• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
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  • 优先权
    • 专利摘要:
    Disclosed herein is a method of culturing Aspergillus terreus strain NRRL 11156 to produce an antineoplastic compound designated as quadrone and having the following structure: <IMAGE>
    公开号:SU969140A3
    申请号:SU792747552
    申请日:1979-03-30
    公开日:1982-10-23
    发明作者:Джин Калтон Гейри；Альвин Эспеншейд Марлин；Лео Раниери Ричард
    申请人:В.Р.Грейс Энд Ко (Фирма)；
    IPC主号:
    专利说明:

    one
    This invention relates to the chemical and pharmaceutical industry and relates to the manufacture of drugs.
    The aim of the invention is to obtain a quadracyclic lactone of the formula
    YU
    7 8
    About possessing antitumor activity.
    The goal is achieved by the fact that the strain Aspergillus terrens NRRL 11156 is cultivated under aerobic conditions at depth. 22-30 ° C during the day. in a nutrient medium containing carbohydrate, protein base, and iron sulfate, and isolate the desired product from the culture fluid. In this case, cultivation was carried out at 25 ° C for 12 days.
    five , -
    The strain Aspergillus terrens used to produce a quadracyclic lactone was isolated from a soil sample from Tipperary, Northern Territory,
    10 Australia and is stored in the collection of the United States Agricultural Research Service, Department for Research and Development for use in the northern areas of the US Department of Agriculture G.Peori, Illinois, under the number NRRL 11156.
    Morphological signs. Conidial head 25-50
    20 X 100-175 microns, uniform in diameter. Very young heads with 1-5 ko-, nidi mi in a chain, spherical in shape, with the approach of maturity become columnar. 396 Conidiophores smooth 4-5 / 1 x 150200 JU. Bubbles 7–17 µm in diameter, sphere-1eichnye, some with a slightly pear-shaped contour. Sterigms have a primary batch size of 5-6 X 2-3 microns, bubble shape. Conidia of a spherical shape with a smooth wall, diameter 2-2, S microns. Cultural characteristics after 8 days of growth with soaking water agar: colony size 30-35 mm, brownish yellow; . potato dextrose agar: colony size 25– + 0 mm, brownish yellow; . Dextrose agar Saburo: Colony size 50-60 mm, tan color; malt-yeast agar: colony size 0 mm, yellowish-brown. The method is carried out as follows. The strain Aspergillusterrens NRRL111 is cultivated under aerobic deep conditions at 22-30 ° C in a nutrient medium containing a carbohydrate-based substrate and ferrous sulphate. Suitable carbohydrate sources are molasses, corn syrup glucose, whey, soluble collapse of small maize, potatoes, co (and other legumes), cassava, bananas, and grains. Suitable sources of nitrogen-containing nutrients are proteins, for example, soybean flour, corn, corn seeds and cottonseed, yeast extracts, meat extracts (from beef, pork, fish, chicken),. fishmeal, dairy products (. casein skimmed milk) and in their decay products. In addition to the solvent extraction method, other methods are used to isolate the lactone from the culture fluid. For example, the culture fluid can be passed through macromolecular (polystyrene divinylbenzene resin. An example of such a resin is XAD-2 with a helium porosity of 40 vol., A specific surface area of 300 and an average pore diameter of approximately. The lactone is sorbed by the resin surface, after it is removed by desorption with lower alcohols, for example, methanol or water methanol solutions. Quadracyclic lactone can be isolated by thin layer chromatography. Lactone can be detected by using a modified A light yellow reagent with Rf 0.6-0.7. After that, the lactone extract in the solvent is subjected to chromatography: on a silica gel plate. The lactone crystals are obtained by washing with methanol (or similar solvents) from silica and subsequent evaporation of methanol to obtain a saturated solution. The solution is slowly cooled, during which crystals are formed. The lactone can be washed out of silica gel plates and other solvent systems (amino acids and other compounds containing nitrogen). Fermentation takes place under aerobic depth conditions. The microorganisms in the course of fermentation in an aqueous medium are suspended. During aerobic fermentation, the fermentation medium is stirred to introduce air into it. This is achieved both by simple mixing and air bubbling. The air is stirred from 100 to tOO rpm. Air can be blown through the culture fluid in an amount of 0.025-1 volume of air per 1 minute per 1 volume of medium, i.e. aeration coefficient corresponds to 0.025: 1. The aeration rate can be increased in order to achieve maximum yield. The quadracyclic lactone is separated from the culture liquid by extraction