SU1253432A3 - Method of producing oxyacids or lactones thereof - Google Patents

Description

The invention relates to a method for obtaining new medicinal products.

substances that have hypocholesteric activity, the general formulas

about

Shz

ABOUT

III

These compounds inhibit cholesterol biosynthesis and can be used for the treatment of atherosclerosis, hyperlipemia and other similar diseases, as well as anticoesional agents.

Hydroxy acids or their lactones are obtained by fermentation of the nutrient medium by the strain Aspergillus terreus ATCC No. 20542, followed by separation of the compounds from the fermentation mixture by solvent extraction and sequential chromatography.

The strain Aspergillus terreus (designated MF-4845) in the collection of cultures (Merck & Co company, Sc, Revay, New York) was permanently stored in the American Collection of Species of Cultures, 12301, Moon Park, Rockville, Meridpeed 20852 and received catalog number ATSS number 20542.

The morphological characteristics of microorganisms ATSS No. 20542 are similar to the morphological characteristics of the genus Aspergillus. As a result of comparison with known species, it was found that the strain belongs to the species Aspergillus terreus.

The cultivation of this strain in order to obtain new compounds is carried out in an aqueous medium, which is common for the production of other fermentation products. This medium contains the sources of carbon, nitrogen and inorganic

Kie salts, KOToi iie, are established by microgens, by organisms.

Carbohydrates, such as sugars, such as glucose, fructose, maltose, sucrose, xylose, mannitol, etc., starches, such as starch grains, such as oats, rice, corn starch, coarse corn flour, etc., can use, both individually and in combination. as sources of absorbed carbon in the nutrient medium. The exact amount of source or source of carbohydrates that is used in the medium depends in part on the other ingredients contained in the medium, but in general, the amount of carbohydrates usually ranges from 1 to 6% by weight of the medium. Carbon sources can be used separately or several of these carbon sources are used in combination. Protein materials can be used as sources of nitrogen in the fermentation process. Relevant nitrogen sources include, for example, yeast hydrolysates, primary yeasts, coarse meal of soybeans, cotton seed flour, casein hydrolysates, saturated corn liqueur, soluble substances from a distiller or tomato paste, and so on. Nitrogen sources are used either individually or in combination in amounts of from 0.2 to 6 wt.% Based on the weight of the aqueous medium.

The nutrient inorganic salts that can be used in the medium for growing a culture refer to common salts containing sodium, potassium, ammonium, calcium, phosphate sulfate, chloride, carbonate, and other similar ions. They may also contain traces of metals such as cobalt, manganese, iron and magnesium.

Fermentation takes place at 20-37 seconds. However, in order to obtain optimal results, fermentation is carried out at 22 ° -EO ° C. The pH of the nutrient medium intended for the cultivation of Aspergillus and the preparation of new compounds can vary from 6.0 to 8.0.

Although new compounds are produced by the culture both on the surface and in the submerged state, the fermentation process is carried out in the submerged state. On a small scale, fermentation is generally carried out with the help of adding Aspergillus culture to the appropriate nutrient medium and then, after transferring the culture medium with the grafted culture to the production medium, the fermentation is carried out at a constant temperature of about 28 ° C for several days. with periodic shaking

Fermentation is initiated in a sterilized flask with medium using one or several stages of seed development. The nutrient medium for the seed stage can be any suitable combination of carbon and nitrogen sources. The flask with seeds is removed in a chamber at a constant temperature of about 28 ° C for two days with occasional shaking or until growth reaches a satisfactory level and part of the resulting second stage or graft production. The flasks with progeny of the intermediate stages, if used, are cultured in essentially the same way, i.e. a portion of the contents of the flask from the last progeny stage is used for grafting the production medium. Grafted flasks are shaken at a constant temperature for several days, and at the end of the incubation period the contents of the flasks

centrifuged or filtered.

