Preparation of a macrocyclic lactone

专利摘要:
The present invention relates to a method of forming a compound of formula III by contacting a compound of formula II with a biological catalyst that is capable of specifically oxidizing an alcohol at the 4 " Reacting a compound of formula III with an amine of formula HN (R 8 ) R 9 , wherein R 8 and R 9 are as defined for the compound of formula I, in the presence of a reducing agent (2) Including, To a compound of formula (I) and, optionally, E / Z isomers thereof, mixtures of E / Z isomers and / or tautomers thereof, in free or salt form, respectively. Formula I In Formula I above, R 1 to R 9 are independently of each other hydrogen or a substituent, m is 0, 1 or 2, n is 0, 1, 2 or 3, A, B, C, D, E and F represent a bond, and independently of each other, two adjacent carbon atoms are bonded by a double bond, a single bond, a single bond and an epoxide bridge, or a single bond and a methylene bridge Indicates. Formula II In Formula II above, R 1 to R 7 , m, n, A, B, C, D, E and F are as defined for compounds of formula (I). Formula III In Formula III above, R 1 to R 7 , m, n, A, B, C, D, E and F are as defined for compounds of formula (I).
公开号:KR20020063890A
申请号:KR1020027006098
申请日:2000-11-10
公开日:2002-08-05
发明作者:파흐라트코요하네스파울；핏테르나토마스；융만폴커
申请人:신젠타 파티서페이션즈 아게；
IPC主号:

专利说明:

Preparation method of macrocyclic lactone
[1] The present invention relates to a process for preparing macrocyclic lactones, to a process for preparing intermediate compounds, and to intermediate compounds used in the process. More particularly, the present invention
[2] Contacting a compound of formula II with a biological catalyst that is capable of specifically oxidizing an alcohol at the 4 "position to form a compound of formula III (1)
[3] Reacting a compound of formula III with an amine of formula HN (R ₈ ) R ₉ , wherein R ₈ and R ₉ are as defined for the compound of formula I, in the presence of a reducing agent (2) Including,
[4] In each case, the compounds of formula (I), their E / Z isomers or their tautomers, respectively, in free or salt form, respectively, obtainable according to the method or by using other methods, respectively, in the free form Or converting to different compounds of formula (I), E / Z isomers or tautomers thereof in salt form, separating the E / Z isomer mixture obtainable according to the process and isolating the desired isomers, and / Or converting the compound of formula (I), its E / Z isomers or its tautomers into salts in free form obtainable according to the method or using other methods or according to the method or using other methods Obtained compounds of the formula (I), their E / Z isomers or salts of the tautomers thereof are obtained in the free form of the compounds of the formula (I), their E / Z isomers, Or converting to a different salt, respectively, to prepare a compound of formula (I) and, optionally, an E / Z isomer, an E / Z isomer mixture thereof and / or a tautomer thereof, in free or salt form, respectively. It is about a method.
[5]
[6] In Formula I above,
[7] R ₁ to R ₉ are independently of each other hydrogen or any substituent,
[8] m is 0, 1 or 2,
[9] n is 0, 1, 2 or 3,
[10] A, B, C, D, E and F represent a bond and, independently of each other, two adjacent carbon atoms are double bond, single bond, single bond and chemical formula Epoxide bridges, or single bonds with It is bonded by the methylene bridge of.
[11]
[12] In Formula II above,
[13] R ₁ to R ₇ , m, n, A, B, C, D, E and F are as defined for compounds of formula (I).
[14]
[15] In Formula III above,
[16] R ₁ to R ₇ , m, n, A, B, C, D, E and F are as defined for compounds of formula (I).
[17] Methods for the synthesis of compounds of formula (I) are described in the literature. However, the process known in the literature has been found to cause significant problems during manufacturing, since it is basically low in yield and complicated in manufacturing. Therefore, the known method is not satisfactory in this respect, and an improvement on the method for preparing the compound is required.
[18] The compounds of formulas (I), (II) and (III) may be in tautomeric forms. Accordingly, it is to be understood herein that, where appropriate, compounds of formulas (I), (II) and (III) include, although the corresponding tautomers are not specifically mentioned in each case.
[19] Compounds of formulas (I), (II) and (III) may form acid addition salts. These salts include, for example, inorganic strong acids, such as mineral acids such as perchloric acid, sulfuric acid, nitric acid, nitrous acid, phosphoric acid or hydrohalic acid; Strong organic carboxylic acids such as unsubstituted or substituted, for example halo-substituted, C ₁ -C ₄ alkancarboxylic acids such as acetic acid, saturated or unsaturated dicarboxylic acids such as oxalic acid, Lonic acid, succinic acid, maleic acid, fumaric or phthalic acid, hydroxycarboxylic acids such as ascorbic acid, lactic acid, malic acid, tartaric acid or citric acid, or benzoic acid; Or organic sulfonic acids, for example unsubstituted or substituted, for example halo-substituted, C ₁ -C ₄ alkanes- or aryl-sulfonic acids, for example methane- or p-toluene-sulfonic acid To form. In addition, compounds of formulas (I), (II) and (III) having one or more acidic groups may form salts with bases. Suitable salts with bases are, for example, metal salts, for example alkali metal salts or alkaline earth metal salts, for example sodium salts, potassium salts or magnesium salts, or salts with ammonia or organic amines, for example For example, morpholine, piperidine, pyrrolidine, mono-, di- or tri-lower alkylamines, for example ethyl-, diethyl-, triethyl- or dimethyl-propyl-amine or mono-, di Or tri-hydroxy-lower alkylamines, for example mono-, di- or tri-ethanolamine. In addition, corresponding internal salts may also be formed. Preference is given to agriculturally advantageous salts within the scope of the present invention. Given the close relationship between the compounds of the formulas (I), (II) and (III) in free form and in the form of salts, where reference is made herein to the free compounds of formulas (I), (II) and (III), if necessary, It is to be understood that the corresponding salts or free compounds of the compounds of I, II and III are also included. This also applies to the tautomers of the compounds of the formulas I, II and III and their salts. In each case, the glass form is generally preferred.
[20] Within the scope of the present invention, n is 1, m is 1, A is a double bond, B is a single bond or a double bond, C is a double bond, D is a single bond, E is a double bond, F is a double bond, a single bond and an epoxy bridge, or a single bond and a methylene bridge, R ₁ , R ₂ and R ₃ are H, R ₄ is methyl, R ₅ is C ₁ -C ₁₀ -alkyl, C ₃ -C ₈ -cycloalkyl or C ₂ -C ₁₀ -alkenyl, R ₆ is H, R ₇ is OH, R ₈ and R ₉ are independently of each other H, C ₁ -C ₁₀ -alkyl or C ₁ Preferred is a process for the preparation of compounds of formula (I), which are —C ₁₀ -acyl or together form — (CH ₂ ) _q —, wherein q is 4, 5 or 6.
[21] Within the scope of the present invention, n is 1, m is 1, A, B, C, E and F are double bonds, D is a single bond, R ₁ , R ₂ and R ₃ are H, R and ₄ is methyl, R ₅ is 2-tert-a-butyl or isopropyl, R and ₆ are H, R ₇ is OH, and, R ₈ is methyl, R ₉ a method for producing a compound of formula I H is particularly preferred Do.
[22] Very particular preference is given to the process for the preparation of emamectins, more particularly preferred is the benzoate salt of emamectin. Emamectin is a 4 "-deoxy-4" -N-methylamino avermectin B _1a / B _1b mixture, described in US Pat. No. 4,874,749 and described in Journal of Organic Chemistry, Vol. 59 (1994), 7704-7708, are described as MK-244. Anemectin salts of particular agricultural interest are described in US Pat. No. 5,288,710. Compounds of formula (I) are particularly important pesticides for controlling pests and representative ticks. The pests mentioned include, for example, those described in lines 55 to 58 of page 5, lines 6 and 1 to 21 of page 7 of EP 736 252. Pests mentioned herein are incorporated by reference in the subject matter of the present invention.
[23] General terms used herein have the following meanings unless stated otherwise.
[24] Each of the carbon-containing group and the carbon-containing compound contains 1 to 8, preferably 1 to 6, in particular 1 to 4, more particularly 1 or 2 carbon atoms.
[25] Alkyl is straight chain, ie methyl, ethyl, propyl, butyl, pentyl or hexyl, or side chains, ie isopropyl, isobutyl, secondary-butyl, tert-butyl, isopentyl, neopentyl or isohexyl.
[26] Alkenyl as a group itself and alkenyl as structural elements of other groups and compounds, such as haloalkenyl and arylalkenyl, in each case, may be a straight chain, taking into account the number of carbon atoms contained in that group or compound. For example vinyl, 1-methylvinyl, allyl, 1-butenyl or 2-hexenyl, or side chains such as isopropenyl.
[27] C ₃ -C ₆ -cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, in particular cyclohexyl.
[28] A further aspect of the invention is that
[29] Compounds of formula III as defined above,
[30] A process for the preparation of a compound of formula III starting from the compound of formula II according to step (1) above or
[31] A process for the preparation of compounds of formula (I) starting from compounds of formula (III) according to step (2) above.
[32] Within the scope of the present invention, the term "biological catalyst"
[33] (a) viable microorganisms, for example in the form of feeder cells, dormant cells or lyophilized cells,
[34] (b) spores of the microorganism,
[35] (c) a killed microorganism, preferably in partially degraded form, ie, the cell wall / cell membrane is permeable mechanically, chemically or by spray drying,
[36] (d) crude extracts of the cell contents of the microorganisms, and
[37] (e) enzymes for converting the compound of formula II to the compound of formula III.
[38] Bacteria and fungi are particularly suitable microorganisms for the process according to the invention. In particular, suitable bacteria are representative bacteria of Actinomycetes, in particular Streptomyces. Streptomyces tubercidicus, Streptomyces chattanoogensis, Streptomyces lydicus, Streptomyces saraceticus and Streptomyces cassis Preferred are Streptomyces strains selected from the group consisting of Streptomyces kasugaensis. Strain Streptomyces R-922 (Streptomyces tubercidicus) and especially Streptomyces I-1529 (Streptomyces tubercidicus) are region specific of the hydroxy group at the 4 ″ position of the compound of formula II. proved to be particularly suitable for regiospecific oxidation.
[39] Streptomyces strains Streptomyces I-1529 and Streptomyces R-922, under the Treaty of Budapest, on Nov. 5, 1999, under Accession Nos. DSM-13135 and DSM-13136, des-38124, Braunschweig, Maschader Beck, Germany It has been deposited in DSMZ-Doych Jamrong von Microorgangansmen Unt Chellkulturen GmbH, 1-Be.
[40] For example, Streptomyces strain MAAG-7027 (Streptomyces tubercidicus), Streptomyces strain DSM-40241 (also identified as Streptomyces saraceticus, Streptomyces catanogensis), Streptomyces To perform a region specific oxidation according to the present invention, including Seth strain NRRL-2433 (Streptomyces lydicus subspecies Lydiacus) and Streptomyces strain A / 96-1208710 (Streptomyces kasugaensis) Additional strains have been identified that can be used as appropriate. All of the above strains are closely related to the Streptomyces strains Streptomyces I-1529 and Streptomyces R-922, respectively, which can be proved from the fact that 16s rDNA analysis showed 99.4 to 100% identity.
[41] Compounds of formula (I) are known to be highly active plant pest control agents. For example, in known processes for the preparation of compounds of formula (I) as described in European Patent No. 301 806 and in the microbiological process according to the invention, compounds of formula (II) are used as starting materials.
[42] In a known method, in the first step, the oxygen at position 5 of the compound of formula II is protected using conventional protecting group techniques as described in Green and Wuts, Protective Groups in Organic Synthesis, 1999 It is then oxidized to 4 "-ketone and then converted to an amine and the deprotected masked hydroxy group at the 5 position.
[43] The method according to the invention has the advantage that it comprises only two steps compared to the four steps of known methods. In addition, the method does not use a protecting group and is less ecologically safe because it uses less chemicals. Compounds of the general formula (III) obtained from the biological catalysis step according to the invention are known from EP 401 029.
[44] In certain embodiments, the method according to the invention can be carried out in detail as follows.
[45] In a first step, the compound of formula III is prepared. This can be done by directly contacting a compound of Formula II with a biological catalyst that is capable of specifically oxidizing the alcohol at the 4 ″ position and maintaining this contact for a time sufficient to allow the oxidation reaction to occur.
[46] More readily, the process is carried out using a microbial catalyst capable of carrying out the oxidation reaction according to the present invention as a biological catalyst. Preferably, the microorganism is cultured under controlled conditions in the presence of the compound of formula II in a suitable culture medium that promotes microbial growth, and the microorganism and its substrate for a time sufficient for oxidation to occur, preferably Incubation is continued until 25 to 99.9%, more preferably 50 to 99.9%, most preferably 80 to 99.9% of the added amount of the compound of formula II is converted to the compound of formula III.
[47] Also more preferably, the method comprises first culturing a microorganism capable of carrying out the oxidation reaction according to the invention under conditions controlled in a suitable culture medium that promotes microbial growth, and then, for example, by filtration or centrifugation. It is carried out by collecting the biomass (biomass) of the microorganism using a suitable method such as. The biomass of the microorganism is then used immediately for the reaction as a biological catalyst for converting the compound of formula II to the compound of formula III, or on its own or after freeze drying or spray drying followed by cold storage. Subsequently, the microorganism and the compound of formula (II), which are newly collected or stored as described above, are subjected to a sufficient time for oxidation to occur, preferably 25 to 99.9%, more preferably 50 to 50% of the amount of the compound of formula (II) added. Incubate together in a reaction medium that is not beneficial for microbial growth until 99.9%, most preferably 80-99.9%, are converted to the compound of formula III.
[48] The reaction product of formula (III) obtained in this way can be separated from the starting material of formula (II) without the use of significant techniques by conventional separation methods, for example fractional crystallization or chromatography. Chromatography includes, for example, column chromatography, thick layer chromatography or thin layer chromatography on a mineral carrier material such as silica gel or on an organic ion resin.
[49] Instead of feeder cell constructs, microbial spores can be used, which are specifically collected from microorganisms capable of oxidizing the alcohol at the 4 "position to form a ketone of Formula III and then sufficient to cause a corresponding oxidation reaction. Incubate with the compound of formula II for a time The culturing of spores and substrate is preferably carried out in the absence of culture medium to prevent germination of the spores.
