stirring speed of 300 rpm and with an aeration rate of 0.3 vvm in incubated in a 10 l Giovanola fermenter (leaf stirrer). The connection (2) was in a final concentration of 50-250 mg / l medium in methanolic Lö solution (2.5 g dissolved in 60 ml methanol) was added at the beginning of the fermentation. to Monitoring the biotransformation process served daily samples of 50 ml were taken sterile and examined using analytical HPLC. Under These conditions were, preferably with B. simplex DSM14028, sales rates up to 60% of the connection (2) to the connection (1) is reached.

Example 5 Analytical HPLC

Analytical HPLC methods were used for detection during the biotransformation with UV / visual (HPLC-UV / Vis) and with mass spectrometric detection set.

Before HPLC analysis, the samples were dissolved in MeOH and sterile filtered.

HPLC-MS were carried out on an HP1100 HPLC system, coupled to a Micromass-LCT mass spectrometer (Micromass, Manchester, Great Britain) in ESI + mode. Mass spectra were recorded in the range between 200 and 1200. Compound (1) had a retention time of 4.08 minutes. The HP1100 system used operated with the following parameters: Stationary phase: Waters Symmetry C18, 3.5 by 2.1 × 50 mm column, mobile phase: gradient water (A), acetonitrile (B) + 0.1% formic acid (0-1 minutes 100% A ; 1-5 minutes linear gradient to 10% A / 90% B; 5-6 minutes 10% A / 90% B, 6-6.10 minutes 90% B - 100% B). Compound (1) eluted at 4.08 minutes. The detection was based on the protonated molecular ions (M + H) ⁺ .

HPLC-UV / Vis analyzes were carried out using a Hewlett Packard Series 1100 analytical HPLC system (HP, Waldbronn, Germany), consisting of a G 1312A binary pump system, a G 1315A diode array detector, a G 1316A column temperature control system, a G 1322A degasifier system and a G 1313A auto-injector. 0.01% H ₃ PO ₄ : acetonitrile (ACN) was used as the mobile phase at a flow of 1 ml / minute, while a Merck (Darmstadt, Germany) Lichrospher RP 18 column (125 × 4 mm, particle size 5 μm) was used as the stationary phase served. The samples were separated on a continuous linear gradient (0% ACN to 100% ACN in 10 minutes), followed by isocratic elution (5 minutes at 100% ACN). HPLC-UV chromatograms were recorded at 210 nm (detection of impurities) and 254 nm (optimal detection in the UV maximum of the spinosyn derivatives) with a respective reference wavelength of 550 nm with a bandwidth of 80 nm. The diode array detection in the range of 210-600 nm provided the HPLC / UV / Vis spectra. The data was saved using the HP ChemStation software. The connection (2) here had a retention time of 7.85-7-9 minutes, while the connection (1) eluted with a retention time of 8.21-8.27 minutes.

Internal and external standards of the pure substances were used for the analytical detection of the compound (2) and the compound (1) in fermentation samples, and the detection was carried out with identical retention times in the two different analytical systems, including the respective HPLC-UV / Vis - and HPLC-MS spectra. Fig. 1 shows an HPLC-UV chromatogram of the crude extract at 254 nm and HPLC / UV spectra (diode array; Rt = retention times) of compound (2) and compound (1) from a fermentation sample from S. argillaceus (GS- Medium) after 146 hours of fermentation.

Example 6a) Extraction and preparative presentation of the compound (1) from shake cultures

Ten 150 ml cultures of the strain mixed with 50 mg / l of compound (2) (S. argillaceus DSM 14030) in 1000 ml Erlenmeyer flasks were after 240 hours harvested, and the culture broths were combined and freeze-dried. The after Freeze-drying residues were obtained twice for 30 minutes each Ultrasonic bath extracted. The supernatants were filtered off, combined and in the Vacuum evaporated to dryness on a rotary evaporator. The residues were redissolved in 100 ml water: ethyl acetate (EtOAc) 1: 1. The organic ethyl Acetate phases were separated, dried over anhydrous sodium sulfate and in Rotary evaporator evaporated to dryness. The aqueous phase was still 50 ml of ethyl acetate were added twice and the organic phases were separated off and united with the first phase. From the combined organic phases of the Ex traction were obtained about 300 mg of an oily crude product, which in 2 ml MeOH dissolved and over a Baker (Deventer, Netherlands) Bond Elut C18 1 ml Solid phase extraction cartridge was filtered. The filtrate was immediately used for the preparative HPLC used.