For a larger scale, fermentation is carried out in a suitable tank equipped with a stirrer and means for aeration of the medium (11). According to the proposed method, the nutrient medium is loaded into the tank and sterilized by heating to about 120 ° C. After cooling, the sterilized medium is grafted the pre-purified seeds (offspring) of the producing culture and the fermentation process continues for, for example, 3-5 days. At the same time, stirring and / 1 nutrient medium aeration and temperature maintained at about 28 ° C. This method of biosynthesis of new compounds is intended, in particular, to produce large quantities.

The compounds are recovered in a known manner from the fermentation broth.

Compounds I and II can be hydrolyzed with bases such as NaOH to form salts such as the sodium salts of compounds III and IV. Using bases with other cations acceptable from a pharmaceutical point of view, salts of these cations can be obtained. Accurate acidification of the salts gives hydroxy acids III and IV, which can be converted to compounds I and II at acidic pH values. By treating compounds I and II with acidic or alkaline catalysis with metalmethanol, ethanol, propanol or butanol, or with phenyl, dimethylamino or acetylaminoalkanols, the corresponding esters of compounds III and IV are obtained.

Compounds III and IV (especially III) can be isolated in a known manner without using chromatography in the form of ammonium salts. This isolation method is more convenient and much more adapted for industrial use than chromatography. In addition, the salts of compounds III and IV are much more active than compounds I and II under actual conditions in order to inhibit the process of cholesterol biosynthesis and as anti-mold agents. In fact, hydroxy acids (and their salts) are active forms. Therefore, such salts are more preferable, in particular when used in the form of doses. In addition to ammonium salt; ammonium tetramethyl salts to ethylene diamine, sodium, potassium,, N-methyl glucose, lysine, arginine and ornithine are also salts. These compounds are pharmacologically active compounds and can be used as medicinal preparations in a stomatological or parenteral manner in the form of capsules, tablets, injections, and the like. In general, the dental method is used. Doses can vary depending on age, degree of disease, body weight and other human factors, but the daily dose for an adult patient contains 5: it is in the region from about 2 to 2000 mg (preferably 2-100 mg) 5 which can be divided into two to four separate doys. If necessary, you can use higher doses.

The compounds can also be used as anti-gel agents.

Example 1. Fermentation. Tube with lyophilized culture. Aspergillus tsrreus.ATCC No. 20542 is opened under sterile conditions and the suspension is suspended in a 250 m Erlenmeyer flask without a septum (I1 seed flask) containing 40 ml of medium C, which has the following composition, g:

Saturated corn liqueur 5 Tomato paste 40 Oats for coarse flour 10 Glucose 10

Mixture media ff 2 10 Distilled water 1000 ml pH 6.8 with NaOH. The flask with the grafted cult foy is incubated for 24-48 hours at 28 ° C on a vibrator, making 220 col./min (span or amplitude 5.08 cm). A portion (approximately 0.5 ml) of the contents of this flask is then used to graft a culture in an inclined tube containing medium E, Medium E has the following composition, g: Yeast extract 4 Malt extract 10

,

253432

ten

Dextrose 4

Agar 20

Distilled water 1000 ml

pH 7.0 with NaOH.

The inclined graft culture tube is incubated for 11 days at room temperature. It is then stored for 3-4 months. A portion of the contents of this tube is then suspended in a 250-mid-epilithic Erlenmayer flask without a septum (seed flask 2) containing 40 ml of medium C. The culture flask is incubated for 24 hours at 28 ° C on a vibrator with a 220 count. / min (span, 5.08 cm). Six Zllenmayer flasks with a capacity of 250 ml without a septum containing 40 ml of medium C are prepared. A culture is then grafted into each flask with 2 ml per flask of the contents of the seed flask 2. These six flasks are incubated for 48 hours at 28 ° C on a vibrator that performs 220 col./min (span 5.08 cm). Then, they take six two-liter Erlenmeyer flasks containing medium F in a volume of 500 ml, and a culture is established in each flask with help and maintenance of seed flask 3. Medium F has the following state

25

thirty

Rich Corn 15.

