• 专利附录
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  • 权利要求
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    • 专利摘要:
    Maytansinol, maytanacin and/or maytansinol propionate are prepared by culturing a microorganism belonging to the genus Nocardia, which is able to produce maytansinol, maytanacin and/or maytansinol propionate, with concentration of maytansinol, maytanacin and/or maytansinol propionate, and isolating it from the culture broth. These compounds are useful antitumour agents, i.e. useful cytostatics.
    公开号:SU938746A3
    申请号:SU772533351
    申请日:1977-10-07
    公开日:1982-06-23
    发明作者:Хигасиде Эйдзи；Асаи Мицуко；Танида Сеиити
    申请人:Такеда Кемикал Индастриз Лтд(Фирма)；
    IPC主号:
    专利说明:

    Conidia are assembled into chains, whereas the cellular suspensions obtained from the surfaces of such cultures, observed under a microscope, contain many elongated ellipsoidal (0.8-1.2 x X, 0-6.8 mm and ellipsoidal 0.81, 2 X 1.0–2.0 mm7 of bodies similar to aproporus. The strain grows well in various environments, and the vegetative mycelium is colorless, pale yellow in the initial stages and light yellowish to yellowish in oak bark in the last stages. gives soluble pigments from yellow to yellowish tones of oak bark in various taxonomic environments Sucrose nitrate agar. Growth is exuberant. Substrate mycelium FROM yellow melon to amber. Aerial mycelium poor, white. Soluble pigment is absent or pale yellow, reddish brown. Glucose-aspartic agar. Medium growth. Substrate mycelium per color. Marigold to yellow. Vigorous mycelium scanty, white. Soluble pigment - yellow. Starch agar. Growth is medium. Substrate mycelium is ivory. Aerial mycelium is abundant, ivory. No soluble pigment is present. Nutrient agar. Growth is average. Substrate mycelium from ivory to yellow. Aerial mycelium is scanty, white. There is no soluble pigment. Agar with calcium malate. Growth is average. Substrate mycelium from ivory to wheat. Aerial mycelium is medium, white to ivory. Permissible pigment is absent. Yeast agar with malt. Growth is average. Substrate mycelium from amber to yellow. Aerial mycelium medium, white to ebony. Soluble pigment is missing. Agar with glycerol nitrate. Growth is average. Substrate mycelium is ivory, cortex-like educated bodies. Aerial mycelium medium, white. Soluble pigment is missing. Asparagine agar with glycerin. The growth is medium, the substrate mycelium is ivory, the cortex is similar to the formed bodies. Aerial mycelium scanty, white. Soluble pigment is missing. Agar with oats nkoy. Growth is average. Substrate mycelium from ivory to yellow, cortex-like educated bodies. Aerial mycelium scanty, white to light yellow. Soluble pigment is missing. Peptone yeast extract in strong agar. Growth is average. Substrate mycelium yellow. Aerial mycelium is absent. Soluble pigment is yellow. Tyrosine agar. Growth is average. Substrate mycelium from ivory to yellow melon. Aerial mycelium is medium, from white to ivory. Soluble pigment - camel color, Physiological characteristics. Grows at. The optimal temperature for growth is 20-35 C. It is diluted with gelatin, the starch hydrolyzes. Milk pentonizes but does not coagulate. Casein decomposes. Nitrates liquefy, does not form melanoid pigments. Tyrosine decomposes, Xanthine and hypoxanthin does not decompose. Tolerance is lysim positive. Sodium chloride tolerance - 2%. Very well absorbs xylose, glucose, fructose, mannose, well absorbs arabinose, galactose, rhamnose, trehalose, melibiose, mannitine, starch. Poorly absorbs lactose, inositin, sorbitin, glycerin. Gram stain the vegetative mycelium of strain - positive. The medium used to cultivate a strain capable of producing methanzinol, methanacin or methanzinol propionate can be liquid and solid. In the latter case, it must contain a nutrient medium, which the strain can utilize, although liquid medium is preferable for highly productive processes, and, as was found, the medium is a mesoform, while the spots found correspond to galactose and arabinose. The medium contains carbon and nitrogen sources, which can be assimilated and digested by strain No. C-15003, inorganic material, nutrient traces, etc. Glucose, lactose, sucrose, maltose, dextrin, starch, glycerin, mannitin sorbitin, etc., fats and oils (for example, soybean oil, lard, chicken fat, etc.) and others can be used as carbon sources. . Nitrogen sources can be, for example, mace extract, starch extract, dry starch, soybean flour, grains submerged in liquid, peptone, casein, cotton seed flour, molasses, urea, ammonium salts (for example, sulfate, chloride, nitrate ammonium acetate, etc.) and others. The medium may also contain salts of sodium, potassium, calcium, magnesium, etc., salts of iron, manganese, zinc, cobalt, nickel, etc., salts of phosphoric boric acids, etc., salts of organic