with solvents, such as n-butanol, chloroform, ethyl acetate, diethyl ether, and T (trafidrofuran. After extraction, the quadracyclic lactone is separated from the extract by known methods, for example, the extract can be evaporated: and crystallize from methanol (water or other solvent systems), other systems can be used. 5 For example, ethanol (water, methanol, this NOL, chloroform), cyclohexane, Cyclohexane or ethyl acetate, for example, ethanol or chloroform.Crystals can also be obtained by other methods, for example, methanol extract can be evaporated and the material obtained is redissolved in methanol / water system, followed by cooling to form crystals.Quadcyclic lactone, obtained by the proposed method is a substance with a molecular weight, determined by the mass spectral method; this corresponds to the empirical formula of 15%. The infrared spectrum shows adsorption at cm, indicating the presence of a five-membered ke tone or b-lactone, and at 1390 and 1375 cm, indicating the presence of double-dimethyl group, lactone UV spectrum dormancy binds weak absorbance at 280 nm. The proton NMR spectrum confirms the presence of a double dimethyl group with two 3-proton singlets at 1.2 and 1.28. A two-, single-prong doublet of doublets centered at (Y 4.23 (j 12.5 Hz and, b5 (, j 12.1 Hz)) are characteristic of non-equivalent methylene protons that are attached to the c / lactonic ring. Example 1. Production of quadracyclic lactonol. A lyophilized sample of Aspergil1u5 terreus ((11165) is dissolved in 2 ml of 2-malt extract solution. The resulting suspension is spread onto the surface of oblique potato-dextrose agar, 16x125 mm in size, and incubated at 30 ° s for 7 days. The resulting culture is suspended in 10 ml of water in order to obtain a su A bottle containing about 10 ° Spores / ml 20 ml of the prepared suspension is used to inoculate them in 1 liter of medium in a four-liter vessel. The composition of the medium is as follows, g / l: Glucose Flour of cotton seeds Flour from sprouts corn Oil for flour from soybean K / 2IP04 HgSO.-742.0 0 CACCO3 NaCl0.5 To this medium, sufficiently deionized water is added to produce 1 l of broth. After inoculation, the broth is incubated at 25 ° C for 3 days at constant stirring (250 rpm). 2 L of this broth is used to inoculate 20 L of broth. At the same time, flour from corn sprouts or soy flour was not used, and the amount of FeS04-7H20 was reduced to 0.001 g / l. Thus, the only source of nitrogen is cotton seed flour, the broth pH is set to 6, and incubated at 25 ° C. After 7 days, the final pH is 6.0-6.5. In addition, the broth contains 0.5-2 wt.% Of glucose. During both incubation stages, a sufficient amount of a block copolymer of oxyethylene oxypropylene (Pluronic PL-61) is used in order to minimize foaming. As a rule, the amount of surfactant is 0.15%. In the second stage of incubation, the broth is continuously stirred at a speed of 350 rpm. In addition, about 2 L / min of air is blown through the broth. Example 2. The selection of quadracyclic lactone. Filter 20 l of the developed broth in order to remove the mycelial mass (about 1 l). 4 equal parts of n-butanol are added to the filtrate in a volume ratio j :. Thus, the lactone dissolved in n-butanol is isolated from the medium; after the compound, the extracts in n-butanol are evaporated to dryness over a water bath at (approximately 1 mm / Nd i) using a thin film rotary evaporator. The resulting resin is triturated with 0.5 l of chloroform, which is then distilled off with steam. As a result, a thick brownish oil is obtained. This oil is dissolved in a mixture of methanol and water, from which the lactone crystals are crystallized by seed. Using the procedure described, the yield of lactone is about 0.2 mg / ml of the original enzyme broth.
    权利要求:
    Claims (2)
    [1]
    Claim
    1. A method for producing a quadricyclic lactone of the formula having antitumor activity, characterized in that the strain Aspergί11 us terrens NRRL 11156 is cultivated under aerobic conditions at 22-30 ° C for 4-15 days in a nutrient medium containing a carbohydrate, a protein base and sulfate is the desired liquid product.
    [2]
    2. The method according to claim 1, p and th with the fact that the cultivation is carried out at 25 ° C for 12 days.
    iron, and vydeiz kulturnogo otlichayuVNIIIPI Order 8195/81 Circulation 714
    Branch of the PPP Patent, Uzhgorod, Project 4,
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    同族专利:
    公开号 | 公开日
    BE869439A|1978-12-01|
    US4147798A|1979-04-03|
    引用文献:
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    US82108777A| true| 1977-08-02|1977-08-02|
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