[50] The compound of formula II is used as the substrate for oxidation reaction according to the present invention. Such compounds are known (see German Patent No. 2717040) or can be prepared from known compounds in analogy to known methods. They are suitable for controlling pests of animals and plants and are also important starting materials or intermediates in the preparation of compounds of formula (I). In addition, the compounds of formula III are not the microorganisms themselves, but the active components derived from those microorganisms that are capable of specifically oxidizing the alcohol at the 4 ″ position to form ketones of formula III [definitions (b) to (e above). Can be prepared by oxidizing the compound of formula II.
[51] Thus, a further aspect of the present invention provides the immobilization of trophic microbial cells, cell-free extracts, spores, enzymes and specifically alcohols at the 4 "position to form ketones of formula III. It relates to the use of an enzyme mixture of the microorganism that can be.
[52] Immobilization of such biological catalysts can be carried out analogously to methods known in the art. Within the scope of the present invention, adsorbing, ionic or covalent bonding, crosslinking of biological catalysts with bifunctional or multifunctional agents, and matrix encapsulation of the biological catalysts generally to water-insoluble solid carrier materials Mention may in particular be made on the basis of membrane separation or combinations of two or more of the above mentioned methods.
[53] Adsorption bonding to the water insoluble carrier (adsorbent) is achieved in particular by the force of Van der Waals. Many inorganic and organic compounds, and synthetic polymers, are suitable as adsorbents.
[54] Such immobilization methods of microorganisms are described in Bickerstaff (Ed.), 1997, Immobilisation of Enzymes and Cells, van Haecht et al. (1985) (yeast cells / glass), Black (1984) (yeast cells / tablet steel, polyester), Wigel and Diykstra (1984) (Chlostridia / Cellulose, Hemicellulose), Forberg and Hag Haggstrom (1984) (Clostridia / wood shavings) and by Erhardt and Rehm (1985) (Pseudomonas / Activated Carbon). Corresponding details on the use of enzymes immobilized by adsorption bonds are described in particular in Krakowiak et al. (1984) (glucoamylase / aluminum oxide), Cabral et al. (1984) (glucoamylase / titanium- Activated glass), by Miyawaki and Wingard (1984) (glucose oxidase / activated carbon) and Kato and Horikoshi (1984) (glucose transferase / synthetic resin) It is described. Ionic bonding is based on the electrostatic attraction between the carrier material (eg, commercial ion exchangers based on polysaccharides or synthetic resins) and the oppositely charged groups of the biological catalyst to be bound.
[55] Methods for immobilization of microorganisms based on ionic bonds include DiLuccio and Kirwan (1984) (Azotobacter spec./Cellex E (Cellose)) and Giart. (Giard) et al. (1977) (Animal Cells / DEAE-Sephadex). Immobilization of the corresponding enzymes is described by Angelino et al. (1985) (aldehyde oxidase / octylamino-Sepharose 4B), Hofstee (1973) (lactate dehydrogenase / Octylamino-sephadex), Kuhn et al. (1980) (glucose oxidase / DEAE-sepadex, DEAE-cellulose) and others as set forth in detail.
[56] Further immobilization methods are based on the use of covalent bonds to immobilize biological catalysts or immobilize biological catalysts and carrier materials. Suitable carrier materials are porous materials such as glass, silica or other insoluble inorganic materials.
[57] Within the scope of the process according to the invention, microorganisms are, for example, Messing and Oppermann (1979) (Enterobacteria / borosilicate glass; Yeast Cells / Zirconium Oxide], Romanovskaya et al. (1981) (Methane Bacteria / Silochrome) and Navarro and Durand (1977) (Yeast Cells / Porous Silica) It can be immobilized similarly to the method of.
[58] Immobilization of enzymes can be performed according to the methods described by Wetall and Mason (1973) (Papine / porous glass) and Monsan et al. (1984) (invertase / porous silica).
[59] In the process according to the process of the invention, not only the carrier materials already mentioned, but also a series of all natural or synthetic polymers, for example cellulose, dextran, starch, agarose and the like or, for example, generally reactive copolymers Polymers based on acrylic acid and methacrylic acid derivatives used in the preparation are also suitable for immobilization. Suitable reactive groups for forming a bond to the biological catalyst are reactive dinitrofluorophenyl or isothiocyanate groups, or in particular oxirane and acid anhydride groups. Additionally, chloride activation of resins having carboxy groups commercially available, for example, under the trade names Amberlite ^R XE-64 and Amberlite IRC-50 can be performed.
[60] Immobilization of microorganisms using natural or synthetic carrier materials is described by Chipley (1974) (Bacillus subtilis / agarose), Gainer et al. (1980) (Azotobacter spp./cellulose). , Jack and Zajic (1977) [Micrococcus / Carboxymethylcellulose], Jirku et al. (1980) (Yeast Cells / Hydroxyalkylmethacrylates) and Schmidz ( Shimizu) et al. (1975) (bacterial cell / ethylene maleic anhydride copolymer). Immobilization of enzymes is particularly well described by Canon et al. (1984) (lactate oxidase / cellulose), Dennis et al. (1984) (chymotrypsin / sepharose), Ibrahim et al. (1985) (epoxy hydride). Lolase / dextrin), Beddows et al. (1981) (α-galactosidase / nylon-acrylate copolymer) and Raghunath et al. (1984) (urease / methacrylate-acrylate Can be performed similarly to
[61] In the crosslinking process, the biological catalysts are bound to each other by bifunctional or multifunctional agents, for example glutaraldehyde and diisocyanate, and form general insoluble gelatin aggregates which are characteristically high molecular weight.
[62] Immobilization of these microorganisms can be performed similarly to the method of De Rosa et al. (1981) (co-crosslinking with egg albumin by bacterial cells / glutardialdehyde). Methods of immobilization of enzymes that can be used within the scope of the present invention include Barbaric et al. (1984) (crosslinking with invertase / adipic acid dihydrazide), Talsky and Gianichopulose. (Gianitsopoulos) (1984) (peptide binding between enzyme molecules in the absence of chymotrypsin / crosslinker), Workman and Day (1984) (with enzyme-containing cells of inulinase / glutardialdehyde) Crosslinking), Khan and Siddiqi (1985) (crosslinking with pepsin / glutardialdehyde), Bachmann et al. (1981) (glucose isomerase / glutardialdehyde) Cross-linking with gelatin) and Kaul et al. (1984) (co-crosslinking with egg albumin by α-galactosidase / glutaldehyde).
[63] Matrix encapsulation generally involves enclosing the biological catalyst in natural or synthetic polymers having a gelatinous structure. Particularly suitable matrix materials for encapsulation of cells, organelles and spores are natural polymers such as alginate, carrageenan, pectin, agar, agarose or gelatin, because these compounds are nontoxic and protect cells during handling. Also suitable are synthetic resins such as, for example, polyacrylamides and photocrosslinked resins. Matrix encapsulation forms can be varied within a wide range and include, for example, spherical, cylinder shaped, fibrous and sheet shaped. Immobilization of microorganisms using natural or synthetic matrix materials is described by Mazumder et al. (1985) (bacterial cells / photocrosslinked resins), Bettmann and Rehm (1984) (bacterial cells / poly Acrylamide hydrazide), Umemura et al. (1984) (bacteria cells / carrageenan), Karube et al. (1985) (bacteria protoplasm / agar-acetylcellulose), cantarella, etc. (1984) (yeast cells / hydroxyethyl methacrylate), Qureshi and Tamhane (1985) (yeast cells / alginate), Deo and Gaucher (1984) [ Hypomycetes / carrageenan, Eikmeier and Rem (1984) (Hipomycetes / alginate), Bihari et al. (1984) (Hypomycetes conidial) / Polyacrylamide], Pogel and Brodelius (1984) (plant cells / alginates, agarose) and Nakajima et al. (1985) (plants PO / agar, alginate, carrageenan) may be performed as described.
[64] Immobilization of the enzyme can be performed similarly to the method of Mori et al. (1972) (aminoacylase / polyacrylamide).
[65] Membrane separation involves the formation of specific confined regions in which the reaction proceeds. The basic modification of membrane separation is modified as follows.
[66] (a) microencapsulation,
[67] (b) liposome technology and
[68] (c) Use of biological catalysts in membrane reactors.
[69] The immobilization method described above can be combined with each other, for example, such as adsorption and crosslinking. In this case, the enzymes are first adsorbed to the carrier and then crosslinked with each other by the bifunctional agent.
[70] Specifically incubating the biological catalyst used within the scope of the present invention with the compound of formula II to oxidize the alcohol at the 4 ″ position to form a ketone of formula III is carried out using methods conventional in applied microbiology. In addition to the use of shaking cultures, mention may be made of various fermenter systems which have been established, particularly in microbiological research and industrial production for a long time.
[71] The main role of the bioreactor is to create optimal hydrodynamic conditions to reduce the apparent Michaelis constant and increase the reaction rate.
[72] Essentially this is achieved by maintaining a suitable relative shift between the biological catalyst and the surrounding medium to increase the external mass shift to a degree that is no longer substantially inhibited.
[73] Suitable reactor types for the process are, for example, stirred vessel reactors, loop reactors, needle reactors, fluidized bed reactors, membrane reactors and many specific types of reactors, for example, especially sieve-stirring. Reactors, rhomboid reactors and tube reactors. See, for example, W. Hartmeier, Immobilisierte Biokatalysatoren, 1986; W. Crueger and A. Crueger, Biotechnologie-Lehrbuch der angewandten Mikrobiologie, 1984; P. Prave et al., Handbuch der Biotechnologie, 1984]. Within the scope of the present invention, preference is given to using stirred vessel reactors.
[74] Stirred vessel reactors are the most commonly used reactor type for fermentation in the field of biotechnology. This type of reactor has a high agitation capacity and high oxygen delivery capacity to quickly and uniformly mix the substrate and the biological catalyst.
[75] The advantages of stirred vessel reactors are in their simple economic structure and well studied properties.
[76] In principle, when using a stirred vessel reactor, two kinds of operations are possible: firstly a "batch" working process, a so-called "batch" process and secondly a continuous process.
[77] In a "batch" process, once the process is terminated, the biological catalyst is separated or filtered, discarded (nutrition cells) or reused in a second batch (immobilized biological catalyst).
[78] If a continuous process is used, the new substrate for the final product of the reaction is exchanged permanently and continuously. Appropriate means (sieve, filter and recovery device) should be used to prevent the biological catalyst from leaving the reactor.
[79] Cultivation of proliferative microorganisms within the scope of the present invention is generally carried out according to conventional methods known in the art, and preferably, since the liquid nutrient medium is practical.
[80] The composition of the nutrient medium depends on the microorganism used. In general, complex media comprising readily assimilable carbon (C) and nitrogen (N) sources with unclear boundaries are preferred, for example, as is commonly used for antibiotic preparation.
[81] In general, however, vitamins and essential metal ions contained in suitable concentrations as constituents or impurities in the complex nutritional medium used are also essential. If desired, such constituents, such as in particular essential vitamins, Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , NH ₄ ⁺ , (SO ₄ ) ^2- , Cl ⁻ and (CO ₃ ) ^2- Ions and trace elements cobalt, manganese and zinc can be added in salt form. Apart from yeast extracts, yeast hydrolysates, yeast autolysates and yeast cells, particularly suitable nitrogen sources are in particular soybean meal, corn meal, oat meal, edamine (enzymatic lactate albumin), peptone, casein hydrolyzate, corn steep liquor And meat extracts.
[82] Preferred concentrations of this N-source are from 0.1 to 6 g / l. Suitable carbon sources are in particular glucose, lactose, sucrose, dextrose, maltose, starch, cellulose, cellulose, mannitol, malt extract and molasses. The preferred concentration range of the carbon source is 1.0 to 25 g / l. Particularly where the microorganisms used are representative microorganisms of the genus Streptomyces, the use of D-glucose, soluble starch or malt extract and selose is advantageous for the oxidation process described below. Thus, for example, the following culture media are well suited for representative microorganisms of the genus Streptomyces:
[83] Badge 1
[84] 1.0 g of soluble starch
[85] 0.2 g peptone
[86] Yeast Extract 0.2g
[87] Adjust to 1 L with distilled water, adjust to pH 7 with NaOH and autoclave.
[88] Badge 2
[89] D-glucose 4.0 g
[90] Malt Extract 10.0g
[91] Yeast Extract 4.0g
[92] Adjust to 1 L with distilled water, adjust to pH 7 with NaOH and autoclave.
[93] Badge 3
[94] Glycerol 10.0 g
[95] Dextrin 20.0g
[96] Soytone [Difco Manual, 9th ed.,
[97] Detroit, Difco Laboratories, 1969] 10.0 g
[98] (NH ₄ ) ₂ SO ₄ 2.0g
[99] CaCO ₃ 2.0g
[100] Adjust to 1 L with distilled water, adjust to pH 7 with NaOH and autoclave.
[101] Badge 4
[102] D-glucose 10.0 g
[103] Malt Extract 10.0g
[104] Yeast Extract 3.0g
[105] Pharmamedia [Traders Protein,
[106] Southern Cotton Oil Co.
[107] Memphis, Tennessee, USA] 10.0g
[108] Meat Extract 1.0g
[109] Adjust to 1 L with distilled water, adjust to pH 7 with NaOH and autoclave.
[110] Badge 5 (ISP-2 Agar)
[111] Yeast Extract [Oxoid Ltd.]
[112] Hashshire Basingstoke, UK] 4g
[113] 4 g of D (+)-glucose
[114] Bacterial malt extract [Difco No.0186-17-7] 10g
[115] Agar (Differco No. 0140-01) 20g
[116] The components are dissolved in 1 L of demineralized water and the pH is adjusted to 7.0. The solution is sterilized at 121 ° C. for 20 minutes and cooled to maintain at 55 ° C. for the short time required to prepare the agar plate immediately.
[117] Badge 6 (PHG Badge)
[118] 10 g of peptone (Sigma 0521)
[119] Yeast Extract (Deepco) 10g
[120] 10 g of D (+)-glucose
[121] NaCl 2g
[122] MgSO ₄ × 7H ₂ O 0.15 g
[123] NaH ₂ PO ₄ × H ₂ O 1.3 g
[124] K ₂ HPO ₄ 4.4 g
[125] The components are dissolved in 1 L of demineralized water and the pH is adjusted to 7.0.
[126] In addition, the above-mentioned medium is excellently suitable for culturing representative microorganisms of Streptomyces genus and performing oxidation reactions. Both the above general data on the medium composition and the medium described in detail herein are merely illustrative of the invention and do not limit the invention.
