Fungus strain aspergillus terreus 44-62 as producer of lovastatin, industrial method for isolation of lovastatin and method for lactoninization of statins

FIELD: biotechnology, organic chemistry, microbiology, pharmacy.

SUBSTANCE: invention describes a new highly productive strain of fungus Aspergillus terreus № 44-62 that is deposited in the Collection of the company Metkinen Oy, Littoinen, Finland, producing lovastatin. Also, invention relates to a method for isolation of lovastatin and a method for lactonization of statins, such as lovastatin and simvastatin. Method for isolation of lovastatin involves its extraction from raw obtained in culturing the above said fungus-producer, concentrating extract, lactonization of lovastatin in the absence of solvent, clearing and crystallization of the end product. The lactonization process of statins carrying out in the absence of solvent provides preparing their lactones in crystalline form directly and practically without impurities of dimmers and acid form. Invention provides highly profitable manufacturing lovastatin and allows preparing the end product corresponding to Pharmacopoeia purity, higher yield (above 70%) and low cost.

EFFECT: improved preparing method and enhanced quality of product.

48 cl, 2 dwg, 10 ex

 

The technical field, which includes inventions

The invention relates to biotechnology and organic chemistry relate to microbiological synthesis of lovastatin is an inhibitor of 3-hydroxy 3-methylglutaryl-coenzyme a (HMG-CoA) reductase and are producing strains of lovastatin, is an industrial method of its selection and separate stage of this process, namely the way lactonization statins.

The invention can be used in the chemical-pharmaceutical, pharmaceutical, perfumery and cosmetic industry, medicine, veterinary medicine and agriculture.

The level of technology

Lovastatin along with other statins finds very wide application as the active principle of the pharmaceutical drugs hypocholesterolemic actions. Drugs of this class find high osteo-and antithrombotic clinical efficacy (Meier G.R., Schlienger R.G., Kraenzlin M.E., et al. HMG-CoA reductase inhibitors and risk of hip fractures. JAMA. 2000; 283:3205-3210.; Wang P.S., Solomon D.H., Mogan J. HMG-CoA reductase inhibitors and risk of hip fractures in elderly patients. JAMA. 2000; 283:3211-3216.; EP 1291017 A3; Grady d, Wenger N.K., Herrington d, et al. Postmenopausal hormone therapy increases risk for venous thromboembolic disease: the Heart and Estrogen/progestin Replacement Study. Ann Intern Med. 2000; 132: 689-696).

Because atherosclerosis is the main cause of death in economically developed countries, and fractures associated including osteo what orosa, and venous thrombosis are major causes of morbidity of the population, the need for the above drugs is constantly increasing.

In addition, lovastatin and its chemical analogues are used as starting substance to obtain derivatives of the same biological effect, as well as other well-known statins, particularly simvastatin and pravastatin, by chemical and biological transformation (JP 2003012607; EP 1284264 A1; US 6.472.542; JP 2001286293).

In connection with the foregoing special significance of obtaining highly productive industrial strains - producers of lovastatin and developing cost-effective, technologically advanced and environmentally friendly methods of its allocation, as well as individual operations of this process, providing a commercial product Pharmacopoeia purity with maximum output and low cost.

Known strains-producers of lovastatin from the genus Aspergillus: Asp. terreus AD 43, DS 28373, CBS 456.95 (Cenrraal Bureau voor Schimmelcultures, Delfr, The Netherlands; EP 0877089 Al); Asp. terreus CCM 8236 (WO 99/19458; WO 00/63411); Asp. terreus ATCC 20541 (US 4.231.938); Asp. terreus ATCC 20542 (EP 0022478); Asp. oryzae ATCC 74135 (EN 2114912; US 5.362.638); Asp. obscurus - MV-1 holotype strain (NCAIM (P) F 001189; WO 94/10328).

However, the productivity of known strains do not exceed 3 g/l of culture fluid, which causes a lack of profitability of production of lovastatin.

Known methods of allocation lowest is in the lactone form of raw materials, obtained by the cultivation of mushrooms-producers of the genus Aspergillus: (EN 2114912; WO 00/63411; EP 0877742 B1; WO 94/10328; EP 0 089 877 A1; WO 97/20834; WO 00/17182; WO 98/50572; EP 0 556 699; US 5.362.638).

The disadvantages of the known methods are:

- insufficient purity of the target product due to contamination, in particular, difficult to separate such impurities as dimers and acid form of lovastatin;

- insufficient yield of the target product, in some cases, not exceeding 50-60%;

- low industrial and environmental safety due to use of large volumes of toxic organic solvents, acids, catalysts, etc.;

- high energy consumption due to the distillation process of the high-boiling organic solvents;

- low efficiency and manufacturability due to the complexity and mnogostadiinost number of processes, and the use of special expensive equipment.

The closest analogue of the claimed allocation method lovastatin is a way of separating it from the culture fluid obtained by cultivation of mushroom-producing species of Aspergillus terreus, including the acidification of the culture liquid of hydrochloric acid, extraction of lovastatin with ethyl acetate, concentrating the obtained extract by distillation in vacuum, lactonization acid form of lovastatin by boiling is oncentrate in toluene at 106° With in two hours, two-stage cleaning lactone form of lovastatin from impurities by chromatography on silica gel and purification activated carbon and, finally, the crystallization of the desired product (EP 0033536 B1; Compound III a, pp.3-4; steps B1-B3).