The preparative HPLC was operated at 22 ° C. The system used by the company Gilson Abimed (Ratingen, Germany) consisted of Gilson Unipoint Software, a 306 binary pump system, a 205 fraction collector, an 119 UV-Vis Detector, an 806 manometric module and an 811C dynamic mixer. A Merck (Darmstadt, Germany) LichroSorb RP18 column (particle size 7 µm; Column dimensions 250 × 25 mm) was used as the stationary phase. As a mobile Phase served a gradient of 0.1% aqueous trifluoroacetic acid against acetonitrile (ACN) with a continuous flow of 10 ml / minutes, under the following Elution profile: linear gradient from 20% to 50% ACN in 35 minutes; after that isocratic conditions at 50% ACN for 25 minutes followed by a linear one Gradients from 50% ACN to 100% ACN in 40 minutes. Aliquots of 10 ml were  the fractionated and combined depending on UV adsorption at 210 nm. retention times (Rt) were 77-82 minutes for compound (2) and 91-97 minutes for the conn dung (1).

Example 6b) Extraction and isolation of compound (1) from stirred fermenter cultures (10th Liter scale)

The mycelia from fermentations of the B. simplex DSM 14028 strain in 10 liters The supernatant was measured by centrifugation (10 minutes at 1000 × g) separated and extracted directly with 2 × 200 ml of methanol. The methanol extracts were evaporated to dryness and provided an oily intermediate. This Product was as described below using flash chromatography on silica gel Further processed material.

The culture supernatant obtained after centrifugation was over a 6 × 18 cm Lewapol (500 ml) OC 1064 (Bayer AG, Leverkusen, Germany) adsorber resin column given. This column was washed with 1 liter of water and successively with 2 l of 70% methanol and 1 l of acetone eluted. The organic eluates were up to an aqueous residue on a rotary evaporator. The mass of the pro duktes was found in the acetone fraction, which was then flash Chromatography - as described below - was further processed.

The oily intermediates from the extractions of mycelium and supernatant were the dissolved in as little tert-butyl methyl ether (TMBE) as possible and in 400 ml Kieselgel 60 (0.040 - 0.063 mm, Merck Darmstadt, Germany) bound. The TMBE was carefully evaporated on a rotary evaporator. Then were the intermediates bound to the carrier material dry on 100 ml of pebble gel packed in a 500 ml SIM module (Biotage). It gradually became isocratic with 2 liters of cyclohexane (CH) followed by 1.6 liters of a linear gradient of cyclo hexane eluted against TBME.  

The fractions eluted with CH: TBME contained the highly enriched product in addition to residual amounts of unreacted aglycon and other ingredients. This Fractions were concentrated on a rotary evaporator and to isolate the ver Binding (1) added to the preparative HPLC.

The compound (1) was isolated using the method described in Example 6a preparative HPLC system using an MZ analysis technology Kromasil 100 C8 (7 µm, 250 × 40 mm) column as stationary phase and 0.1% Trifluoroacetic acid: acetonitrile (ACN) as the mobile phase in a continuous Flow of 10 ml / minutes in the following gradient: 30% ACN for 20 minutes, ge follows from linear gradient (30% - 50% ACN in 60 minutes), then again linear gradient from 50% ACN to 100% ACN in 42 minutes.

Aliquots of 10 ml were fractionated and subsequently added depending on the UV absorption 210 nm combined. Retention times (Rt) were 105-111 minutes for the compound (2) and 115-119 minutes for compound (1).

The processing and isolation from fermentations was carried out on a larger scale analogous to this method, with extraction, elution and column bed volumes increased accordingly (i.e. adjusted to the volumes of the production cultures) and the Intermediates may have been aliquoted prior to the final HPLC separation.  