acids, such as acetates and propionates. The medium may also contain as additives various amino acids (for example, glutamic, aspartic acids, alanine, glycine, lysine, methylated, proline, etc.), peptides, for example, dipeptides, tripeptides, etc., vitamins, for example. nicotinic acid, Cj2, C, E, etc.), nucleic acids (for example, purine, pyramidine and their derivatives, etc.). To create a certain pH value, organic or inorganic acids, and fir trees, buffers, etc. are added. Soluble quantities of oils, fats, surfactants, etc. can also be added as antifoam agents. Cultivation is carried out under stationary, vibrational, submersible, aerobic, and other conditions. For highly productive processes, submersible aerobic cultivation is preferred. Cultivation depends on the conditions and composition of the medium, strain, cultivation method and other factors, and incubation is preferably carried out at 25-30 ° C with an initial pH value in the range 7. In the intermediate cultivation stage, the temperature is 23-30 ° C with an initial pH of 6, . Since the incubation time varies depending on the factors mentioned, it is desirable to continue the incubation until the titer of the proposed antibiotic is maximized. When cultured under the conditions of aerobic immersion in a liquid medium, the required time interval is usually 8-1 hours. Methanacin, methanzinol propionate or methanzinol is obtained and accumulated in the resultant cultivated broth both extracellularly and intracellularly. None of these substances shows a distinct antibiotic activity, so they were detected by thin layer chromatography. The enzyme broth is decomposed in the cells. And the filter is filtered or centrifuged and the filtrate is extracted with the same volume of ethyl acetate. To the cells, the same amount of 70% acetone solution in water as to the filtrate is added, and after stirring for one hour, the suspension is filtered. The acetone is removed from the filtrate and the aqueous filtrate is extracted with ethyl acetate. Each of the extracts was concentrated to 1/100 in volume and analyzed by thin layer chromatography on a silicate gel-glass plate (solvent chloroform: methanol 9: 1. Activity is judged by the intensity of the spots determined by irradiation with ultraviolet light at 2537 A. Methanecin , methanzinol propionate or methanzinol, which are thus obtained in cultured, dipophilic and neutral, they can be easily regenerated by separation and purification, which are usually carried out to obtain such microbiologists. For example, an operation that uses the difference in solubility between the antibiotic and impurities, methods that use the fractional absorption efficiency of various absorbents, such as activated carbon, microporous non-ionic resins, silica gel, etc., can be carried out. removing impurities through ion exchange resins, etc. These methods can be applied separately, in combination with each other, one or more times.
    Methanacin, methanzinol or methanzinol propionate are obtained in the filtrate, antibiotics are separated and purified by absorbents. Antibiotic is obtained either directly or after screening.
    solvent extraction in the case of a filtrate, or after solvent extraction in the case of microbial cells. Solvent extraction can be carried out from cultured bouillon to separate cells and from the filtrate obtained after filtration, centrifugation or other processes. To extract the filtrate and cells independently of each other, solvents can be used to extract the filtrate — water emulsion organic solvents such as fatty acid esters, for example ethyl acetate and amyl acetate, alcohols, for example butanol, halogenated hydrocarbonates, for example chloroform, ketones, for example methyl isobutyl ketone . Extraction was performed at pH, close to neutral. Preferably, the cultured liquid, adjusted to pH 7, is extracted with an ethyl volume. The extract is washed with water and concentrated under reduced pressure. Then, a non-polar solvent, such as petroleum ether or hexane, is added to the concentrate, and the crude product 1, containing the active compounds, is regenerated. Since a number of other spots are found on the thin-layer chromatogram, besides methanacin, methanesinol, methazinol propionate, product 1 is purified. A number of methods, such as absorption chromatography, are used for purification using one of the common absorbents such as silica gel , aluminum, macroporous non-ionic resin. Silica gel is particularly suitable for cleaning the crude product. 1 Then petroleum ether and hexane are used with the addition of polar solvents such as ethyl acetate, acetone, ethanol or methanol. Silica gel is used as a carrier, the chromatographic column is filled with successively increasing ratios of hexane to ethyl acetate. The sample is examined by thin layer chromatography, and the fractions containing the active