The known method has a number of disadvantages:

- target product has no pharmacopeial purity due to contamination of its dimers formed during the high temperature lactonization in the presence of a solvent;

- low industrial and environmental safety of the process due to the use of large volumes of toxic organic solvents (toluene, methylene chloride, etc.);

- high consumption of the process because of the duration and the high-temperature regime stage lactonization;

- low efficiency and manufacturability of the process in connection with the necessity of recycling and/or recovery of large amounts of silica and organic solvents, and the use of special expensive equipment.

Despite the fact that physiologically active is the acid form of the statin, the latter, as a rule, get in the lactone form, which is a commercial product, as the lactone easily crystallizes, it cleanses away impurities and subject to long-term storage and ser is eficacia.

Therefore, the key stage of selection of lovastatin and other statins is the process of lactonization their acid form.

There are various ways of lactonization acid form of the statin, which consists in conducting the process in toluene at a temperature 106-110°within two to six hours (EP 0033536 B1; WO 98/50572; US 5.362.638); the implementation of lactonization simultaneously concentrated in butyl acetate under reduced pressure and a temperature of 55-60°WITH (EN 2114912, US 5.712.130); boiling in chloroform and other halogenated solvents (WO 97/20834); heated in water to a temperature of 50-60°in the presence of large quantities of mineral acid as a catalyst for 24 h (WO 02/0065); vacuum distillation in an organic solvent at a temperature of 60°in the presence of 0.1-1.0%acetic acid (WO 00/63411); by heating ammonium salt of lovastatin or simvastatin in acetic acid to a temperature of 35-40°C in nitrogen atmosphere (EP 0955297 A1); by heating ammonium salt of lovastatin or simvastatin to a temperature of 100-110°in toluene in a stream of nitrogen in the presence of dehydrating agents within 3 h (EP 1288212 A1); conducting lactonization in an organic solvent in the atmosphere of nitrogen at room temperature in the presence of agents lactonization, binding to the insoluble complex is formed in which the process of the reaction water (US 6.562.984 B2).

The disadvantages of the known methods are:

- the formation of by-products, in particular dimers, with the exception of lactonization in the regime of high temperatures and the presence of solvents and/or use as catalysts in a strong mineral acid, which prevents the obtaining of target products Pharmacopoeia purity and leads to the need for additional purification (US 20020002288; US 6.521.762);

- incomplete lactonization (acid form is transformed into the lactone only 50-90%) and the junk duration of the process (up to 24 h) at moderate temperatures and the use of acids as catalysts;

- the necessity of division and/or allocation of the target product from the reaction mixture;

- high energy consumption and insufficient technological and environmental safety of the process when using the high-boiling toxic solvents (toluene, halogenated solvents, etc.);

- the need for recycling and/or recovery of used in the process, as a rule, large amounts of solvents, precipitators, catalysts, etc.;

- the need for pre-allocation lovastatin or simvastatin in the form of ammonium salts.

The process of lactonization in the absence of solvent are not described in literature.

Brief description of figures

Further the invention are explained Phi is urami, thus

Figure 1 - illustrates the1H NMR spectrum allocated stated how substances - lovastatina in solution CDCl3regarding tetramethylsilane;

Figure 2 illustrates the IR spectrum allocated stated how substances - lovastatina in KBr.

The essence of inventions

The basis of the inventions based on the task of getting a new high-yielding strain of Aspergillus terreus producer of lovastatin and development of high-tech, economical and environmentally safe method of its selection, as well as a separate operation of this method, namely the process of lactonization statins.

The task of the invention in terms of strain is solved by the fact that the new strain of Aspergillus terreus No. 44-62 (deposited in the collection of the company Medinan, Littoinen, Finland) - producer of lovastatin. Technical results of the claimed invention are:

- high productivity (6-8 g/l of culture fluid);

genetic stability;

- stability in the conditions of production (declared strain does not lose the initial productivity for 4-5 cycles of cultivation).

The claimed strain obtained by multistage combined method induced mutagenesis of the original strain Asp. terreus stored in American Collections Typed Cultures under ATS 20542.

The strain deposited at the collection company is mkinen, Littoinen, Finland number 44-62.

The strain is characterized by the following features:

- cultural-morphological

On agar medium 12-day colony has a rounded irregular shape with irregular cut edges, volcanic profile. Possible crater in the center. The colony diameter of 20-30 mm; the color of the colony brownish-red. Mycelium dense, compact, without air fluffy layer. Hyphae short, branched, consisting of extended cells irregular in shape, with a diameter of from 5 to 30 μm. Substrate mycelium grows deeply into the nutrient medium, forming an air cavity at the bottom of the Cup.

In the liquid nutrient medium is growing in an open filamentous and compact spherical shape. Hyphae mostly short, silica branched, screwed, consisting of irregular extended short cells.

- physiological

The strain is a strict aerobe, when fermentation is in need of intensive aeration. Grows on many organic substrates, such as oatmeal, rice, corn flour. Growth Petelino environment and fermentation can occur at a temperature of 20-37°C, while the temperature optimum is 25-27°C, the optimum pH value of 5.5 to 6.5.

biochemical

Strain in a nutrient medium absorbs as a source of carbohydrates glitch the memory, sucrose, fructose, maltose and glycerol; as a source of nitrogen uses peptone, yeast extract, casein hydrolysate; inorganic salts preferably uses sodium chloride, magnesium sulfate and potassium phosphate.