[127] Apart from the medium composition, the methods used to prepare the medium, for example dissolution or suspension order, sterilization of the entire nutrient solution or sterilization of each component, and prevention of contamination also play an important role and thus Should be optimized.
[128] It should also be noted that sterilization can change the pH value of the nutrient medium and cause precipitation.
[129] In addition, the remaining culture methods correspond to the methods commonly used for culturing microorganisms.
[130] On a small scale, fermentations performed within the scope of the present invention, including any pre-culture, are generally in the form of shake cultures, in which case from 0.1 to 5 L containing 0.05 to 2 L, preferably 0.1 to 2 L of nutrient medium. It is advantageous to use glass flasks, preferably with a volume of 0.5 to 5 liters. Preferably a baffle is fitted to the flask. After autoclaving and adjusting the pH to a value of 4 to 8, in particular 7.0 to 7.5 (bacteria) or 6 to 7.5 (fungi), the microbial culture is inoculated into the flask under sterile conditions. In general, the inoculating material used is a preculture prepared from conserved inoculum according to the data described below.
[131] Advantageously, the culture comprising any preculture is about 25 to about 37 with continuous shaking at about 80 to about 300 rpm (rpm), preferably about 100 to 250 rpm, especially about 120 rpm, on a rotary shaker. Incubate under aerobic conditions at a temperature of about 26 ° C., preferably about 26 ° C. to about 30 ° C., particularly preferably about 28 ° C. Under the conditions mentioned above, for streptomyces, optimum oxidative activity is reached after incubation for 1.5 to 7 days.
[132] Once the catalytic capacity of the cell is high enough to carry out the desired oxidation reaction, preferably after 40 hours, the substrate (compound of formula II) can be added to contact the microorganisms and the material to be oxidized in any way. For practical reasons, it has proved advantageous to add the substrate, ie the compound of formula II, to the microorganisms in the nutrient solution.
[133] Substrates to be oxidized are, for example, in powder form or in suitable solvents such as dimethylformamide, acetone, dimethyl sulfoxide, N-methyl-2-pyrrolidone, alcoholic solvents such as methanol , Ethanol, isopropanol or tert-butanol, ether solvents such as tetrahydrofuran or 1,4-dioxane (0.5-15% by volume, preferably 2% by volume), ester solvents such as ethyl It may be used in the form of a solution in an acetate or hydrocarbon solvent, for example octane, cyclohexane, toluene or xylene, or a mixture of a suitable solvent and a suitable surfactant. The term "surfactant" includes ionic surfactants, nonionic surfactants and zwitterionic surfactants, and should also be understood to include mixtures of surfactants.
[134] Both water soluble soaps and water soluble synthetic surfactant compounds are suitable anionic surfactants. Suitable soaps can be obtained from alkali metal salts, alkaline earth metal salts or unsubstituted or substituted higher fatty acid (C ₁₀ -C ₂₂ ) ammonium salts, for example oleic acid, stearic acid, or for example coconut oil or tallow. Which is the sodium salt or potassium salt of the natural fatty acid mixture. Further suitable surfactants are also fatty acid methyltaurine salts. More frequently, however, so-called synthetic surfactants are used, in particular fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates. Fatty sulfonates or sulfates are generally in the form of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and contain C ₁₀ -C ₂₂ -alkyl radicals containing alkyl moieties of acyl radicals, for example Sodium salt or potassium salt of lignosulfonic acid, dodecyl sulfate or a mixture of fatty alcohol sulfates obtained from natural fatty acids. These compounds also include salts of sulfided and sulfonated fatty alcohol / ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain two sulfonic acid groups and one fatty acid radical having 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of condensates of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid or naphthalenesulfonic acid and formaldehyde. Suitable anionic surfactants are also bile acid salts, for example sodium salts of cholic acid or deoxycholic acid. Also suitable are the phosphate ester salts of the p-nonylphenol adducts with the corresponding phosphates, for example 4 to 14 mol of ethylene oxide, or phospholipids.
[135] Suitable cationic surfactants are tetraalkyl ammonium salts such as cetyl trimethylammonium bromide.
[136] Suitable neutral surfactants are alkyl glycosides, for example alkyl-β-D-maltosides containing C ₆ -C ₁₂ -alkyl radicals, alkyl-β-D-glucopyranosides or alkyl-β-D- Thioglucopyranoside. Further suitable neutral surfactants are glucamides such as N, N-bis (3-D-gluconamidopropyl) -colamide, N, N-bis (3-D-gluconamidopropyl ) -Deoxycolamide or fatty acid N-methylglucamide containing C ₇ -C ₁₂ -alkyl radicals. Further suitable neutral surfactants are monodisperse and polydisperse polyoxyethylenes such as BRIJ ^R , GENAPOL ^R , LUBROL ^R , PLURONIC ^R , Tesit. (THESIT ^R ), TRITON ^R and TWEEN ^R.
[137] Suitable zwitterionic surfactants are N, N, N-trialkyl glycine, for example Nn-dodecyl-N, N-dimethylglycine. Further suitable zwitterionic ionic surfactants are ω-N, N, N-trialkylammonium alkyl sulfonates, eg, 3- (N-alkyl-N, N containing C ₈ -C ₁₆ -alkyl radicals -Dimethyl) -1-propane-sulfonate. Further suitable zwitterionic surfactants are 3-[(3-colamidopropyl) -dimethylammonio] -1-propane-sulfonate and 3-[(3-colamidopropyl) -dimethylammonio] -2-hydroxy-1-propane-sulfonate.
[138] Surfactants commonly used in the field of solubilization and formulation are described in particular in Bhairi S.M. (1997) "A guide to the Properties and Uses of Detergents in Biology and Biochemistry", Calbiochem-Novabiochem Corp. San Diego CA; "1999 International McCutcheon's Emulsifiers and Detergents" The Manufacturing Confectioner Publishing Co., Glen Rock, New Jersey, U.S.A.].
[139] Chromatographic methods commonly used in microbial studies monitor the reaction process continuously.
[140] The invention also specifically relates to the cultivation of microorganisms capable of oxidizing the alcohol at the 4 ″ position to form ketones of formula III and to the compounds in bioreactors, in particular bioreactors of the stirred vessel reactor type. In order to optimize the rate of product formation in the actual production fermenter, it is recommended, first of all, to grow the microorganisms in the preculture The number of fermenter precultures is optimal for each particular case. Advantageously, depending on the microorganism used, the following concentrations of inoculum are produced for the fermentation step: 0.1 to 3% bacteria, 5 to 10% fungi, Actinomycetales 5 To 10%.
[141] Inoculation of small fermenters (up to 20 liters in volume) is generally carried out using shake flask precultures. In this case, the entire flask contents are used to inoculate the fermentor. The starting material used for the preparation of the preculture is generally a conserved inoculum material, which may be, for example, in the form of a lyophilizate or in the form of a frozen or cold stored material. Preserved inoculum materials used within the scope of the invention are preferably substances stored at -80 ° C.
[142] Preferably the propagation of the inoculum is carried out in a liquid medium in a glass flask on a rotary shaker or on a solid nutrient substrate if spores are used. Nutritional substrates and culture parameters, such as, in particular, the conditions relating to temperature, pH and oxygen introduction, should be optimized according to the microorganism or method used. Growth time for preserved inoculum material may be from several hours to several days depending on the starting material used.
[143] Freeze Dried 3-10 Days
[144] Cryopreserved Cultures
[145] Bacteria 4-18 hours
[146] Actinomycetales 1 to 5 days
[147] Fungi 1-7 days
[148] Cold stored culture
[149] Bacteria 4 to 24 hours
[150] Actinomycetales 1-3 days
[151] Fungus 15 days
[152] When using spores as the inoculum, the spores are first grown on the solid nutrient substrate under standard conditions (sterile aeration, climatic chamber) from the preserved inoculum. When using porous nutrient substrates based on peat, bran, rice or barley, the culture is constantly shaken uniformly to obtain high density spores. In addition, the preserved inoculum can be cultured on nutrient media solidified by agar or other conventional gelling agents, in which case it is preferred to use nutrient media that initiate induction of sporulation.
[153] Germination time is 7 to 30 days depending on the microorganism used and the nutrient medium used.
[154] To inoculate a preculture fermentor or preparative fermenter, the spores are a surfactant, for example a Tween 80 [surfactant commercially available from Sigma-Aldrich Co. (Montana, USA)] solution. Suspension is used to put into the fermentor with the nutrient medium, or when using a solid nutrient medium, it is also washed and separated from the solid nutrient substrate using the above surfactant. The spore containing solution thus obtained is then inoculated into the fermentor. Preferably, both the recovery of spores and the inoculation of the fermentor are performed under sterile conditions.
[155] To prepare the compounds of formula III within the scope of the present invention, bioreactors of various dimensions can be used which accommodate a capacity of 0.001 to 450 m ³ , depending on the required amount of product.
[156] When using a stirred vessel bioreactor, the following fermentation parameters should be considered important for the optimal reaction process:
[157] 1. Temperature: Within the scope of the process according to the invention the oxidation reaction with a biological catalyst is preferably carried out in the intermediate temperature range (temperature range of 20 to 45 ° C). The optimum temperature range for growth and product formation is 20 to 32 ° C., in particular 24 to 30 ° C.
[158] 2. Aeration: The aeration rate is 0.1 to 2.5 vvm (air volume per volume of liquid per minute), preferably 0.3 to 1.75 vvm. The aeration rate, if appropriate, is adapted to the oxygen demand obtained during the fermentation process.
[159] 3. Pressure: Stirred vessel reactors are generally operated under slight overpressure of 0.2 to 1.0 bar, preferably 0.5 to 0.7 bar to reduce the risk of contamination.
[160] 4. pH value: The pH value can be changed within a certain range depending on the microorganism used. When using microorganisms of the Actinomycetes group, the initial pH value is 6-8, preferably 6.5-7.5.
[161] When fungi are used, the initial pH of the culture is preferably 4 to 8, in particular 6 to 7.5.
[162] 5. Stirring: The stirring speed depends on the type of stirrer used and the fermenter size. Within the scope of the present invention, a stirrer equipped with a disc-shaped impeller is preferred, for a stirred vessel reactor of size 0.002 m ³ , 150 It is operated at a speed of from 550 rpm, in particular from 200 to 500 rpm.
[163] Within the scope of the present invention, the fermentation time may be from 20 hours to 10 days depending on the microorganism used. The reaction by the biological catalyst is about 25 to about 99.9%, more preferably about 50 to about 99.9%, most preferably about 80 to about 99.9% of the initial addition of the substrate (compound of formula II) Stop when converted to compound.
[164] To ascertain the optimal time to terminate the oxidation reaction, the overall fermentation state of the reaction process is monitored by conventional analytical methods, in particular by chromatographic methods such as HPLC or thin layer chromatography.
[165] In the modified method of the method outlined above, the bioreactor can only be used to produce biomass, which is then collected by filtration or centrifugation. The biomass of the microorganism is then immediately used as a biological catalyst for converting the compound of formula (II) to the compound of formula (III), or stored by itself or cold storage after freeze drying or spray drying. The newly collected or stored microorganisms as described above are then further distributed to another vessel, for example a flask equipped with a baffle or a stirred vessel reactor, where a biological conversion takes place. A substrate (compound of formula II) is added to contact the microorganism and the substance to be oxidized with each other in any way. For practical reasons, it has proven advantageous to add a substrate, ie, a compound of formula (II), to a microorganism in a buffer that is not beneficial for growth. The substrate to be oxidized (compound of formula II) can be used, for example, in powder form or in solution in a suitable solvent as described above.
[166] In a preferred embodiment of the present invention, the substrate (compound of formula II) is first prepared with a suitable solvent such as dimethyl sulfoxide, Tween 40 [Sigma-Aldrich Campani, St. Louis, Montana, USA]. ] Or a mixture thereof and added to a baffle-dalen flask containing a buffer, preferably a phosphate buffer, more preferably 0.07 M phosphate buffer at pH 7.0. The solution obtained is then sterilized and then a biological catalyst (microbial biomass) is added. The reaction mixture is then incubated at room temperature and shaken at 100 to 150 rpm, preferably about 120 rpm for about 2 to 7 days, depending on the microbial strain.
[167] In a further preferred embodiment of the invention, the substrate (compound of formula II) is first commercially available in a suitable solvent, for example dimethyl sulfoxide, Tween 40 [Sigma-Aldrich Campani, St. Louis, Montana, USA]. Surfactant] or a mixture thereof, and added to a baffle-dalen flask containing culture medium which promotes the growth of microorganisms for use in carrying out the desired oxidation reaction. The solution obtained is then sterilized and then a biological catalyst (microbial biomass) is added. The reaction mixture is then incubated at room temperature and shaken at 100 to 150 rpm, preferably about 120 rpm for about 2 to 9 days, depending on the microbial strain.
[168] In a further particular embodiment of the invention, after washing in a suitable buffer and resuspended in a disruption buffer, for example, by mechanical means 2 to 15 ℃, preferably 3 to 6 ℃, most preferably Cell-free extracts were prepared using wet cells disrupted at a temperature of 4 ° C. The suspension obtained is centrifuged and the supernatant cell-free extract is collected.