Krahma-l СРС 20

Maize flour 1

Crushed soybeans 4

Hl; okoza 5

Soybean oil 2.5

(Prince)

KN, RO

., / .. so,

0.3

6

12 .

. CaCO3, Cystriched water 1000 ml

Ji with HELP NaOH.

Graft culture flasks are incubated for 11 days. without stirring at 28 ° C. After incubation for 11 days. the broth is subjected to extraction.

Extraction

The entire broth pH 6.0 is mixed in a Waring blender in order to break the heavy layer of the mycelium, centrifuged, and the transparent upper layer is decanted. After filtration, the filtrate is extracted with a help of 3 l of ethyl acetate, resulting in 1820 ml of clear extract. Second extracts

7

Dosing with 3 L of ethyl acetate gives 3350 M; I clear extract. The broth solids are extracted by stirring for 1 hour using 2 liters of methanol and, after filtration, 2100 ml of filtrate is obtained.

Portions of these extracts are dried and sent for analysis. The filtrates are tested as HMG-GoA reductase enzyme inhibitors by the method described by Beg, Stonik and Brever. The enzymes obtained according to the description given by Kleinzek, Rangatam and Porter are used. The positive test — inhibited is more than 90% at 20 µg / ml. ICg is 2.3 µg / ml, which indicates the presence of a very strong inhibitor of cholesterol synthesis acting on the HliG-CoA reductase content.

The results of the analysis of the extract are given in table.1. about.

Table

Gel filtration. All solid material obtained from the first two extracts is combined together, dissolved in methanol and filtered to remove insoluble solids, 30 ml of the filtrates are loaded into a gel filtration column.

(2, 200 cm, 980 ml), filled with Sephadex LH-20j material and the sample is fractionated according to the size of the molecules with methanol, which is used as a solvent. Using the refractive index and UV analysis, the best fractions are determined by bio-assay (see table 2).

table 2

20

25

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Isolation and purification. A sample from fraction 2 is pre-filtered through a layer of 1 g of material from Bondapac C18 / Porosil 5 B and eluted with five volumes of methanol. The methanol eluate is concentrated to 0.5 ml. This sample was subjected to chromatography several times on a Waters C 18 material, 10 of which a column was made (3.9 mm 20 cm), and a mixture of methanol was used as a developing solvent: a 0.05 M ammonium phosphate solution, pH 2.9, (75:25). 15 The fractions are examined on a Beckmann spectrophotometer and those fractions for which the maximum absorption is corresponding. 236 nm with bends at 229 and 245 nm, combined and concentrated under reduced pressure to an aqueous solution. The pH of the concentrate is adjusted to 6.5 with a 2 M solution of potassium hydroxide and the active components are extracted with ethyl acetate. The organic layer is dried, concentrated to dryness and the residue is dissolved in 0.3 ml of methanol. The methanol solution undergoes chromatography and is recycled. The fractions containing the previously eluted component are combined, concentrated to an aqueous solution, and extracted with chloroform. The residue from chloroform is placed in methanol and the solvent is evaporated under nitrogen atmosphere. 3.5 mg of the dried product thus obtained are identified as hydroxy acid (compound III). The fractions containing the second component are combined and extracted with chloroform. 0.87 mg of dried product was obtained, which was identified as a lactone (compound I).

45 The physicochemical properties of Compound I (MSD-803) are as follows: mp. 170-171 ° C., molecular weight (mass spectroscopy) 404; the formula is C HjgOs (found using 405.555 mass spectroscopy, calculated 404, 2563), UV spectrum (in acetonitrile), nm: 230.5 with Е% 505.7; 237.5 with E% 576.6, 246 with E% 395.2.