- biotechnology

The maximum productivity of the strain is 6-8 g/l of culture fluid for 200-240 hours at a temperature of 26°and With the following composition environment for fermentation (g/l):

peptone 10 g; soya flour 40 g; malt extract, 5 g; sucrose 200 g; KN2PO40.5 g; NaCl, 2 g; MgSO4·7H2O, 0.5 g; distilled water to 1 l; to sterilization, the pH is 6.0.

At the same time declared the strain does not lose above productivity for 4-5 cycles of cultivation.

the pathogenicity of the strain

Experimentally investigated the pathogenic properties of the microorganism, its influence on the integral indicators of the state of the organism of experimental animals and the intestinal microflora, immunotoxic properties and the possibility of its dissemination to internal organs with the aim of establishing a limiting criteria for adverse actions (LCVD) and justification MACZ.and MACAV.

Intraperitoneal injection of high doses of micromycete not identified virulent and toxigenic properties of the studied strain of mold fungus. He has no ability to dissemina the AI in the blood and internal organs of animals after a single intraperitoneal injection of large doses of the producer strain. Irritant effect of the suspension of the fungus in a single drawing on the conjunctiva of the rabbit is also missing.

Examination of experimental animals in the chronic experiment showed that the impact of the producer strain in two concentrations (3.5·103and 3.5·104CL/m3within 1 month did not lead to the change of integral indicators of the state of the organism of experimental animals, which were estimated on the dynamics of body weight during the experiment and during the rehabilitation period. The obtained data indicate the absence of General toxicity producer strain on the organism of rats under chronic exposure of his in the studied concentrations.

As a result of studies on the immunotoxic properties of the microorganism found that the coefficients of the weight of thymus and spleen in experimental animals when exposed to higher concentrations did not differ from those in animals of the control group.

Thus, the experimental results indicate that Aspergillus terreus No. 44-62 (deposited in the collection of the company Medinan, Littoinen, Finland) has not pathogenic for warm-blooded animals.

The technical results of the claimed invention in terms of allocation method lovastatin are:

- Poluchenie target product Pharmacopoeia purity without a mixture of dimers and acid form of lovastatin while increasing its output (over 70%);

- improving industrial and environmental safety of the process by eliminating the use of large volumes of toxic organic solvents (toluene, methylene chloride) at high temperature;

- lower energy process;

- increase efficiency and technological process due to its simplicity, lack of special expensive equipment and use of closed production cycle (one solvent, in particular ethyl acetate, the entire production).

The allocation method lovastatin according to the invention comprises the following stages:

a) extraction from a raw material obtained by the cultivation of mushroom producer lovastatin;

b) concentrating the extract;

c) lactonization in the absence of solvent;

d) two-stage cleaning from impurities, namely washing with an organic solvent or mixture of organic solvents and bleaching;

e) crystallization of the desired product.

In private cases, implementing the inventive method as a mushroom producer can be used strains of Aspergillus terreus, Aspergillus orysae or Aspergillus obscurus, preferably Asp. terreus No. 44-62 (deposited in the collection of the company Medinan, Littoinen, Finland); raw material obtained by the cultivation of the fungus producing the, can serve as a culture fluid, filtrate or mycelium.

Extraction of lovastatin, it is advisable to carry out an organic solvent after adjusting the pH of the culture fluid to a value in the range of 2-5 using hydrochloric, phosphoric or sulfuric acid or alkali. As the organic solvent, preferably ethyl acetate or butucea, and as the alkali is ammonium hydroxide. The concentration of the extract lovastatin preferably carried out under reduced pressure.

Lactonization acid form of lovastatin is preferably carried out in the temperature range of 70-75°With under reduced pressure in the absence of other reagents and/or components in the reaction medium.

However, this process can be carried out in a stream of nitrogen in the presence of dehydrating agents - salt or high-molecular solid dehydrating adsorbents.

In the first stage of purification from impurities - washing with organic solvent can be used benzene or toluene, as well as mixtures of organic solvents is a mixture of nonpolar and polar solvent. Preferably used for this procedure, a mixture of hexane - ethyl acetate (3:1), petroleum ether - ethyl acetate (4:1), heptane - ethyl acetate (2:1), hexane - butyl acetate (3:1), petroleum ether - butyl acetate (4:1), hepta the - butyl acetate (2:1) and others

In the second stage of purification from impurities - lightening is advisable to use as adsorbent alumina or activated carbon or other adsorbent in series.

Crystallization of the desired product can be made from ethanol or aqueous solution.

The technical results of the claimed invention in terms of the way lactonization acid form of the statin are:

- the almost complete absence in the product lactonization impurities in the form of dimers, since the high viscosity of the reaction medium and crystallization from it lactones as their education prevents intermolecular interactions, leading to the formation of dimers (minimum dimers in the product lactonization is 0.13-0.17 wt.% to lovastatin or simvastatin, EP 1288212 A1 and 0.06-0.1 wt.% for simvastatin, US 6.562.984 B2);

the reaction until almost the end that provides 100%output lactone form of the statins due to serial shift the equilibrium towards the formation of lactone as its crystallization;

- obtain the lactone form of statins at the end of the process directly in the form of crystals, as a result it eliminates the need for separate and/or selection of the target product from the reaction mixture;

- cost reduction is the product of lactonization due to the exclusion of the cost of the solvents, catalysts, precipitators, dehydrating agents, etc. and their subsequent disposal and/or regeneration;

- industrial and environmental safety of the process due to the exclusion from it of organic solvents, acids, precipitators and the like, and high-temperature regimes.