[169] To the cell-free extract thus obtained is added a solution comprising a suitable fractional amount of foreign electron supply system such as perredoxin and perredoxin reductase, and a substrate. After addition of the substrate, the mixture is preferably uniformly mixed and aerated immediately. Appropriate fractions of NADPH are then added and the mixture is incubated at a temperature of 15-40 ° C., preferably 20-35 ° C., most preferably 30 ° C.
[170] Processing of the fermentation liquid medium to recover the oxidation product (compound of formula III) can be carried out by methods commonly used in the field of fermentation. W. Crueger and A. Crueger, 1984; P. Prave, 1984].
[171] First, the granular components are removed from the filtrate by removal from the reaction liquid medium using a filter, centrifuge or separator.
[172] If feeder or dead cells are used as biological catalysts and if some of the reaction product (compound of formula III) is present inside the cell, the cells must be digested and then extracted. For this purpose, various cell degradation methods based on mechanical, thermal, chemical or enzymatic methods can be used.
[173] Suitable mechanical methods for use in the process according to the invention are, for example, grinding in stirred ball mills or colloid mills, pressure and relaxation in homogenizers and cell degradation by ultrasonic action. Non-mechanical methods include cell lysis by drying, cell lysis by osmotic shock, chemical autolysis and cell lysis by enzymes.
[174] Once the granular component is removed, the reaction product is concentrated by extracting the culture and the isolated cell components using a suitable solvent. For extraction, a number of commercial auxiliaries commonly used in the field of fermentation are used, for example, mixer-settlers, countercurrent columns and extraction centrifuges.
[175] Further, for example, the reaction product can be concentrated, for example, by membrane filtration, ultrafiltration, freeze concentration and ion exchange methods.
[176] Further processing of the crude product obtained after extraction can be carried out by methods well established in microbial and chemical research and industrial use.
[177] These methods are, for example, chromatographic methods, for example, adsorption chromatography, ion exchange chromatography, molecular sieve chromatography, affinity chromatography, hydrophobic chromatography, partition chromatography, covalent chromatography, and the like. Crystallization methods.
[178] Suitable solvents for extraction, as such or as mixtures, are aromatic hydrocarbons such as toluene, xylene mixtures or substituted naphthalenes, phthalates such as butyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as hexane Isomers of heptane, octane or paraffin or cyclohexane, alcohols, glycols and ethers and esters thereof, such as methanol, ethanol, 2-propanol, 1-butanol, tert-butanol, ethylene glycol, methyl tertiary- Butyl ether, ethyl acetate, ethylene glycol monomethyl or monoethyl ether, ketones such as acetone or 2-butanone or cyclohexanone, chlorinated hydrocarbons such as dichloromethane, chloroform or carbon tetrachloride.
[179] In addition, the term "surfactant" is understood to include surfactant mixtures. Both water soluble soaps and water soluble synthetic surfactant compounds are suitable anionic surfactants. Suitable soaps can be obtained from alkali metal salts, alkaline earth metal salts or unsubstituted or substituted higher fatty acid (C ₁₀ -C ₂₂ ) ammonium salts, for example oleic acid, stearic acid, or for example coconut oil or tallow. Which is the sodium salt or potassium salt of the natural fatty acid mixture. Further suitable surfactants are also fatty acid methyltaurine salts. More frequently, however, so-called synthetic surfactants are used, in particular fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates. Fatty sulfonates or sulfates are generally in the form of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts, and contain C ₁₀ -C ₂₂ -alkyl radicals containing alkyl moieties of acyl radicals, for example Sodium salt or potassium salt of lignosulfonic acid, dodecyl sulfate or a mixture of fatty alcohol sulfates obtained from natural fatty acids. These compounds also include salts of sulfided and sulfonated fatty alcohol / ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain two sulfonic acid groups and one fatty acid radical having 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of the dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid or a condensate of naphthalenesulfonic acid and formaldehyde. Also suitable are the phosphate ester salts of the p-nonylphenol adducts with the corresponding phosphates, for example 4 to 14 mol of ethylene oxide, or phospholipids. Surfactants commonly used in the field of formulations are described in particular in literature ("1986 International McCutcheon's Emulsifiers and Detergents", The Manufacturing Confectioner Publishing Co., Glen Rock, New Jersey, USA).
[180] A preferred embodiment of the present invention provides a 4 "-oxo-avermectin produced after contacting a biological catalyst, for example, abermectin with a microorganism capable of converting avermectin to 4" -oxo-avermectin. To a 4 "-oxo-avermectin by separating from the reaction mixture.
[181] Aspects of the invention
[182] Cells are produced by inoculating a pre-culture of microorganisms capable of converting a compound of formula (II) into a compound of formula (III), preferably avermectin to 4 "-oxo-avermectin, in a nutrient medium that promotes cell growth. Step (1),
[183] Collecting cells after growth (2),
[184] Dissolving the compound of formula II, preferably avermectin in a suitable solvent (3),
[185] Adding the solution obtained from step (3) to a reaction medium that does not promote cell proliferation (4),
[186] (5) adding the cells of step (2) to the reaction medium of step (4),
[187] Shaking (6) or stirring the reaction mixture of step (5) in the presence of air,
[188] Separating the cells from the medium (7),
[189] Extracting the supernatant and the cells using a suitable solvent (8),
[190] Concentrating the organic solvent phase from step (9) (9) and
[191] The compound of formula III, preferably comprising purification (10) of the compound of formula III contained in the extract from step 9, preferably 4 " -oxo-avermectin, by chromatography or crystallization. Relates to a process for preparing 4 ″ -oxo-avermectin.
[192] Further preferred embodiments of the present invention
[193] Cells are produced by inoculating a pre-culture of microorganisms capable of converting a compound of formula (II) into a compound of formula (III), preferably avermectin to 4 "-oxo-avermectin, in a nutrient medium promoting cell growth Step (1),
[194] Collecting cells after growth (2),
[195] Dissolving the compound of formula II, preferably avermectin in a suitable solvent (3),
[196] Adding the solution obtained from step (3) to a reaction medium that does not promote cell proliferation (4),
[197] (5) adding the cells of step (2) to the reaction medium of step (4),
[198] Shaking (6) or stirring the reaction mixture of step (5) in the presence of air,
[199] Extracting the whole gravy using a suitable solvent (7),
[200] Separating the phases (8),
[201] Concentrating the solvent phase from step (8) (9) and
[202] The compound of formula III, preferably comprising purification (10) of the compound of formula III contained in the extract from step 9, preferably 4 " -oxo-avermectin, by chromatography or crystallization. Relates to a process for preparing 4 ″ -oxo-avermectin.
[203] Further preferred embodiments of the present invention
[204] Dissolving a compound of formula II, preferably avermectin in a suitable solvent (1),
[205] (2) adding the solution obtained from step (1) to a nutrient medium that promotes cell proliferation,
[206] Inoculating the nutrient medium of step (2) with a preculture of microorganisms capable of converting a compound of formula II to a compound of formula III, preferably avermectin to 4 "-oxo-avermectin (3) ),
[207] (4) culturing a microorganism capable of converting a compound of formula II to a compound of formula III, preferably capable of converting avermectin to 4 "-oxo-avermectin,
[208] Separating the cells from the medium (5),
[209] Extracting the supernatant and cells using a suitable solvent (6),
[210] Vacuum concentrating the organic solvent phase from step (7) and
[211] The compound of formula III, preferably comprising purification (8) of the compound of formula III contained in the extract from step (7), preferably 4 " -oxo-avermectin, by chromatography or crystallization. Relates to a process for preparing 4 ″ -oxo-avermectin.
[212] Further preferred embodiments of the present invention
[213] Dissolving a compound of formula II, preferably avermectin in a suitable solvent (1),
[214] (2) adding the solution obtained from step (1) to a nutrient medium for promoting cell growth,
[215] (3) inoculating a nutrient medium of step (2) with a preculture of microorganisms capable of converting avermectin to 4 "-oxo-avermectin,
[216] (4) culturing a microorganism capable of converting a compound of formula II to a compound of formula III, preferably capable of converting avermectin to 4 "-oxo-avermectin,
[217] Extracting the whole gravy using a suitable solvent (5),
[218] Separating the phases (6),
[219] Vacuum concentrating the solvent phase from step (7) and
[220] The compound of formula III, preferably comprising purification (8) of the compound of formula III contained in the extract from step (7), preferably 4 " -oxo-avermectin, by chromatography or crystallization. Relates to a process for preparing 4 ″ -oxo-avermectin.
[221] In another pure chemical step, the compound of formula (III) thus obtained is prepared in the presence of a reducing agent, as known, in the presence of an amine of the formula HN (R ₈ ) R ₉ , wherein R ₈ and R ₉ As defined above).
[222] The reaction component can be reacted in the absence of a solvent, but preferably in the presence of a solvent. In addition, especially in liquid amines, the reaction can be carried out using an excess of one of the reactants. However, it is generally advantageous to add inert liquid solvents or diluents. Examples of such solvents or diluents are aromatic, aliphatic and cycloaliphatic and halogenated hydrocarbons such as benzene, toluene, xylene, mesitylene, tetralin, chlorobenzene, dichlorobenzene, bromobenzene, petroleum ether, hexane, Cyclohexane, dichloromethane, trichlormethane, tetrachlormethane, dichlorethane, trichlorethane or tetrachlorethane; Esters such as acetic acid ethyl ester; Ethers such as diethyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, tert-butylmethyl ether, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol dimethyl ether, dimethoxydi Ethyl ether, tetrahydrofuran or dioxane; Ketones such as acetone, methylethylkenone or methyl isobutyl ketone; Alcohols such as methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol or glycerin; Amides such as N, N-dimethylformamide, N, N-diethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone or hexamethyl phosphate triamide; Nitriles such as acetonitrile or propionitrile; Sulfoxides such as dimethyl sulfoxide; Organic acids such as acetic acid and water.
[223] Preferred solvents are ethers such as tetrahydrofuran and ethylene glycol dimethyl ether, in particular tetrahydrofuran; Alcohols such as methanol, ethanol or isopropanol; Halogenated solvents such as dichloromethane or dichloroethane; Aromatic solvents such as benzene or toluene; Nitriles such as acetonitrile; Amides such as N, N-dimethylformamide; Carbonic acid such as acetic acid; Water and mixtures thereof.
[224] Very particularly preferred solvents are methanol, ethanol or mixtures thereof.
[225] The reaction is preferably carried out at a pH between 0 and 14, in particular between pH 2 and 10, in most cases between pH 6 and 9, very particularly at pH 9.
[226] Preferably the reaction is carried out in a temperature range of -80 to 140 ° C, preferably -30 to 100 ° C, in most cases -10 to 80 ° C, in particular 0 to 50 ° C.
[227] Preferred reducing agents are hydrides such as borides; Borane; Formic acid; Formate or hydrogen. Especially preferred are hydrides such as sodium borohydride, zinc borohydride, lithium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride or tetramethyl-ammonium triacetoxyborohydride. Sodium borohydride is particularly preferred.
[228] The reaction can be carried out-if applicable-in the presence of certain further chemicals, for example homogenizing or non-homogenizing catalysts or acids. Acids such as hydrochloric acid, p-toluenesulfonic acid, acetic acid, propionic acid, tartaric acid or phthalic acid; Lewis acids such as titanium tetrachloride, titanium tetraisopropylate or zinc chloride; Salts such as magnesium perchlorate, sodium acetate, sodium potassium tartrate, ytterbium chloride or pyridinium-p-toluenesulfonate; Absorbents such as sodium sulfate, molecular sieve or silica gel; Or mixtures thereof are particularly suitable. Preferred further agents are acids, for example acetic acid, propionic acid or tartaric acid, with acetic acid being preferred. When carrying out the reaction with hydrogen, it is advantageous to add one or several suitable homogeneous or heterogeneous catalysts. Preferred such catalysts are heterogeneous metal catalysts known in the art, preferably Ni-, Pt- or Pd-catalysts, in particular Raney-nickel and Lindlar catalysts (Pd-CaCO ₃ -PbO). Suitable homogeneous catalysts are in particular rhodium complexes such as the Wilkinsons catalyst (chloro-tris-triphenyl-rhodium).
[229] The compounds of formula I, respectively in free or salt form, may be in the form of one of the possible isomers or in the form of a mixture thereof, for example depending on the number of asymmetric carbon atoms in the molecule and its absolute and relative structure and / or in the molecule Depending on the structure of the non-aromatic double bonds, they are in the form of pure isomers, for example optically symmetric and / or diastereomeric forms or in the form of isomer mixtures, for example enantiomer mixtures, for example racemates. , Diastereomeric mixtures or racemic mixtures. The present invention relates to pure isomers and to all possible isomeric mixtures and should be understood as above, although in each case stereochemical details are not mentioned in detail.
[230] The diastereomeric mixtures and racemic mixtures or salts thereof of the compounds of formula (I), which can be obtained according to this method or by other means, depending on the starting materials and methods selected, are based on the physicochemical differences between the components. Can be separated into pure diastereomers or racemates by known methods such as fractional crystallization, distillation and / or chromatography.