 5imp chemical shifts. Spectrum 5 was obtained in a CDCl solution (20.1 mg in 0.35 ml). Chemical shifts are given relative to internal tetramethylsilane at zero ppm, with

thirty

35

40

Experimental conditions A solvent pulse (CDCl) takes place in the region of 70.0 ppm (parts per million). 24 carbon atoms were found, which is consistent with the data of mass spectroscopy, they have the following chemical shifts, ppm: 11.5; 13.6; 16.0, 22.6; 24.1; 26.6; 27.2; 30.5; 32.5; 32.8; 35.9; 36.4; 37.1; 38.4; 41.3;

62.4; 67.8; 76.4; 128.4; 129.7;

131.7; 133.2; 170.8 and 177.2.

Optical rotation.

Characteristic optical rotation td. 320.7 is determined on a solution of 5.30 mg / ml CHjCN. This value is obtained by measuring the sodium-D line wavelength.

Based on these and other data, it can be assumed that the product has the following chemical stereostructure

ct

/BUT/

Shz

The corresponding hydroxy acid dilination III) has the structure

 P

GHs.i sns 1n.n

Example 2. A tube with the lyophilized culture of Aspergillus terreus ATCC No. 20542 is opened under sterile conditions and the contents are suspended in a 250 ml Erlenmeyer flask without partitions (seed flask) containing medium C. The flask with the grafted culture is incubated for 30 hours with on the vibrator, making 220 col./min (span 4.08 cm). A 250 ml Erlenmeyer flask without a septum containing 40 ml of medium G is inoculated with a culture using 2 ml per flask of the contents of a replacement flask. Wednesday G has the following composition.

Dextrose 45 Petonized milk 24 Autolyzed yeast 2.5 Polygolicol P2000 2.5 ml Distilled water 1000 ml pH 7.0 with NaOH.

The flask with the Grafted culture is incubated for 120 hours at 28 ° C on a vibrator, making 220 col./min g (span 5.08 cm). After 120 hours of incubation, the contents of the flask are extracted in accordance with the procedure of Example 1. The total capacity is 21 5 500 U / ml.

Example 3. Fermentation The ATCC f 20542 lyophilized culture test tube is opened under sterile conditions and the contents suspended in a 250 ml Erlenmeyer flask without a septum (seed flask) containing approximately 10 ml of medium which has the following composition, g

Rich corn liquor 5 5 Tomato paste. 40 Oats on wholemeal flour. 10 Glucose10 Trace trace solution 10 0 Distilled water 1000 ml. pH 6.8 is provided with NaOH.

The trace element, mg: FeSO -7H201000

MnSO, 4H, 0 1000

25 100 56

(NH) g Mo. 4Н, о: 19 Q ZnSO -7H20200

Distilled water (de-ionized) 1000 m

The flask with the grafted culture is incubated for 24 hours at 28 ° C on a vibrator making 220 col./min (span 5, 08 cm). Then, in a two-liter Erlenmeyer flask without a septum containing 500 ml of medium, the culture is grafted with 10 ml of the contents of the first stage of fermentation from seed mixture. This flask is also on the vibrator for 24 hours at 28 ° C.

A fermentation tank of 200 gallon (757 l) stainless steel is loaded with 485 l of cellulose containing 4.5% w / v, peptonicized milk, 2.5% w / v, auto5

CuClj 2H20 CaClj 2H20 NWO3

eleven

yeast 0.25 w / v., polyglycol - R2000 0.25% v / v. The pH of the medium is adjusted to 7.0. Content is sterilized for 15 minutes at 121 ° C. Then one liter of contents from the second stage is poured into the tank and the mixture is incubated on a vibrator, performing 85 col./min, for 12 hours, then on a vibrator, performing 130 col./min. For 84 hours at 28 ° C with air supply at a rate of 5 0.142 for 12 hours, and then at a rate of 10 0.34 for 84 hours

Extraction

The two simultaneously loaded portions are combined in one hundred gallon (378 L) of broth, acidified with stirring to pH 4.1 with the careful addition of 800 ml of concentrated hydrochloric acid and extracted with 75 gallon (284 L) of ethyl acetate, then stirring is continued for 2 hours