Lactonization acid form of the statin according to the invention is carried out in the absence of solvent.

The process of lactonization statins preferably takes place in the temperature range of 60-80°With under reduced pressure in the absence of other reagents and/or components in the reaction medium. However, the claimed method lactonization can be carried out in a stream of nitrogen in the presence of dehydrating agents - salt or high-molecular solid dehydrating adsorbents.

Information confirming the possibility of inventions

EXAMPLE 1

In the example used a strain of Aspergillus terreus No. 44-62 (deposited in the collection of the company Medinan, Littoinen, Finland). Cultivation was performed in a bioreactor with a volume of 10 DM34.5 DM3the reaction medium containing carbohydrates, nitrogen sources (soy, oat flour, gelatin) and inorganic salts. The parameters of the process of cultivation: temperature 28°C, mixing of 3.6 to 13.3 sec-1the aeration of 0.13-0,90 vol/vol/min, the concentration of dissolved oxygen 30%. In PR is the process of fermentation with increasing viscosity of the culture fluid was performed supplements the sucrose solution and distilled water. The duration of the process is 180-240 hours, the Culture fluid at the end of the fermentation process contains 8.7 g/DM3lovastatin (the sum of the acid and lactone forms).

5 DM3the culture fluid containing 43,5 g of lovastatin, acidified with diluted phosphoric acid to pH 3.5 and incubated for 30 minutes Biomass is filtered off, washed with acidified water and wring out. Lovastatin twice extracted from the raw biomass with ethyl acetate. United an ethyl acetate extracts are concentrated by evaporation under reduced pressure. The solvent is distilled off, the residue is heated under reduced pressure and/or in a stream of nitrogen for 3 h at 70-75°for full lactonization acid form of lovastatin. The reaction mass is washed with 80 cm3a mixture of hexane - ethyl acetate in the ratio of 3:1 to remove lipids and non-polar impurities. The precipitate is filtered off and washed with 20 cm3this same mixture. Received technical lovastatin (34.4 g of 96% purity) dissolved in ethyl acetate and the solution is lighten, passing through the layer of neutral alumina (150 g) or activated charcoal (50 g), or both sequentially. Alumina and/or activated carbon is washed with ethyl acetate and the combined an ethyl acetate solution evaporated to dryness. Exhaust ethyl acetate regenerate and re-use for which straccia lovastatin.

Get 33 g of lovastatin 98% purity. The target product is recrystallized from aqueous ethanol and dried. The output of lovastatin is 70% (30,4 g), the content of the basic substance (HPLC) - 99,5%. The dimers are absent. The chromatographic mobility of the compounds on the plates Silufol (Czech Republic) in the system chloroform - acetone (3:1) Rf˜0,45. The structure of the selected substances - lovastatin confirmed by NMR spectroscopy (figure 1) and IR spectroscopy (figure 2).

In the ultraviolet region of the spectrum for a solution of the selected substance in methanol absorption maxima are observed at 246,0; 237,5 and 230,0 nm. For maximum 237,5 nm A1% in methanol is 594.

EXAMPLE 2

For biosynthesis of lovastatin used a strain of Aspergillus terreus No. 43-16 (deposited in the collection of the company Medinan, Littoinen, Finland). The culture fluid containing lovastatin, received in the bioreactor with a volume of 10 DM36 DM3environment similar to the environment in example 1. The fermentation process was performed with the following parameters: temperature 28°With a stirring 5,0-14,0 s-1the aeration of 0.16-10,8 vol/vol/min, the concentration of dissolved oxygen at 10%. In the fermentation process was added to the sucrose solution in the amount of 20% by volume environment. The duration of the process is 192 hours Culture liquid after the fermentation process contains 8.5 g/DM3lovastatin is a (the amount of the lactone and acid forms).

5,5 DM3the culture fluid containing 46,75 g of lovastatin, acidified with diluted hydrochloric acid to pH 4.0. The mycelium is filtered off, washed and squeezed. Lovastatin from the mycelium is extracted with chloroform. The chloroform solution is concentrated on a rotary evaporator. The chloroform is distilled off completely, and to the residue add a dehydrating agent is magnesium sulfate (7.5 g) or calcium chloride (6 g) and the resulting mixture is stirred, then heated at 70-75°for full lactonization acid form of lovastatin. Butter crystals are drained, and the residue is washed with a mixture of petroleum ether - ethyl acetate in the ratio of 4:1. Technical product is dissolved in chloroform, filtered and the filtrate lighten, passing through the layer of aluminum oxide or activated carbon. Aluminum oxide or activated carbon is washed with chloroform until the complete washout of lovastatin. The chloroform solutions are combined, the solvent is distilled off. The residue is washed with a small amount of hexane, recrystallized from ethanol and dried. The output of lovastatin is 55% (25,7 g) with a basic substance content of 99% (HPLC). The dimers do not exceed 0.01%. The structure of the selected substances - lovastatin confirmed as described in example 1.

EXAMPLE 3

In the example used a strain of Aspergillus terreus No. 44-62 (deposited in the collection of the company IU kinen, Littoinen, Finland). Cultivation was performed in a bioreactor with a volume of 250 DM3with 120 DM3the environment is identical to the composition of the medium of example 1. The parameters of the fermentation process: temperature 28°C, mixing the 1.6-7.5 sec-1the aeration of 0.08 to 1.2 vol/vol/min, the concentration of dissolved oxygen 50%. During the fermentation process conducted supplements the sucrose solution and distilled water, the total amount of additives was 60 DM3. The duration of the process is 216 hours the Culture fluid at the end of the process contains 5,1 g/DM3lovastatina.