Example 7 Structure elucidation of the connection (1)

The structural elucidation of compound (1) was carried out using the 1D and 2D core resonance spectroscopy (NMR) and positive electrospray mass spectrometry (+ ESI-MS) carried out. NMR spectra were recorded on a Bruker DMX500 Spectrometer measured at 302 K in DMSO. MS spectra were recorded on an LCT ESI-TOF device from Micromass measured.

The mass determined by (Es + I) -MS was 400 (C ₂₄ H ₃₂ O ₅ ) (corresponding to a measured molar mass of 401 (m / z + H)). The mass determined by MS high resolution (ESI +) was 401.2340 (calculated: 401.2328). The NMR data are summarized in the following table, and the proton spectrum is shown in Fig. 2.

¹ H and ¹³ C chemical shifts of compound (1) in DMSO-d ₆ measured at 302 K.

The optical rotation is determined on a Perkin-Elmer polarimeter Model 341 at 20 ° C and 589 nm (10 cm cuvette length, solvent methanol). The Optical rotation angle of compound (1): (NaD, 589 nm) in MeOH: -272.2 °.

Example 9 Characterization of the strains used a) Streptomyces argillaceus DSM 14030

This strain was produced as a mithramycin and as a type strain of the new Streptomyces Art S. argillaceus at the American Type Culture Collection at the accession number ATCC 12956 from Pfizer Inc., New York, USA (S. argillaceus ATCC 12956). The strain description can be found in US-A 3,646,194  be removed. The culture was again on February 8, 2001 under the Filing number DSM 14030 at the German Micro Collection organismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, in accordance with the provisions of the Budapest contract deposited.

b) Streptomyces griseus BS 2134

The Actinomyceten strain BS 2134 was from a in Rhineland-Palatinate (Germany) collected soil sample isolated. The culture was on 08.02.2001 under the deposit number DSM 14029 with the German Collection of microorganisms and cell cultures GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, in accordance with the provisions of the Budapest Treaty.

c) Actinomycetales sp. BA 312

The Actinomycetes strain BA 312 was from a in Rhineland-Palatinate (Germany) collected soil sample isolated. The culture was on 02/27/2001 under the deposit number DSM 14077 with the German Collection of microorganisms and cell cultures GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, in accordance with the provisions of the Budapest Treaty.

d) Actinomycetales sp. BA 579

The Actinomycetes strain BA 579 was from a in Rhineland-Palatinate (Germany) collected soil sample isolated. The culture was on 02/27/2001 under the deposit number DSM 14078 with the German Collection of microorganisms and cell cultures GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, in accordance with the provisions of the Budapest Treaty.  

e) Streptomyces sp. WS 2199

The Streptomycetes strain WS 2199 was made from an aqueous sample (Germany) isolated. The culture was deposited on February 27, 2001 number DSM 14079 at the German Collection of Microorganisms and Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, in accordance with the provisions of Budapest Ver deposited.

f) Bacillus simplex DSM 14028

Bacillus simplex DSM 14028 was introduced as Bacillus megaterium NRS 610 in the Collection of Nathan R. Smith, U.S. Department of Agriculture, Washington, D.C., USA and published by Priest et al. as Bacillus simplex rewritten. It was created by the German Collection for Microorganisms included in the collection as DSM 1318 (Priest, F. G., Goodfellow, M., Todd, C., A numerical classification of the genus Bacillus. J. Gene. Microbiol. 134: 1847-1882, 1988). The trunk was again on 08.02.2001 under the deposit number DSM 14028 with the German Collection of microorganisms and cell cultures GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, in accordance with the provisions of the Budapest Treaty.

g) Bacillus megaterium DSM 333

Bacillus megaterium DSM 333 was deposited by D. Claus number DSM 333 deposited with the German Collection for Microorganisms and by Hunger et al. (Hunger, W., Claus, D .: Taxonomic studies on Bacillus megaterium and on agarolytic Bacillus strains, pp. 217 - 239, In Berkeley, R. C. W., Goodfellow, M (eds.) The aerobic endospore-forming bacteria: classifi cation and identification. Academic Press, London 1981).  