[231] The enantiomer mixtures, such as racemates, thus obtained are subjected to known methods such as recrystallization from optically active solvents, chromatography on chiral adsorbents, for example high pressure liquids on acetyl cellulose. Chromatography (HPLC), with the aid of a suitable microorganism, for example, using chiral crown ethers to form encapsulated compounds and cleavage by specific immobilizing enzymes, in which only one enantiomer forms a complex Or, for example, reacting the racemate, which is a basic end product, with an optically active acid, such as a carboxylic acid, such as camphoric acid, tartaric acid or malic acid, or sulfonic acid, such as camphorsulfonic acid. To convert to diastereomeric salts and, for example, by fractional crystallization based on their different solubility, Dividing the body mixture to diastereomers and Yen desired therefrom suitable anti Omer agents, for example, the optical body may be divided symmetrically by the method to be removed by the action of a basic agent.
[232] Apart from the separation of the corresponding isomeric mixtures, according to the invention, for example, by using generally known diastereoselective or enantioselective synthesis methods, for example, having a correspondingly suitable stereochemistry By carrying out the process according to the invention using starting materials, pure diastereomers or enantiomers can be obtained.
[233] In addition, the compounds of the formulas (I) and (III), acid addition products and salts thereof may comprise solvents which can be obtained in the form of their hydrates and / or used in the crystallization of other solvents, for example the compounds produced in solid form.
[234] The present invention provides that, in any step of the process, a compound obtainable as starting material or intermediate is used as starting material and all or part of the remaining steps are carried out, or the starting material is a derivative or salt and / or racemate thereof Or an optical symmetric form or in particular formed under reaction conditions.
[235] Compounds of formulas (I) and (III) obtainable by this method or by other means can be converted to different compounds of formulas (I) and (III) by methods known per se.
[236] In the process of the invention, preferably, the respective starting materials and intermediates in each case, in free or salt form, are used, which form compounds of the formulas (I) and (III) or salts thereof which are described at the outset as particularly important.
[237] When R ₉ is hydrogen, reaction step (2) can be divided into two separate steps, wherein in the first step the compound of formula III is a compound of formula H ₂ N (R ₈ ), wherein R ₈ is To form a compound of formula IV, and in a second step the compound of formula IV is reduced according to the method of step (2) above. The two separate steps can be carried out by one pot synthesis without separating the compound of formula IV. However, it may be advantageous, for example, to separate the compound of formula IV for purification purposes. Compounds of formula IV are novel compounds and one embodiment of the present invention.
[238]
[239] In Formula IV above,
[240] R ₁ to R ₈ , m, n, A, B, C, D, E and F are as defined for the compound of formula (I).
[241] The invention is further described with reference to the following detailed examples. These examples are provided solely for the purpose of illustrating the invention and do not limit the invention unless otherwise noted. The present invention particularly relates to the production method described in the Examples.
[242] Example 1: Cell Production
[243] 1.1 Streptomyces tubercidicus strain I-1529
[244] Precultures of strain I-1529 (Streptomyces tubercidicus; DSM-13135) were stored in 20 500 mL doses of Blendallin Erlenmeyer flasks containing 100 mL of medium 2, respectively. Grow at 120 ° C. for 3 days, rotating at 120 rpm.
[245] These cultures are inoculated in a 50 L fermentor containing 40 L of medium 4. Cells are grown at 28 ° C. with aeration at a rate of 0.7 vvm (= 30 L / min). Stirrer speed pO ₂ - to and sensed by the detector 200 to maintain the 300rpm prevents the pO ₂ is reduced to less than 25%. After growing for 2 days, cells are collected by centrifugation using a flow-through centrifuge. 4.2 kg (wet) of cells are obtained.
[246] 1.2 Streptomyces tubercidicus strain R-922
[247] Streptomyces tubercidicus strain R-922 (DSM-13136) is grown on ISP-2 agar in Petri dishes. The cultures are used to inoculate four 500 ml volumes of baffle shake shake flasks each containing 100 ml of PHG medium (Medium 6). The preculture was grown on an orbital shaker at 120 rpm for 96 hours at 28 ° C. and then used to inoculate a 10 L capacity fermenter equipped with a mechanical stirrer containing 8 L PHG medium. The main culture is grown at 28 ° C. with stirring at 500 rpm under aeration rate of 1.75 vvm (= 14 L / min) and pressure 0.7 bar. At the end of the exponential growth after about 20 minutes, cells are collected by centrifugation. The yield of wet cells is 70-80 g / liter. For further processing, the wet cells can be stored at 4 ° C., preferably for up to one week.
[248] Example 2: Reaction Process
[249] 2.1 Dormant culture
[250] 2.1.1 reaction conditions
[251] 35.5 g (average) of avermectin are dissolved in 1.05 L of dimethyl sulfoxide / Tween 40 (1: 1). The solution is dispensed by adding 25 ml of fractions to 42 3 l bafflelin Erlenmeyer flasks, each containing 1 l of reaction medium. The solution is sterilized at 121 ° C. for 20 minutes. After cooling to room temperature, 100 g of wet cells (fresh or stored at 4 ° C. for up to 4 days), as prepared in Examples 1.2 and 1.2, are added, respectively. The reaction mixture is then shaken at 120 rpm for 4-5 days at room temperature.
[252] Reaction medium:
[253] 0.5g molasses
[254] 0.5 g MgCl ₂
[255] ZnCl ₂ 12.5mg
[256] MnCl ₂ 4H ₂ O 12.5mg
[257] CoCl ₂ 6H ₂ O 25mg
[258] NiCl ₂ 6H ₂ O 12.5mg
[259] CuCl ₂ 2H ₂ O 2.5mg
[260] NaMoO ₄ 2H ₂ O 6.3 mg
[261] 0.15 ml of 1M HCl
[262] Adjust to 1 L with 70 mM phosphate buffer, pH 6.0, then autoclave.
[263] 2.1.2 Post Processing
[264] The reaction product is centrifuged in a 500 ml polypropylene centrifuge flask at 13000 x g for 15 minutes at 4 ° C. The supernatant from 40 liters of the reaction mixture is pooled and extracted twice with methyl tert-butyl ether (0.5 vol equivalent, 0.4 vol equivalent). The pooled methyl tert-butyl ether phase is then reextracted three times with 0.185 volume equivalents of distilled water. The methyl tert-butyl ether phase is concentrated in vacuo on a rotary evaporator. Drying of the residue gives 10-12 g of extract S. Discard the aqueous phase. Centrifuged cells from 120 to 132 centrifuge flasks are extracted as follows: Cells obtained from 24 centrifuge flasks are transferred to a 2 L Erlenmeyer flask. To each Erlenmeyer flask is added 80 g of diatomaceous earth (Hyflo Supercell, purified) and 1.2 L of acetone. After manual mixing, the mixture is homogenized using a large magnetic stir bar. The pulp obtained is vacuum filtered through paper on a Buchner funnel having a diameter of 20 cm and washed with acetone until colorless eluting. This affords filtrate C1 and filter cake C1.
[265] Filtrate C1 is concentrated in vacuo on a rotary evaporator to remove acetone. The aqueous phase obtained is then extracted three times with 0.7 liters of toluene. The combined toluene phases are dried over anhydrous sodium sulfate. Filtration and vacuum evaporation in a rotary evaporator yields extract C1.
[266] The filter kit C1 is transferred to a 2 L Erlenmeyer flask and manually mixed with 1.5 L toluene. Homogenize the mixture using a large magnetic stir bar. The pulp obtained is vacuum filtered through paper on a Buchner funnel having a diameter of 20 cm and washed with toluene until colorless eluting. This affords filtrate C2 and filter cake C2. Filter cake C2 is discarded.
[267] Filtrate C2 is concentrated in vacuo on a rotary evaporator to yield extract C2 which is dried under high vacuum.
[268] The combined extracts C1 and C2 obtained from 40 liters of the reaction mixture are dried under high vacuum to afford 30-35 g of extract C. 45 g of the combined extracts S and C, N ₂ pressure 0.5 on a column packed with 1.5 kg of silica gel (Merck 60, 0.040 to 0.063 mm), similar to that described by Clark-Still et al. Flash chromatography on bar eluting with ethyl acetate: hexanes (3: 2) and monitoring by thin layer chromatography. The yield of purified 4 "-oxo-avermectin is 5.6 g.
[269] 2.2 Growth Cultures
[270] 2.2.1 Reaction Conditions
[271] 1 g of avermectin (industrial) is dissolved in 50 ml of dimethyl sulfoxide / twin 40 (1: 1). A 2.5 mL fraction is dispensed by adding 20 500 mL baffle Erlenmeyer flasks, each containing 100 mL of medium 4. The solution is sterilized at 121 ° C. for 20 minutes. After cooling to room temperature, 5 ml of preculture as prepared in Examples 1.1 and 1.2 are added, respectively. The inoculated culture is then incubated at 28 ° C. for 7 days with rotary shaking at 120 rpm.
[272] 2.2.2 Post-processing
[273] The reaction mixture is centrifuged in a 500 ml capacity polypropylene centrifuge flask at 13000 xg for 15 minutes at 4 ° C. and similarly processed as described in Example 3. 252 mg of purified 4 "-oxo-avermectin is obtained.
[274] 2.3. Action of Cell-Free Biological Catalysts
[275] 2.3.1 Preparation of Cell-Free Extracts
[276] Stock:
[277] PP-Buffer: 50 mM K ₂ HPO ₄ / KH ₂ PO ₄ (pH 7.0)
[278] Disintegration Buffer: 50 mM K ₂ HPO ₄ / KH ₂ PO ₄ (pH 7.0)
[279] 5 mM Benzamidine
[280] 2 mM dithiothreitol
[281] 0.5 mM Pefabloc
[282] [Manufacture of Roche Diagnostics]
[283] Substrate: 10 mg of avermectin is dissolved in 1 ml of isopropanol.
[284] 6 g of wet cells washed in PP-buffer are resuspended in 35 ml of decay buffer and disintegrated at 4 ° C. in a French press. The suspension obtained is centrifuged at 35000 x g for 1 hour. The supernatant, cell-free extract, is collected.
[285] 2.3.2 Progress of Enzyme Activity Assay
[286] Stock:
[287] Ferredoxin: 5 mg solution of 1-3 mg / ml of ferredoxin (from spinach) in Tris / HCl-buffer (manufactured by Fluka),
[288] 5 mg solution of 1-3 mg / ml of perredoxin (obtained from Clostridium pasteurinum) in Tris / HCl-buffer (manufactured by Fluka) or
[289] 5 mg solution of 1-3 mg / ml of perredoxin (obtained from porphyra umbilicalis) in Tris / HCl-buffer (manufactured by Fluka)
[290] Ferredoxin reductase: 1 mg of a solution of 3.9 U / mg freeze-dried ferredoxin reductase (from spinach) in 1 ml of H ₂ O (manufactured by Sigma)
[291] NADPH: 100 mM NADPH in H ₂ O (Roche Diagonistics)
[292] (All stocks are stored at -20 ℃ and kept on ice when in use)
[293] HPLC conditions:
[294] HPLC instrument: Merck-Hitachi
[295] HPLC-column: 70 × 4 mm, Kromasil 100 C18,
[296] 3.5μ [Macherey-Nagel (Switzerland) manufacture]
[297] Solvent A: Acetonitrile containing 0.0075% of trifluoroacetic acid
[298] Solvent B: water containing 0.01% trifluoroacetic acid
[299] Flow rate: 1.5 ml / min
[300] Detection: UV 243nm
[301] Sample: 30 μl
[302] Retention time: Avermectin B1a 3.18 minutes
[303] 4 "-Oxo-Avermectin B1a 4.74 min
[304] Pump table: 0.0 min 75% A 25% B
[305] Straight gradient up to 7.0 minutes 100% A 0% B
[306] 9.0 minutes 100% A 0% B
[307] Gradient gradient up to 9.1 minutes 75% A 25% B
[308] 12.0 min 75% A 25% B
[309] To 475 μl of cell-free extract, the following solution is added: 10 μl of ferredoxin, 10 μl of ferredoxin reductase and 1 μl of substrate. After addition of the substrate, the mixture is immediately uniformly mixed and aerated. Then 5 μl of NADPH is added and the mixture is incubated at 30 ° C. for 30 minutes. 1 ml of methyl tert-butyl ether is then added to the reaction mixture and mixed uniformly. The mixture is centrifuged at 14000 rpm for 2 minutes and the methyl-tert-butyl ether phase is transferred to a 10 ml flask, followed by vacuum evaporation using a rotary evaporator. The residue is dissolved in 200 μl of acetonitrile and transferred to an HPLC sample vial. When 30 μl of sample was injected, a peak appeared at 4.74 minutes, indicating that 4 ″ -oxo-avermectin Bla was present. HPLC-mass spectrometry indicated that the peak was assigned a mass of 870 Da, which is 4 " Corresponds to the molecular weight of oxo-avermectin.
[310] When analyzing product formation by HPLC and HPLC-mass spectrometry, another peak appears at 2.01 minutes, corresponding to ketohydrate 4 "-hydroxy-avermectin. This cell-free extract is avermectin. Is converted to 4 "-hydroxy-avermectin from which 4" -oxo-avermectin is formed by dehydration.
[311] Spinach ferredoxin can be replaced with, for example, a bacterium Clostridium pasteurinum or a red alga porperura umbilicalis, in which case avermectin is Is converted to 4 "-oxo-avermectin.
[312] Example 3: Streptomyces Strain
[313] The relationship to the Streptomyces strain that can be used in the method according to the invention and its Streptomyces tubercidicus strains I-1529 and R-922 based on 16s rDNA analysis is shown in the table below.
[314] Strain numberNearest GenBank Target16s rDNA identity (%) I-1529 Streptomyces tubercidicus DSM 40261 strain100 R-922Streptomyces tubercidicus DSM 40261 strain100 MAAG-7027Streptomyces tubercidicus DSM 40261 strain100 DSM-40241Streptomyces Saraceticus = described as ATCC 25496 Streptomyces catanogensis Streptomyces Lydicus ATCC 25470 = NRRL 243310099.8 A / 96-1208710 Streptomyces kasugaiensis strain DSM 40819 strain Streptomyces kasugaiensis M338-M199.599.4 NRRL-2433(Strain type Streptomyces lydicus subspecies lydicus = described as ATCC 25470 = CBS 703.69 = DSM 40461)
[315] Example 4 Preparation of 4 "-Deoxy-4" -Epi- (methylamino) -Avermectin B1 of Formula
[316]
[317] Where R is methyl or ethyl
[318] 3 ml of acetic acid in 30 ml of methanol are cooled to 0-5 ° C. Gas phase methylamine is added to bring the pH of the solution to 9.
[319] To 8.25 mL of this methylamine solution is added a solution of 1.0 g of 4 "-oxo-avermectin B1 in 6 mL of methanol at 0 ° C. The mixture is allowed to warm to ambient temperature and then stirred for an additional 50 minutes at room temperature. Subsequently, 90 mg of sodium borohydride in 2.5 ml of ethanol is added and the resulting mixture is stirred for an additional 45 minutes, 10 ml of ethyl acetate is added to the reaction mixture, the organic phase is extracted three times with saturated aqueous sodium hydrogen carbonate solution, and then the organic phase. Is separated and dried over sodium sulfate. The solvent is distilled off to obtain 4 "-deoxy-4" -epi- (methylamino) -avermectin B1. The purity is at least 90%.
[320] references
[321]
[322]
[323]
[324]
[325] Cited Patent Documents:
[326] U.S. Patent 5,288,710
[327] European Patent Publication No. 736 252
[328] European Patent No. 301,806
[329] European Patent No. 401,029
[330] German Patent No. 2,717,040