8 DM3the culture liquid is acidified with dilute sulfuric acid to pH 2-3 and incubated for 1 h Biomass is filtered off and washed on the filter with acidified water. Get 2 kg of raw biomass, containing 40 g of lovastatin. Lovastatin twice extracted from the biomass with ethyl acetate. The extract was concentrated on a rotary evaporator to complete the distillation of ethyl acetate. The residue is heated at 70-75°C for 3 hours, the Crystals are washed with a mixture of heptane - ethyl acetate 2:1 ratio. Technical lovastatin dissolved in ethyl acetate and the resulting solution is lighten, stirring 30 min with 100 g of aluminum oxide and filtering through a layer of aluminum oxide. The clarified solution is evaporated to dryness. The residue is recrystallized from aqueous alcohol. Get to 25.2 g of lovastatin what is 63%. The purity 99% (HPLC). The dimers are absent. The structure of the selected substances - lovastatin confirmed as described in example 1.

EXAMPLE 4

In the example used a strain of Aspergillus oryzae ATCC 74135.

Cultivation was performed in a bioreactor with a volume of 10 DM36 DM3environment similar to the environment in example 1 under the conditions shown in example 2.

The obtained culture liquid (5 l)containing 1.3 g/DM3lovastatin (the sum of the acid and lactone forms) in the filtrate and 0.12 g/DM3in mycelium, filtered. The mycelium was removed, and to the filtrate add 2 liters of butyl acetate and extracted with lovastatin for 1 h, the Extract is cooled, then on a rotary evaporator, the solvent is distilled off completely under reduced pressure. The residue is heated for 3 hours in a stream of nitrogen at 75°C. the Fluid is drained and the remaining crystals are washed with a mixture of cyclohexane and benzene (10:1). Next, the washed crystals are processed as described in example 1.

The output of lovastatin in the lactone form is 3.2 g (49%) with a purity of 98.7 per cent (HPLC). The dimers are absent. The structure of the selected substances - lovastatin confirmed as described in example 1.

EXAMPLE 5

In the example used laboratory strain of Aspergillus obscums VJ-1.

The process of biosynthesis of lovastatin were performed as described in example 1. 5 DM3the culture fluid containing 6.5 g lovastatin is a, acidified with H3PO4to pH 3-5, incubated 30 min and implement a dual extraction of lovastatin toluene wrung from raw biomass.

Next, the solvent from the extract is distilled off completely and the residue is heated for 6 h at 60°With under reduced pressure.

From the remainder of the separated liquid phase and the crystals washed with a mixture of toluene-petroleum ether (1:10).

The washed crystals are processed as described in example 1.

The output of lovastatin was 5 g (77%) with a purity of 98% (HPLC). The dimers do not exceed 0.01%.

The structure of the selected substances - lovastatin confirmed as described in example 1.

EXAMPLE 6

In the example used a strain of Aspergillus terreus No. 44-62 (deposited in the collection of the company Medinan, Littoinen, Finland). Cultivation was performed in a bioreactor with a volume of 10 DM36 DM3environment similar to the environment in example 1 under the conditions shown in example 2.

The culture fluid contained 8.5 g/DM3lovastatin (the amount of the lactone and acid forms).

To 5.5 DM3the culture fluid containing 46,75 g of lovastatin, add 0,5 DM3a 10%aqueous solution of NH3and extracted for 3 h at room temperature. Then the biomass is filtered off, washed with 0.5%aqueous solution of NH3and squeezed.

Extract and rinse unite and concentrate what parinayam under reduced pressure to remove water.

The residue is heated under reduced pressure at 70-80°C for 3-4 hours for a full lactonization lovastatin.

Liquid crystals merge, and the crystalline residue is processed as described in example 1.

The output of lovastatin is 80% (or 37.4 g) with a basic substance content of 98.5% (HPLC). The dimers are absent.

The structure of the selected substances - lovastatin confirmed as described in example 1.

EXAMPLE 7

A chloroform solution containing lovastatin or simvastatin in acid form, concentrated in vacuo at 30-40°C. the Solvent is distilled off completely.

The residue is heated under reduced pressure or in a stream of nitrogen at 70-75°for 2.5 hours During this time the acid is completely converted into the lactone (control by TLC on plates of silica gel in the system chloroform - acetone 3:1; Rfthe lactone 0,45-0,50; Rfacid 0,15-0,20). The dimer formation is not more than 0.01% (HPLC).

The lactones of statins at the end of the process get in crystalline form.

EXAMPLE 8

An ethyl acetate solution containing lovastatin or simvastatin in acid form, concentrated in vacuo at 40-45°C. the Solvent is distilled off completely and the residue is heated under reduced pressure and/or in a stream of nitrogen at 65-70°C for 3 hours, the Acid is completely transformed into the lactone (control by TLC, as described in the example ). The formation of dimers is not observed (HPLC). The lactones of statins at the end of the process get in crystalline form.

EXAMPLE 9

2 g of ammonium salt of lovastatin or simvastatin (99% purity, HPLC) is heated under reduced pressure and/or in a stream of nitrogen at 70 to 80°C for 3-4 hours

At the end of the process get to 1.82 g of lovastatin or of 1.83 g of simvastatin in the lactone form in crystalline form with a purity of 99% (HPLC).