h) Bacillus megaterium DSM 339

Bacillus megaterium DSM 339 was developed by the Institute of Microbiology at the Univer sity Göttingen under the deposit number DSM 339 with the German Collection for microorganisms deposited. The taxonomy of the tribe is from Hunger et al. (Hunger, W., Claus, D .: Taxonomic studies on Bacillus megaterium and on agarolytic Bacillus strains, pp. 217-239, In Berkeley, R.C.W., Goodfellow, M (eds.). The aerobic endospore-forming bacteria: classification and identification. Academic Press, London 1981).

i) Zygorhynchus moelleri Vuill. strain WP 0796

The strain was created by Dr. H.-G. Wetzstein (Bayer AG) 1994 isolated from a soil sample collected in Germany and by Dr. P. Hofmann (DSMZ) characterized morphologically. This taxonomy was examined by means of a microscopic control of the culture deposited at the DSMZ with the help of the relevant key in KH Domsch et al. (1980), Compendium of soil fungi Vol. 2. ICW Verlag (under Zygorrhynchus moelleri) confirmed. The currently valid generic name is, however, according to Hawksworth, DL et al. (eds.): Ainsworth &Bisby's Dictionary of the Fungi, CAB International, London, 8 ^th edition (1996) as Zygorhynchus (instead Zygorrhynchus) again woks. The culture was deposited on March 21, 2001 under the deposit number DSM 14198 at the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, in accordance with the provisions of the Budapest Treaty.

j) Mucor circinelloides Van Tiegh strain WP 0799

The strain was created by Dr. H.-G. Wetzstein (Bayer AG) 1994 from an in Germany collected soil sample isolated and by Dr. P. Hofmann (DSMZ) characterized morphologically. Using a microscopic control of the This taxonomy was deposited with the aid of the culture deposited with the DSMZ relevant determination key in K.H. Domsch et al. (1980) Compendium of soil fungi Vol. 2. ICW Verlag (under Mucor circillenoides) confirmed. The  Culture was filed on March 21, 2001 with the deposit number DSM 14199 the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, in In accordance with the provisions of the Budapest Treaty.  

Example 10 Chemical representation of compound (1) by pyridinium dichromate oxides tion

The compound (1) was prepared by pyridinium dichromate oxidation from the compound (2): 46.55 g (115.6 mmol) of the compound (2) were in inert gas in 1100 ml of abs. Dissolved dichloromethane and mixed with 43.51 g (115.6 mmol) pyridinium dichromate. After 4 hours of stirring at 25 ° C. and addition of 900 ml of diethyl ether, the chromium salts which had precipitated were filtered off and the filtrate was concentrated in vacuo. Column chromatography on silica gel (eluent: cyclohexane / ethyl acetate 1: 1, then 100% ethyl acetate) gave 3.68 g of 9,17-diketo-spinosyn-aglycone and 23.40 g of an approx. 9: 1 mixture of the compound (2) and 17-ketospinosyn -Aglycon in addition to 11.74 g of recovered compound (2). The compound (1) is enriched to <98% by recrystallization of the mixture in cyclohexane / ethyl acetate. 20.78 g of compound (1) are obtained as colorless crystals. - Compound (1): TLC: R _f (SiO ₂ , ethyl acetate) = 0.44 - ¹ H NMR: CDCl ₃ , δ = 6.77 (s, 13-H); 5.97 (d, 6-H); 5.88 (m, 5-H); 4.72 (m, 21-H); 3.69 (m, 17-H) et al. - LC / ESI-MS: m / z = 401 (25%) [M] ⁺ , 289 (100%). The compound (1) represented in this way is identical in all spectroscopic data to the compound (1) represented by bioconversion. - 17-Ketospinosyn aglycone: TLC R _f (SiO _2, ethyl acetate) = 0:40 - ¹ H-NMR: _CDCl3, δ = 6.97 (s, 13-H); 5.90 (d, 6-H); 5.79 (m, 5-H); 4.85 (m, 21-H); 4.45 (m, 9H); 4.25 (q, 16-H) inter alia - LC / ESI-MS: m / z = 401 (100%) [M + H] ⁺ , 273 (70%). - 9,17-diketospinosyn aglycone: TLC: R _f (SiO ₂ , ethyl acetate) = 0.64. - ¹ H NMR: CDCl ₃ , δ = 6.92 (s, 13-H); 5.97 (d, 6-H); 5.87 (m, 5-H); 4.85 (m, 21-H); 4.25 (q, 16-H) inter alia - LC / ESI-MS: m / z = 399 (100%) [M + H] ⁺ .