权利要求:
Claims (14)
[1" claim-type="Currently amended] Contacting a compound of formula II with a biological catalyst that is capable of specifically oxidizing an alcohol at the 4 "position to form a compound of formula III (1)
Reacting a compound of formula III with an amine of formula HN (R ₈ ) R ₉ , wherein R ₈ and R ₉ are as defined for the compound of formula I, in the presence of a reducing agent (2) Including,
In each case, the compounds of formula (I), their E / Z isomers or their tautomers, respectively, in free or salt form, respectively, obtainable according to the method or by using other methods, respectively, in the free form Or converting to different compounds of formula (I), E / Z isomers or tautomers thereof in salt form, separating the E / Z isomer mixture obtainable according to the process and isolating the desired isomers, and / Or converting the compound of formula (I), its E / Z isomers or its tautomers into salts in free form obtainable according to the method or using other methods or according to the method or using other methods Obtained compounds of the formula (I), their E / Z isomers or salts of the tautomers thereof are prepared in the free form of the compounds of the formula (I), their E / Z isomers, Or converting to a different salt, respectively, to prepare a compound of formula (I) and, optionally, an E / Z isomer, an E / Z isomer mixture thereof and / or a tautomer thereof, in free or salt form, respectively. Way.
Formula I

In Formula I above,
R ₁ to R ₉ are independently of each other hydrogen or a substituent,
m is 0, 1 or 2,
n is 0, 1, 2 or 3,
A, B, C, D, E and F represent a bond and, independently of each other, two adjacent carbon atoms are double bond, single bond, single bond and chemical formula Epoxide bridges, or single bonds with It is bonded by the methylene bridge of.
Formula II