    ingredients, get and concentrate under reduced pressure. Then, petroleum ether or hexane is added to the concentrate, whereby it is obtained that it contains more impurities, it is purified. For example, product 11 can be purified using a second column with cyan gel, using a differential product P. Since this product is a solvent system. A solvent system for this purpose may consist of a halogenated hydrocarbon such as dichoromethane or chloroform, with the addition of a polar solvent such as an alcohol, such as ethanol or methanol, ketone, such as acetone or methyl ethyl ketone, and the like. In this way, methanacin, methanzinol or methanzinol propionate is isolated. So that the solvent system for the first and second silica gel columns can be regenerated, conventional organic solvents can be used with those mentioned, if necessary. When the macroporous absorption resin is used as a means to purify the crude product 11, the purification of methanacin, methanzinol or methanzinol propionate is performed in a mixture of water with a lower alcohol, a lower ketone or ether. The lower alcohol may, for example, be methanol, ethanol, propanol or butanol, and the lower ketones may be, for example, acetone or methyl ethyl ketone. The ester may, for example, be ethyl acetate. In a typical operation, the crude product 11 is dissolved in a 60% solution of methanol in water and absorbed on a DIaion-HRIO column. The column is washed, and then with a solution of methanol in WAD. Thus, methanacin, methanzinol propionate or methanzinol are separated from the column. The fractions containing these components are combined and concentrated under reduced pressure. To the dry product add 5-8 volumes of ethyl mixture to stand. Then, acetate, crystals of methanacin, methanzinol propionate, and methanzinol are separated using absorbents. Thus, using silica gel or macroporous non-ionic tuony, and solvent as the absorbent, the described compounds are isolated. When, for example, silica gel is used, hexane, ethyl ethyl ethyl acetate is used.
    acetate or chloroform-methanol mixture, using 60% methanol-water and 95 at the same time separating methanine-methanol-water propionate with the addition of 5 chloride la-methanacin. After detection with sodium, methanacin and propionate methane by thin layer chromatography of fracisol, it appears in the mentioned order that it is concentrated under reduced pressure. After sampling and determination, and ethyl acetate is added to the concentrate by means of thin layer chromatography with centrate. Thus, with each group of active fractions, the final compound can be obtained centered under reduced pressure and crystallized from ethyl acetate as crystals. Selected, 10 nye crystals include ethyl acetate
    When macroporous as a crystallization solvent and after-ion absorbent resin is used, it can be dried over phosphorus pentoxide with a solvent with an individual personal ratio of alcohol, ketone and chemical properties represented by either ether or water for 8 hours. For example, when is- is in the table.
    Z2. ° 92
    K) 121 + (d) 127-10 + 10 (, 35; sleep C) (, 25; SNS
    92
    W 3l3-l (f, 22; СНСЬ) Acid, NeutLygyufil and Lipophil and Neutral Neutral Substance ral or basic substance
    Colored Strip-Positives | p-chi
    on
    Dragendorf
    Belle-Positive Reaction. Positive
    on
    matte The data for elemental analysis, specific rotation, UV, IR and mass spectra are in good agreement with the data for methanacin. and meper 1, Methanzinol, methanacin and methanzinol propionate - Nokardia C-15003 derivatives (IFO 13726; FERM 3992; ATCC 31281), grown on yeast malt extract in agar, are used for grafting 200 h (by volume) of enzymes containing 40 about. h seeds, cultivated in the medium (2 glucose, 3 solutions of starch flour of raw soybeans, 1% of cereals immersed in liquid, 0.5 polypentone, 0.3 Nad, 0.5 CaCOj, pH 7.0). The cultured medium is incubated at 28 ° C for kQ h prior to the introduction of a 0.5 part by volume column. the solution obtained in this way: from the column was placed in 200 parts by volume enzyme containing kO ob.ch. enzyme medium consisting of 5 dextrin, 3 grains immersed in a liquid, 0.1 polypeptone and 0-, 5% CaCO at pH 7.0, and cultured at 28 ° C for 90 h to obtain a seed culture. By the method of sequential dilution, using Tetrachimenus pyriformis V / and methanzinol propionate as a standard product as the test organism, it was found that the above culture has a titer of 25 mg / ml. Example 2. 10 ob.ch. seed cultures obtained in example 1, put 8 2000 ob.h. enzyme containing 50 ob.h. seeds cultivated in Atakoy medium as mentioned above) and incubated at 28 ° C for 48 hours, 500 ob.h. cultures are transferred to a vessel of steel with a capacity of 50,000 ob.ch ..