The admixture of the acid form of the statin, and dimers are absent (control by TLC as described in example 7; HPLC).

EXAMPLE 10

2 g of ammonium salt of lovastatin or simvastatin (95% purity, HPLC) is heated at 70-80°C for 3-4 h in the presence of dehydrating agent is magnesium sulfate (0.4 g) or sodium sulfate (0.4 g) or calcium chloride (0.3 g).

The reaction mass is recrystallized from ethanol. Get to 1.38 g of lovastatin or 1.40 g of simvastatin in the lactone form with a purity of 99.9% (HPLC).

The formation of dimers is not observed (HPLC), the acid form is not (control by TLC).

1. The strain of the fungus Aspergillus terreus No. 44-62 (deposited in the collection of the firm Metkinen Oy, Littoinen, Finland) - producer of lovastatin.

2. The allocation method lovastatin, including extraction from a raw material obtained by the cultivation of mushroom producer of lovastatin, the concentration of the extract, lactonization acid form of lovastatin and subsequent two-stage cleaning from impurities and crystallization of the desired product, characterized in that lactonization performed in the absence of solvent, and the second purification stage - lighting - carried out after washing with an organic solvent or mixture of organic solvents.

3. The method according to claim 2, characterized in that the raw material obtained by the cultivation of mushroom producer of lovastatin, use the culture fluid, filtrate or mycelium.

4. The method according to any of claim 2 and 3, characterized in that as a producer of lovastatin use fungi of the genus Aspergillus.

5. The method according to any of claim 2 to 4, characterized in that as a producer use fungi belonging to the species Aspergillus terreus or Aspergillus orysae, or Aspergillus obscurus.

6. The method according to claim 5, characterized in that as a producer of lovastatin use a strain of the fungus Aspergillus terreus No. 44-62 (deposited in the collection of the firm Metkinen Oy, Littoinen, Finland).

7. The method according to claim 2, characterized in that the extraction of lovastatin carried out with an organic solvent after adjusting the pH of the culture fluid to a value in the range of 2-5.

8. The method according to claim 7, characterized in that in order to bring the pH of the culture fluid to a value in the range of 2-5 use of mineral acid selected from the group comprising phosphoric, hydrochloric and sulfuric acid.

9. The method according to claim 7, characterized in that the extraction of lovastatin carry out organic races what voditelem, selected from the group comprising esters of organic acids, aromatic and chlorinated hydrocarbons.

10. The method according to claim 9, characterized in that as esters of organic acids using ethyl acetate, butyl acetate or methyl acetate, as the aromatic hydrocarbon is toluene, as well as chlorinated hydrocarbons (chloroform, methylene chloride or dichloroethane.

11. The method according to claim 2, characterized in that the extraction of lovastatin carry out alkali.

12. The method according to claim 11, characterized in that as the use of alkali hydroxide ammonium.

13. The method according to claim 2, characterized in that the concentration of the extract lovastatin carried out at reduced pressure.

14. The method according to claim 2, characterized in that lactonization carried out in the temperature range 60 - 80°C.

15. The method according to any of claim 2 and 14, characterized in that lactonization carried out under reduced pressure.

16. The method according to any of claim 2 and 14, characterized in that lactonization carried out in a stream of nitrogen.

17. The method according to item 15, wherein lactonization carried out in a stream of nitrogen.

18. The method according to any of claim 2 and 14, characterized in that lactonization carried out in the presence of a dehydrating agent.

19. The method according to item 15, wherein lactonization carried out in the presence of a dehydrating agent.

20. The method according to item 16, characterized in that lactonization carried out in the presence of a dehydrating agent.

21. The method according to p, characterized in that as a dehydrating agent using agent selected from the group comprising magnesium sulfate, sodium sulfate, calcium chloride and high-molecular solid dehydrating adsorbent and a combination of both.

22. The method according to claim 19, characterized in that as a dehydrating agent using agent selected from the group comprising magnesium sulfate, sodium sulfate, calcium chloride and high-molecular solid dehydrating adsorbent and a combination of both.

23. The method according to claim 20, characterized in that as a dehydrating agent using agent selected from the group comprising magnesium sulfate, sodium sulfate, calcium chloride and high-molecular solid dehydrating adsorbent and a combination of both.

24. The method according to any of PP-23, characterized in that the high molecular solid dehydrating adsorbent using silica gel, Sephadex or molecular sieve.

25. The method according to any of claim 2 and 14, characterized in that lactonization lovastatin carried out in the absence of other reagents and/or components in the reaction medium.

26. The method according to item 15, wherein lactonization lovastatin carried out in the absence of other regentova/or components in the reaction medium.

27. The method according to claim 2, characterized in that the bleaching is carried alumina or activated carbon, or both sequentially.

28. The method according to claim 2, characterized in that washing with organic solvent used toluene or benzene.

29. The method according to claim 2, characterized in that for washing as a mixture of organic solvents, a mixture of nonpolar and polar solvent.

30. The method according to clause 29, characterized in that as a non-polar solvent used solvent selected from the group comprising petroleum ether, heptane and hexane.

31. The method according to clause 29, characterized in that the polar solvent used solvent selected from the group comprising ethyl acetate, acetone, butyl acetate, chloroform, dichloroethane and methylene chloride.

32. The method according to claim 2, characterized in that the crystallization of the desired product is carried out on ethanol or aqueous solution.