In Formula II above,
R ₁ to R ₇ , m, n, A, B, C, D, E and F are as defined for compounds of formula (I).
Formula III

In Formula III above,
R ₁ to R ₇ , m, n, A, B, C, D, E and F are as defined for compounds of formula (I).
[2" claim-type="Currently amended] Contacting a compound of formula (II) with a biological catalyst that is capable of specifically oxidizing the alcohol at the 4 "position, and maintaining this contact for a time sufficient to allow oxidation to occur, followed by separation and purification of the compound of formula (II) A process for preparing the compound of formula III, including (1).
Formula II

In Formula II above,
R ₁ to R ₇ , m, n, A, B, C, D, E and F are as defined for compounds of formula (I) in claim 1.
Formula III

In Formula III above,
R ₁ to R ₇ , m, n, A, B, C, D, E and F are as defined for compounds of formula (I) in claim 1.
[3" claim-type="Currently amended] The compound of claim 1, wherein n is 1, m is 1, A is a double bond, B is a single bond or a double bond, C is a double bond, D is a single bond, E is a double bond, and F Is a double bond, a single bond and an epoxy bridge, or a single bond and a methylene bridge, R ₁ , R ₂ and R ₃ are H, R ₄ is methyl, and R ₅ is C ₁ -C ₁₀ -alkyl, C _3- C ₈ -cycloalkyl or C ₂ -C ₁₀ -alkenyl, R ₆ is H, R ₇ is OH, R ₈ and R ₉ are independently of each other H, C ₁ -C ₁₀ -alkyl or C _1- C ₁₀ -acyl or together form-(CH ₂ ) _q -wherein q is 4, 5 or 6.
[4" claim-type="Currently amended] The compound of claim 1, wherein n is 1, m is 1, A, B, C, E and F are double bonds, D is a single bond, R ₁ , R ₂ and R ₃ are H, and R ₄ Is methyl, R ₅ is secondary-butyl or isopropyl, R ₆ is H, R ₇ is OH, R ₈ is methyl and R ₉ is H.
[5" claim-type="Currently amended] The method of claim 1, wherein the 4 ″ -deoxy-4 ″ -N-methylamino avermectin B _1a / B _1b benzoate salt is prepared.
[6" claim-type="Currently amended] The method of claim 1 or 2, wherein the biological catalyst is a microorganism.
[7" claim-type="Currently amended] The method of claim 1 or 2, wherein the biological catalyst is
(a) viable microorganisms in the form of feeder cells, dormant cells or lyophilized cells,
(b) a killed microorganism, preferably in partially degraded form, ie, the cell wall / cell membrane permeable mechanically, chemically or by spray drying,
(c) crude extract of the cell contents of the microorganism,
(d) an enzyme that converts a compound of Formula II to a compound of Formula III and
(e) a method selected from the group consisting of microbial spores.
[8" claim-type="Currently amended] The method of claim 3 or 4, wherein the microorganism is a representative microorganism of the genus Streptomyces.
[9" claim-type="Currently amended] The microorganism of claim 8, wherein the microorganism is Streptomyces tubercidicus, Streptomyces chattanoogensis, Streptomyces lydicus, Streptomyces saraceticus Streptomyces saraceticus and Streptomyces kasugaensis.
[10" claim-type="Currently amended] The method of claim 9, wherein the microorganism is strain Streptomyces R-922 deposited with accession number DSM-13136.
[11" claim-type="Currently amended] The method of claim 9, wherein the microorganism is strain Streptomyces I-1529 deposited with accession number DSM-13135.
[12" claim-type="Currently amended] Inoculating a preculture of microorganisms capable of converting a compound of formula II to a compound of formula III into a nutrient medium that promotes cell growth to produce cells (1),
Collecting cells after growth (2),
Dissolving the compound of formula II in a suitable solvent (3),
Adding the solution obtained from step (3) to a reaction medium that does not promote cell proliferation (4),
(5) adding the cells of step (2) to the reaction medium of step (4),
Shaking (6) or stirring the reaction mixture of step (5) in the presence of air,
Separating the cells from the medium (7),
Extracting the supernatant and the cells using a suitable solvent (8),
Concentrating the organic solvent phase from step (9) (9) and
A method of preparing a compound of formula III, comprising the step (10) of purifying the compound of formula III contained in the extract from step (9) by chromatography or crystallization.
[13" claim-type="Currently amended] Dissolving the compound of formula II in a suitable solvent (1),
(2) adding the solution obtained from step (1) to a nutrient medium that promotes cell proliferation,
(3) inoculating a nutrient medium of step (2) with a preculture of microorganisms capable of converting a compound of formula II to a compound of formula III,
(4) culturing a microorganism capable of converting the compound of formula II to the compound of formula III,
Separating the cells from the medium (5),
Extracting the supernatant and cells using a suitable solvent (6),
Vacuum concentrating the organic solvent phase from step (7) and
A process for preparing a compound of formula III, comprising the step (8) of purifying the compound of formula III contained in the extract from step (7) by chromatography or crystallization.
[14" claim-type="Currently amended] The method of claim 12 or 13, wherein the compound of formula II is avermectin and the compound of formula III is 4 ″ -oxo-avermectin.

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1999-11-12|Priority to GB9926887.2

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1999-11-12|Priority to GBGB9926887.2A

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2000-11-10|Application filed by 신젠타 파티서페이션즈 아게

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2000-11-10|Priority to PCT/EP2000/011139

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2002-08-05|Publication of KR20020063890A

优先权:

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申请号 | 申请日 | 专利标题

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GB9926887.2|1999-11-12|

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GBGB9926887.2A|GB9926887D0|1999-11-12|1999-11-12|Organic compounds|

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PCT/EP2000/011139|WO2001036434A1|1999-11-12|2000-11-10|Preparation of a macrocyclic lactone|