Approximately 25 feet (11.34 mkg) of siliceous auxiliary filtering material is then added and the entire slurry is passed through a 61 cm filter press. An additional 75 gallons (284 L) of ethyl acetate is used to rinse the compressed cake and continue extraction with noMoopi reversal of the direction of flow of ethyl acetate through the press. This procedure is carried out four times. Then all of the washing solvent is drained from the press and combined with the first filtrate. The biphasic mixture (filtrate) is allowed to stand and the aqueous layer is removed. The ethyl acetate layer is washed, using 10 gallons (37.85 l) of deionized water, the phases are allowed to separate and the ethyl acetate extracts are concentrated under a vacuum to about 10 gals (37.85 l).

Lactonization.

In order to cyclize any compound IV in the extract to compound II, the following azeotropic treatment is carried out.

Extracts of ethyl acetate from additional: 300 gallons (1135.5 L) of broth are added to the extract and the volume is reduced to about 30 gallons (113.5 L) of broth is added to the extract and the volume is reduced to about 30 gals (113.5 l) at vacuum care

33432. 2

distillation Approximately 50 gallons (189-l) of toluene is added and the contents are concentrated under vacuum to a volume of 32 gals (121 liters), this step 5 is repeated. A sufficient amount of new toluene is then added to bring the volume to 75 gallons (284 liters). Without the use of vacuum, the contents are brought to reflux and this state is maintained for 2 hours at a temperature higher.

This solution is further concentrated under vacuum to a small 15 volume, which is then concentrated to an oily residue in a large rotary evaporator under vacuum. Chromatography on silica gel. The extracts obtained are freed from other solvents by adding 2 gals (7.5 liters) of methylene chloride and repeated concentration to an oil.

The oily residue is dissolved in approximately 5 gal (18.9 l) of this mixture — 5 acetate-methylene chloride d (30/70 v / v), and then the slurry is prepared by adding 2.8 kg of silica gel.

The slurry is applied evenly on the upper part of the Clichagel Q column with dimensions of 30.5 cm 127 cm, filled with the same mixture of solvents.

Elution is performed with a mixture of ethyl acetate-methylene chloride (40/60 v / v) at a rate of 800 ml / min.

5 The head run is 10 gallons (37.85 L), then fractions of 4 gals each each (15.141 L) are collected. Fractions 6 to 10 inclusively are concentrated under vacuum to an oily residue, which dissolves in hot ethyl acetate, is treated with bleached carbon, filtered while hot and then cooled. The MSD 803 crystals (compound I) are isolated by filtration, and the mother liquors are concentrated to an oil and subjected to chromatography.

Repeat chromatography on silica gel.

Residual stock solutions from one and the same broth extract, equivalent in performance to an additional 600 gallons (2271 l)

5 for fermentation, combined according to the methylene chloride solution. Half of this solution is used for far

13

chromatography on silica gel. A small portion study indicates that the total solids content is 325 g. The solution is treated with 40 g of bleached carbon, filtered, and the cake is washed with methylene chloride. The combined filtrate and wash solutions are concentrated in vacuo to an oily residue. The residue is redissolved in 800 ml of a mixture of ethyl acetate / methylene chloride (30-70 v / v,) and the solution is converted to the amm by adding 225 g of silica gel. Ilamm is loaded into the upper part of a layer of silica gel with columns of 14–35 cm filled with the same mixture of solvents. The test is carried out with a mixture of ethyl acetate-methylene chloride (40 / -60 v / v), a head run of 3 liters is discharged, and then fractions of 800 ml each are collected.

Chromatography with reversed-phase filling.