Example 10a) Chemical representation of compound (1) by Swern oxidation

The compound (1) was alternatively by Swern oxidation (variant according to Corey and Kim (1972), J. Am. Chem. Soc., 94: 7586) from compound (2): 133 mg (1 mmol) of N-chlorosuccinimide were dissolved in 10 ml of abs. dichloro suspended in methane and treated with - 70 ° C with 66 mg (1.07 mmol) of dimethyl sulfide. After 30 min. A solution of 402 mg (1 mmol) of compound (1) was stirred in 2 ml abs. Dichloromethane slowly added dropwise. After stirring for 2 hours at - 70 ° C 132 μl (0.95 mmol) of triethylamine were added dropwise and the reaction mixture warmed up to room temperature within 16 hours. After dilution with 100 ml of dichloromethane was successively saturated with 100 ml of water and 100 ml ter aqueous sodium chloride solution, dried over sodium sulfate and concentrated in vacuo. Column chromatography on silica gel (eluent: Cyclo hexane / ethyl acetate 2: 1) gave 246 mg of compound (1) in addition to 37 mg of 9.17- Diketospinosyn aglycone and 120 mg of recovered compound (2). The on Compound (1) shown in this way is in all spectroscopic data identical to that caused by pyridinium dichromate oxidation or bioconversion established connection (1).

Example 10b) Chemical representation of 5,6-dihydro-9-keto-spinosyn aglycone (compound (3))

Compound (3) was prepared analogously to Example 10a) by pyridinium dichromate oxidation from compound (4): 202 mg (0.5 mmol) of compound (4) were dissolved in 5 ml of abs. Dissolved dichloromethane and mixed with 188 mg (0.5 mmol) pyridinium dichromate. After 4 hours of stirring at 25 ° C. and addition of 5 ml of diethyl ether, the chromium salts which had precipitated were filtered off and the filtrate was concentrated in vacuo. Column chromatography on silica gel (eluent: cyclohexane / ethyl acetate 1: 1, then 100% ethyl acetate) gave 24 mg of 5,6-dihydro-9,17-diketo-spinosyn-aglycone and 103 mg of compound (3) in addition to 41 mg of recovered starting material Connection (4). - Compound (3): TLC: R _f (SiO ₂ , ethyl acetate) = 0.44 - ¹ H-NMR: CDCl ₃ , δ = 6.83 (s, 13-H); 4.68 (m, 21-H); 3.69 (m, 17-H) et al. - LC / ESI-MS: m / z = 403 (23%) [M + H] ⁺ , 291 (100%). - 5,6-dihydro-9,17-diketospinosyn aglycone: TLC: R _f (SiO ₂ , ethyl acetate) = 0.64. - ¹ H NMR: CDCl ₃ , δ = 6.99 (s, 13-H); 4.82 (m, 21-H); 4.23 (q, 16-H) inter alia - LC / ESI-MS: m / z = 423 (100%) [M + Na] ⁺ .