    Is positive
    Is positive
    Positive lipophilic and neutral substance containing 30,000 rpm, h. seeds cultivated in the medium, and cultured at 28 ° C, aeration (30,000 rpm / min, agitation of rt (1 / 2DT) and internal pressure (1 kg / cm) to obtain a seed culture This culture was used for seeding 200,000 parts by volume in a vessel made of steel containing 100,000 parts by volume of enzymatic medium, similar to the medium used in Example 1, at an inoculation rate of 10%. The inoculum medium was incubated with aeration (100,000 turns h / min), stirring 200 g. mind, (1 / 2DT) 3 and internal pressure (, 1 kg / cm) for 90 h. Conducted the same operation as in example 1. The resulting culture It has 25 mg / ml titer To 90000 parts by volume of the obtained culture, 2000 hours are added. Hyflo-supertarget P and, after stirring, the mixture is filtered on a filter press to obtain 85000 parts of filtrate and 32000 parts of wet cells. The filtrate 85000 parts by volume is stirred and extracted with 30,000 parts by volume of ethyl acetate. The operation is repeated again. Precipitates of ethyl acetate are combined, washed twice with 30,000 parts by volume of water, dried by adding 500 parts of anhydrous sodium sulfate and concentrated under reduced pressure. up to 200 ob.ch. Petroleum ether is added to the concentrate and precipitation and filtration is carried out. The resulting crude product 1 is mixed with 100 vol. ethyl acetate, and the solution is filtered. The filtrate is mixed with 10 parts of silica gel and the ethyl acetate is removed under reduced pressure. The residue is placed on top of a silica gel column, kOO vol. h) The solution is removed ob.h. hexane, 500 o6.4i mixtures of hexane-ethyl acetate (3: U, 139 500 vol.h., mixtures of hexane-ethyl acetate (1: 1), 500 vol.h. mixture of hexane-ethyl acetate (1: 3, 500 vol.h. ethyl acetate , 1000 parts by volume of a mixture of ethyl acetate-methanol (50: 1) and 1000 parts by volume of a mixture of ethyl acetate-methanol (25: 1) collected in a fraction of 100 parts by volume, One part of the volume of each fraction is concentrated by drying, and 0.1 volume H. Ethyl acetate is added to the concentrate. The mixture gives a spot at a distance of 2.5 cm from the bottom of the tip of the sigmkagel glass plate and grows to about 17 cm with a soluble ethyl acetate-methanol system (19: 1). t definition in ultra violet light (2537 A). Active fractions f 25-30 Rf 0.58-0.63 and fractions f Rf 0.25-0.30 are collected and concentrated under reduced pressure, to about 20 parts by volume. To these concentrates 150 parts by volume of petroleum ether are added to obtain 5 parts of crude product 1 and 2 parts of crude methanzinol. In 10 parts by volume of ethyl acetate, 0.5 parts of the obtained crude product 1 is dissolved and the solution is mixed well with silica gel Ethyl acetate is removed under reduced pressure. The precipitate is placed on top of the column with 300 rpm. silica gel, and the column is first washed with 500 rpm. chloroform and then treated with 500 rpm chloroform-methanol (50: 1) mixtures, 500 ob.h. a mixture of chloroform - methanol (20: 1) and 500 ob.h. chloroform – methanol mixture (10: 1). The solution is collected fraction1; 1 mi to 25 ob.h. 0.5 ob.h. each fraction is concentrated under reduced pressure. 0.05 ob.ch are added to the concentrate. ethyl acetate, and the mixture in the form of a sample is analyzed by thin layer chromatography (chloroform-methanol 9: 1 system). Fraction No. 0, absorbed at 2537A in the zone R 0,, 50, is collected and concentrated by drying under reduced pressure. 0.5 v of ethyl acetate is added to the precipitate, and the mixture is settled, then 0.05 parts of mixed crystals of methanacin and methanzinol propionate are obtained. 