33. The way to obtain the lactone form of the statin of formula I

in which R represents a hydrogen atom or lower alkyl, which lactonization their acid forms of formula II

in which X represents a hydrogen atom, a metal cation or ammonium cation, and R is specified is use, characterized in that the process is conducted in the absence of solvent.

34. The method according to p, characterized in that a compound of formula I is a lactone form of lovastatin or simvastatin.

35. The method according to p, characterized in that a compound of formula II is an acid form of lovastatin or simvastatin, or their ammonium salts.

36. The method according to p, characterized in that the process is conducted in the temperature range 60 - 80°C.

37. The method according to any of PP and 36, characterized in that the process is conducted under reduced pressure.

38. The method according to any of PP and 36, characterized in that the process is conducted in a stream of nitrogen.

39. The method according to clause 37, characterized in that the process is conducted in a stream of nitrogen.

40. The method according to any of PP and 36, characterized in that the process is conducted in the presence of a dehydrating agent.

41. The method according to clause 37, characterized in that the process is conducted in the presence of a dehydrating agent.

42. The method according to § 38, characterized in that the process is conducted in the presence of a dehydrating agent.

43. The method according to p, characterized by the fact that as a dehydrating agent using agent selected from the group comprising magnesium sulfate, sodium sulfate, calcium chloride and high-molecular solid dehydrating adsorbent and a combination of both.

44. The method according to paragraph 41, characterized the clinical topics as a dehydrating agent using agent selected from the group comprising magnesium sulfate, sodium sulfate, calcium chloride and high-molecular solid dehydrating adsorbent and a combination of both.

45. The method according to § 42, characterized in that as a dehydrating agent using agent selected from the group comprising magnesium sulfate, sodium sulfate, calcium chloride and high-molecular solid dehydrating adsorbent and a combination of both.

46. The method according to any of PP-45, characterized in that the high molecular solid dehydrating adsorbent using silica gel, Sephadex or molecular sieve.

47. The method according to any of PP and 36, characterized in that the process is conducted in the absence of other reagents and/or components in the reaction medium.

48. The method according to clause 37, characterized in that the process is conducted in the absence of other reagents and/or components in the reaction medium.



 

Same patents:

The invention relates to a method of transforming isoflavonoid conjugates in isoflavonones the aglycones by processing a mixture containing isoflavone conjugates and water at a pH of about 6-13,5, a temperature of about 2-121oWith over a period of time sufficient to convert most of the conjugates in isoflavone conjugates, then contact with the enzyme capable of decomposing due isoflavonoid glycoside with isoflavonoid the glikona at pH of about 3 to 9 and a temperature of about 5-75oWith over a period of time sufficient to convert these isoflavonoid of glikona in isoflavonones the aglycones, as well as way of turning isoflavonoid conjugates and isoflavonoid of glikona in isoflavonones the aglycones after removal isoflavonoid conjugates and isoflavonoid of glikona from material of vegetable protein

The invention relates to a new microbiological method for the terminal oxidation of the alkyl groups in the carboxyl group

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: the strain-producer is isolated from soft coral Sinularia sp. relating to species Aspergillus fumigatus and deposited in Collection of Marine Microorganisms of Pacific institute of bioorganic chemistry DVO RAN (KMM TIBOKH) at № 4631. Method for preparing indole alkaloids involves surface solid-phase culturing the indicated strain on nutrient medium, milling mycelium and medium, two-fold extraction of prepared mixture with system chloroform - ethanol (2:1), concentrating extract, its chromatography on column with silica gel and the following separation of indole alkaloids by HPLC method. Method based on applying this strain provides enhancing yield of indole alkaloids. Invention can be used in preparing indole alkaloids eliciting an anti-tumor activity.

EFFECT: improved method for preparing, valuable medicinal properties of indole alkaloids.

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FIELD: utilization and rational use of lignin-containing raw material; delignofication of vegetable raw material.

SUBSTANCE: proposed method includes grinding the vegetable raw material followed by ultrasound treatment at frequency of 22.0-24.0 kHz continued for 10-15 min and biological action on vegetable raw material by inoculate of fungus Panus tigrinus BKM F-3616 D continued for 9-14 days at temperature of +24 to +26°C.

EFFECT: facilitated process of bio-conversion; increased food value of vegetable raw material; possibility of utilizing lignin from ligno-cellulose wastes.

1 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to nematophage fungi strain Duddingtonia flagrans useful as base in biological preparations producing. Strain Duddingtonia flagrans F-882 has activity against plant gall nematode and animal parasitic nematode, and stimulates plant growth and development.

EFFECT: strain with high plant growth and development stimulating activity and nematophage effect.

6 tbl, 2 dwg, 7 ex

FIELD: food industry.

SUBSTANCE: the present innovation deals with obtaining gelling concentrate containing structure-forming agents of vegetable and microbial origin. Prepared sugar beet should be deodorated and sterilized with supercritical carbon dioxide followed by extracting residues at mixing at extractional volume water and liquid hydrogen fluoride. Then comes separation of culture liquid, its blending with juice, addition of liquid ammonia and supercritical CO2-extract into the blend out of Mortierella elongata micromycete biomass extracted then according to the preset technique to obtain solid residue treated with liquid ammonia followed by concentrating the blend, treating the concentrate with liquid carbon dioxide, mixing with treated solid residue of Mortierella elongata micromycete biomass and heating the mixture up to 60 C, not less. The innovation provides improved structure-forming capacity and increased thermal stability of gelling concentrate.