40 ml of the 12th fraction of the chromatographic procedure is concentrated to an oil weighing 500 mg and then the oil is redissolved in 5 ml of acetonitrile. This acetonitrile solution is loaded into a chromatographic column of stainless steel with dimensions: outer diameter 1.59 cm, length 1.8 m, filled with preparative reversed phase liquid for chromatography columns Bondapak C 18 / Po razili B, Column: zeriurat with a mixture containing 55% v / v acetonitrile and 45% and 0.05 M ammonium phosphate solution, pH 3, the volume of zylyuvaniya between 1360 and 1700 ml is collected on the basis of the study of the refractive index. The organic solvent is removed. 1 and the residual aqueous solution is extracted with ethyl acetate. After removal under vacuum. 120 mg of the compound, which is crystallized from a concentrated acetonitrile solution, remains in mind, resulting in the formation of MSD 883 crystals (compound II), m.p. 129-131 C, molecule weight 406, found by spectroscopy 406, 2706, calculated 406, 2719.

Optical rotation.

Characteristic optical rotation

p.3 148.6 ° determined on solution32

14

pe 5.23 mg / ml CH, CH. This value is obtained by measuring the sodium-D line wavelength.

 with NMR chemical shift (CDClj), ppm:

11.8i 14.9; 16.5; 21.1; 23,

26.7 (2C); 30.9; 31.3; 33.0; 35.7; 35.9; 37.4; 48.5; 38.6; 41.9 (2C);

62.3; 70.1 / 76.5; 131.0; 132.6,

171.2 and 176.7,

Based on these and other data, it can be assumed that the product has the following chemical stereostructure

BUT

 v

about

° 44

SNS

SNS N

The corresponding hydroxy acids (compound IV) then have the structure

BUT,

BUT

  H

UHJ H / LH

- CHv

Example 4 Inhibition in vitro of HMG coenzyme A reductase

A modified Bega method is used. The modification consists in incubating the enzyme with the inhibitor for 5 min before initiating the reaction with the substrate. When working with such strong inhibitors as the new compounds, the standard procedure, according to which the enzyme is added only to the inhibitor-substrate mixture, gives a non-linear kinetics.

Using a modified procedure, the sodium salt of compound IV gives an ICgo index of inhibition of HMG-CoA reductase equal to 2, compared to 5.4 days ML 236 B.

g 9

10 And for the combination of in-situ inhibition of cholesterol synthesis (compound II).

15

Several groups of male Holtzmann rats are injected with either 5% emulphor in brine or the test compound in the emulphor through a tube inserted into the stomach. After 1 h, VP 80 NCi C acetate / / kg is added. Then, after 50 minutes, he produces blood samples from rats and the content of C cholesterol is determined, as is the IAI cholesterol synthesis:

Dose, mg / kg inhibition,% 38 51 70

5. Comparison of compounds I, II and ML-236B, as inhibitors of cholesterol synthesis in cell culture.

A modified A.U. procedure is used. Adberts et al. For measuring the amount of acetic acid cholesterol biosynthesized from mice cells

0.15 0.6 1: 2 EXAMPLE

Editor N. Bobkov Order 4635/60

Compiled by V. Romanov

Tehred V.KadarKorrektor-M. Pojo

Circulation 490 Subscription

VNIIPI USSR State Committee

for inventions and discoveries 113035, Moscow, Zh-ZZ Raushsk cab., d, 4/5

Production and Printing Department, Uzhgorod, st. Project, 4

sixteen

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L-M in culture. The test compound in 10 µl of dimethyl sulfoxide is added to monolayer cultures with

5 With acetate. After 3 hours of incubation, the cells are oiled and C, the cholesterol is extracted and matched by thin layer chromatography on silica gel using a mixture of petroleum ether - diethyl ethyl acetic acid

acid (75: 25: 1). The area on the swatch, containing one cholesterol, is determined using the residual spot on this area I,

15 and the contents of c are determined using a liquid scintillation counter.

Using this modified procedure, it is established that coupling II gives 1C to inhibit HMG-CoA reductase, equal to 17 da, compared to 22 nm for compound 1 and 46 nm for ML-236B.

Claims (1)

METHOD FOR PRODUCING OXYXIDES OR THEIR LACTONS of the general formulas of the compounds from the fermentation mixture by solvent extraction and sequential chromatography. > CH