0.05 parts of mixed methanacin crystals are dissolved in 5 parts by volume. methanol followed by the addition of 0.1 parts of sodium chloride and 5 vol. water. The column is filled with 200 vol. Diaion HP-10 washed 600 ob.h. 502; methanol-water 61 containing 5 / MaCl. A sample of the solution prepared in this way is passed through the column and the dissolution is completed using 1500 ob.ch. 60 methanol - water containing 5% NaC: 1 and 1500 vol.h...95% methanol - water. The solution is collected in fractions of 15 parts by volume, and each fraction is examined by thin layer chromatography. Fractions 130-135 contain methanocin, and fractions 138-1 2 contain methanzinol propionate. Each group of fractions is concentrated and dissolved by adding 30 ml of water and 50 ml of ethyl acetate. The solution is stirred in a separatory funnel, the aqueous layer is separated, and after washing twice with 30 ml of water, the precipitate of ethyl acetate is dried over anhydrous sodium sulfate, concentrated and settled. Crystals from each group of fractions are obtained as described. : The crystals are collected by filtration and drying. The content of methanacin 0,013 parts, methionzinol propionate 0, 025 parts. In 3 parts by volume ethyl acetate is dissolved in 0.2 parts of the crude methanesinol obtained in this way, and the solution is well mixed with 0.5 parts of silica gel. Ethyl acetate is removed under reduced pressure. The precipitate is placed on top of the column with 80 vol. silica gel, and the column is first washed with 150 rpm. then 150 ob.ch. chloroform-methanol (50: 1) mixtures, 150 ob.h. mix chloroform-methanol (20: 1) and. ZOO ob.ch. chloroform-methanol mixtures (10: 1). The solution is collected in fractions of 10 vol. 0.5 ob.h. each fraction is concentrated under reduced pressure. 0.05 parts by volume is added to the concentrate. ethyl acetate, and the mixture is analyzed by thin layer chromatography (chloroform-methanol 9: 1 dissolving system). Fractions No. 50-52 are absorbed at 2537 A in the zone of 0.33-0.38, collected and concentrated by drying under reduced pressure. To the precipitate add 0.5 ob.h. ethyl acetate, and the mixture is settled, resulting in a gain of 20 parts of methanesinol crystals. Example 3. By mixing 32,000 cell-fragments obtained in Example 2, 50,000 parts by volume were extracted.
    权利要求:
    Claims (3)
    [1]
    Claim
    1. The method of producing alkaloids of methanzinol, methanacin and methanzinol propionate by extraction of the target product with an organic solvent, characterized in that, in order to increase the yield of alkaloids, strain Nocardia C-15003 is grown in a nutrient medium containing carbon and nitrogen sources, mineral salts and growth stimulants , then the culture fluid is separated from the biomass, subjected to their separate extraction and the target product is purified and concentrated.
    [2]
    2. The method of pop. ^ characterized in that the extraction is carried out using a water-immiscible organic solvent.
    product or ”0
    4(!
    [3]
    3. The method of claim 1, wherein the target is purified by precipitation chromatography.
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    同族专利:
    公开号 | 公开日
    ES463206A1|1978-08-16|
    IT7821819D0|1978-03-30|
    DE2746253C2|1986-08-21|
    DK143570C|1982-02-08|
    NL7711273A|1978-10-03|
    CH631740A5|1982-08-31|
    CS214881B2|1982-06-25|
    FR2385798A1|1978-10-27|
    PT67853B|1980-05-05|
    DE2746253A1|1978-10-05|
    ATA736377A|1980-09-15|
    IT1094003B|1985-07-26|
    YU243777A|1982-06-30|
    CA1092999A|1981-01-06|
    PL201539A1|1978-09-25|
    SE7711543L|1978-10-01|
    JPS53121998A|1978-10-24|
    AU2907377A|1979-03-29|
    DK458977A|1978-10-01|
    PT67853A|1978-04-01|
    DK143570B|1981-09-07|
    HU177391B|1981-09-28|
    GB1554395A|1979-10-17|
    AU507869B2|1980-02-28|
    IE45888L|1978-09-30|
    FR2385798B1|1980-04-25|
    IE45888B1|1982-12-29|
    PL108863B1|1980-05-31|
    JPS6010718B2|1985-03-19|
    AT362058B|1981-04-27|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    JPS6010720B2|1977-11-18|1985-03-19|Takeda Chemical Industries Ltd|
    JPS6016236B2|1977-11-18|1985-04-24|Takeda Chemical Industries Ltd|
    JPS55162791A|1979-06-05|1980-12-18|Takeda Chem Ind Ltd|Antibiotic c-15003pnd and its preparation|
    EP0028683A1|1979-09-21|1981-05-20|Takeda Chemical Industries, Ltd.|Antibiotic C-15003 PHO and production thereof|
    US6573074B2|2000-04-12|2003-06-03|Smithkline Beecham Plc|Methods for ansamitocin production|
    US6333410B1|2000-08-18|2001-12-25|Immunogen, Inc.|Process for the preparation and purification of thiol-containing maytansinoids|
    US7432088B2|2003-05-08|2008-10-07|Immunogen Inc.|Methods for the production of ansamitocins|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    JP52037167A|JPS6010718B2|1977-03-31|1977-03-31|
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