EFFECT: higher efficiency.

FIELD: food industry.

SUBSTANCE: the present innovation deals with obtaining gelling concentrate containing structure-forming agents of vegetable and microbial origin. Prepared sugar beet should be deodorated and sterilized with supercritical carbon dioxide followed by extracting residues at mixing at extractional volume water and liquid hydrogen chloride. Then comes separation of culture liquid, its blending with juice, addition of liquid ammonia and supercritical CO2-extract into the blend out of Mortierella reticulata micromycete biomass extracted then according to the preset technique to obtain solid residue treated with liquid ammonia followed by concentrating the blend, treating the concentrate with liquid carbon dioxide, mixing with treated solid residue of Mortierella reticulata micromycete biomass and heating the mixture up to 60 C, not less. The innovation provides improved structure-forming capacity and increased thermal stability of gelling concentrate.

EFFECT: higher efficiency.

FIELD: food industry.

SUBSTANCE: the present innovation deals with obtaining gelling concentrate containing structure-forming agents of vegetable and microbial origin. Prepared sugar beet should be deodorated and sterilized with supercritical carbon dioxide followed by extracting residues at mixing at extractional volume water and liquid hydrogen fluoride. Then comes separation of culture liquid, its blending with juice, addition of liquid ammonia and supercritical CO2-extract into the blend out of Mortierella sarnyensis micromycete biomass extracted then according to the preset technique to obtain solid residue treated with liquid ammonia followed by concentrating the blend, treating the concentrate with liquid carbon dioxide, mixing with treated solid residue of Mortierella sarnyensis micromycete biomass and heating the mixture up to 60 C, not less. The innovation provides improved structure-forming capacity and increased thermal stability of gelling concentrate.

EFFECT: higher efficiency.

FIELD: food industry.

SUBSTANCE: the present innovation deals with obtaining gelling concentrate containing structure-forming agents of vegetable and microbial origin. Prepared sugar beet should be deodorated and sterilized with supercritical carbon dioxide followed by extracting residues at mixing at extractional volume water and liquid hydrogen fluoride. Then comes separation of culture liquid, its blending with juice, addition of liquid ammonia and supercritical CO2-extract into the blend out of Mortierella indohii micromycete biomass extracted then according to the preset technique to obtain solid residue treated with liquid ammonia followed by concentrating the blend, treating the concentrate with liquid carbon dioxide, mixing with treated solid residue of Mortierella indohii micromycete biomass and heating the mixture up to 60 C, not less. The innovation provides improved structure-forming capacity and increased thermal stability of gelling concentrate.

EFFECT: higher efficiency.

FIELD: food industry, confectionary industry.

SUBSTANCE: the suggested concentrate should be prepared due to pressing the juice out of sugar beet prepared with deodorated carbon dioxide in supracritical state. Residues should undergo hydrolysis-extracting with hydrofluoric acid solution obtained directly in the mentioned process at separate supply of water and liquid hydrogen fluoride for this stage. Obtained extract without residues should be mixed with sugar beet juice and supplement the blend with liquid ammonia along with an extract obtained due to extracting Mortierella marburgensis micromycete biomass according to the preset technique. The concentrate should be treated with liquid carbon dioxide and supplemented with a solid residue obtained as a result of extracting Mortierella marburgensis micromycete and treated with liquid ammonia. The mixture should be heated up to 60 C, not less. The innovation provides increased output of the target product and improved structure-forming properties.

EFFECT: increased thermal stability.

FIELD: food industry.

SUBSTANCE: the present innovation deals with obtaining gelling concentrate containing structure-forming agents of vegetable and microbial origin. Prepared sugar beet should be deodorated and sterilized with supercritical carbon dioxide in the field of ultrasound fluctuations, juice pressing should be carried out under aseptic conditions followed by cultivation upon residues of mycellar fungi of Trichoderma and Aspergillus species of acetic acid fermentation. Then comes separation of culture liquid, its blending with juice, addition of liquid ammonia and supercritical CO2-extract into the blend out of Mortierella pusilla micromycete biomass extracted then according to the preset technique to obtain solid residue treated with liquid ammonia followed by concentrating the blend, treating the concentrate with liquid carbon dioxide, mixing with treated solid residue of Mortierella pusilla micromycete biomass and heating the mixture up to 60 C, not less. The innovation provides improved structure-forming capacity and increased thermal stability of gelling concentrate.

EFFECT: higher efficiency.

FIELD: food industry.

SUBSTANCE: the present innovation deals with obtaining gelling concentrate containing structure-forming agents of vegetable and microbial origin. Prepared sugar beet should be deodorated and sterilized with supercritical carbon dioxide in the field of ultrasound fluctuations, juice pressing should be carried out under aseptic conditions followed by cultivation upon residues of mycellar fungi of Trichoderma and Aspergillus species of acetic acid fermentation. Then comes separation of culture liquid, its blending with juice, addition of liquid ammonia and supercritical CO2-extract into the blend out of Mortierella alliaceae micromycete biomass extracted then according to the preset technique to obtain solid residue treated with liquid ammonia followed by concentrating the blend, treating the concentrate with liquid carbon dioxide, mixing with treated solid residue of Mortierella alliaceae micromycete biomass and heating the mixture up to 60 C, not less. The innovation provides improved structure-forming capacity and increased thermal stability of gelling concentrate.

EFFECT: higher efficiency.

New drug substances // 2237657
The invention relates to organic chemistry and can find application in medicine
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