Example 11 Representation of the 9-keto-spinosyn A-9 pseudo-aglycon a) Synthesis of trichloroacetimidate (cf. also method by G. R. Duffin et al. (2000), J. Chem. Soc., Perkin Trans. 1: 2237-2242

200 mg (1,256 mmol) of α-D-forosamine were stirred in 10 ml of methylene chloride, 419.0 mg (2,902 mmol) of trichloroacetonitrile and 108.5 mg (0.333 mmol) of cesium carbonate were added and the mixture was stirred for about two hours at room temperature. The reaction mixture was then diluted with methylene chloride, washed with aqueous sodium hydrogen carbonate solution, the organic phase was dried over magnesium sulfate and the solvent was removed in vacuo. 303 mg (79.5%) of trichloroacetimidate are obtained, which can be used directly for the glycosidation reaction (b).
C ₁₀ H ₁₇ Cl ₃ N ₂ O ₂ (303.6)
LC / MS: m / z = 303 (100%) [M] ⁺

b) Glycosidation of compound (1) with trichloroacetimidate (see also method with G. R. Duffin et al. (2000), J. Chem. Soc., Perkin Trans. 1: 2237-2242

100 mg (0.250 mmol) of compound (1) were stirred in 2 ml of methylene chloride, 50 mg of molecular sieve 4A and 151 mg (0.500 mmol) of fresh trichloroacetimidate (a) were added. 49.4 mg (0.348 mmol) of boron trifluoride etherate were then added, and the reaction mixture was stirred at room temperature for about 18 hours. After the molecular sieve 4A had been separated off, the residue was diluted with a little methylene chloride and washed with aqueous sodium hydrogen carbonate solution. The organic phase was dried over magnesium sulfate and the solvent was removed in vacuo.
C ₃₂ H ₄₇ NO ₆ (541.7)
LC / MS: m / z = 542 (100%) [M] ⁺ (see WO 97/00265 and US-A 6 001 981)

Claims (17)

1. Compounds of formula (I)
in which
R ^{1 represents} methyl or ethyl, and
AB stands for one of the following groups: -HC = CH-, -HC = C (CH ₃ ) -, -H ₂ C-CH ₂ -, -H ₂ C-CH (CH ₃ ) -. 2. Compounds according to claim 1, characterized in that
R ^{1 represents} ethyl. 3. Compounds according to claim 1 or 2, characterized in that
AB stands for the group -HC = CH-. 4. A process for the preparation of compounds according to one of claims 1 to 3, characterized in that compounds of the general formula (II)
wherein
R ¹ and AB have the meanings given in one of claims 1 to 3,
with an oxidizing agent, optionally in the presence of a diluent. 5. The method according to claim 4, characterized in that as an oxide agent uses pyridinium dichromate. 6. The method according to claim 4 or 5, characterized in that as Diluent dichloromethane is used. 7. A process for the preparation of compounds according to one of claims 1 to 3, characterized in that compounds of the general formula (II)
wherein
R ¹ and AB have the meanings given in one of claims 1 to 3,
with a microorganism in an aqueous nutrient medium under aerobic conditions or with an enzyme extract produced therefrom or with one or more enzymes isolated therefrom. 8. The method according to claim 7, characterized in that as a micro organism a microorganism from the genus Bacillus, from the group the Actinomycetes or from the Zygomycetes class. 9. The method according to claim 8, characterized in that one Strain from the species Bacillus megaterium or Bacillus simplex. 10. The method according to claim 9, characterized in that the strain characteristic features of the strains Bacillus megaterium DSM 339, Bacillus megaterium DSM 333 or Bacillus simplex DSM 14028. 11. The method according to claim 8, characterized in that as a micro organism from the group of the Actinomycetes a microorganism of the genus Streptomyces. 12. The method according to claim 11, characterized in that one as a micro organism from the genus Streptomyces a strain from the species Streptomyces argillaceus or Streptomyces griseus. 13. The method according to claim 12, characterized in that the strain distinctive features of the strains Streptomyces argillaceus DSM 14030 or Streptomyces griseus DSM 14029.   14. The method according to claim 8, characterized in that as a micro organism from the class of the Zygomycetes a microorganism from the Genus Zygorhynchus, especially a strain from the Zygorhynchus species moelleri uses. 15. The method according to claim 8, characterized in that as a micro organism from the class of the Zygomycetes a microorganism from the Genus Mucor, in particular a strain from the Mucor circinelloides species starts. 16. The method according to any one of claims 4 to 15, characterized in that the compounds according to one of claims 1 to 3 are isolated. 17. Use of compounds according to one of claims 1 to 3 for Manufacture of spinosyn derivatives.