CS221919B2 - Method of preparation of mixture of hypocholesteremic compounds - Google Patents

Description

The present invention relates to hypocholesteremic mold cultivation products of Aspergillus spp. More specifically, it relates to compounds of formulas I to IV

(II) (III)

HIM

COOH

OH, ch ₃

HIM

COOH

OH, CH ₃ (IV) and their pharmaceutically acceptable salts and alkyl esters or phenyl Nice, dimetylaminonebo ecetylaminosubstituovených alkyl esters of carboxylic acids. The present invention relates to a method of cultivating fungi and isolating compounds of the above structures from the environment. The novel compounds have excellent cholesterol biosynthesis inhibition properties and are useful against hypercholesteremia and hyperlipemia.

Due to the new link between high cholesterol and atherosclerosis, considerable efforts have been made to find ways to lower cholesterol levels in the mammalian body. One of these methods is to inhibit cholesterol synthesis in the mammalian body.

Recently, Endo et al., In U.S. Patent Nos. 4,049,495 and 3,983,140 described a fermentation product obtained by culturing a microorganism of the genus Penicillium and isolating it from the medium.

They called it ML 236B and determined its structure together with two related compounds 236A and 236 C. Their structure under the name compaction was also determined in A.G. Brown, T.C. Smale, T.J. King, J. Chem. Soc. (Perkin I) 1165 (1975). This compound has been found to be an inhibitor of in vivo cholesterol biosynthesis.

It has now been found that, quite unexpectedly, by cultivating a highly differentiated strain than Endo used, namely Aspergillus fungi, novel compounds are produced which are also very effective inhibitors of cholesterol biosynthesis in mammals. It has now been found that these compounds comprise essentially the novel compounds I, II, III and IV of the above structures accompanied by only traces of other compounds. These novel compounds are more potent inhibitors of cholesterol biosynthesis in vivo than ML 236B described by End.

Pharmaceutically acceptable salts of the invention include those with cations derived from sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, ammonia, ethylenediamine, N-methylglucamine, lysine, arginine, ornithine, choline, Ν, Ν'-dibenzylethylenediawine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, 1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole, diethylamine, piperazine, tris (hydroxymethyl) aminomethane and tetramethylammonium.

The compounds of the invention are useful as antihypercholesteremic agents for the treatment of atherosclerosis, hyperlipaemia and the like in humans. They can be administered orally or parenterally in the form of capsules, tablets, injectable preparations and the like. Usually oral administration is desirable. The doses may vary depending on the age, severity of the disease, body weight and other conditions of the patients. The daily dose for adults ranges from about 2 mg to 2000 mg (preferably 2 to 100 mg), which can be administered in two to four divided doses. If necessary, higher doses may be administered.

The compounds of the invention also have useful antifungal activity. For example, they can be used to control strains of Penicillium sp., Aspergillus niger, Cladosporium sp., Cochliobolus miyabeorus and Helminthosporium cynodnotis. For such use, they can be mixed with suitable agents used to prepare compositions such as powders, emulsifying agents or solvents such as aqueous ethanol and then sprayed or dusted onto the plants to be treated.

The present invention provides a process for the preparation of a mixture of hypocholesteremic compounds containing compounds of structural formulas I, II, III and IV

(II) (III)

HCkz-.

| COOH

CH characterized in that the nutrient medium containing assimilable sources of carbon, nitrogen and nutrient inorganic salts is incubated at 20-37 ° C and pH 6.0-8.0 with microorganisms of the genus Aspergillus terreus deposited with the American Type Culture Collection under Nos. 20541 or 20542 and the products are isolated.

Based on toxicology studies, this Aspergillus species has been isolated and identified as an unwritten microorganism and has been designated MF-4833 in the Merck and Co., Inc., Rahway, New Jersey culture collection, and a culture of this type was placed for permanent preservation in the American Type collection. Culture Collection, 12301 Parklawn Drive, Rockwille, Maryland 20852, and designated ATCC 20541. Another sample of the similar organism designated MK-4845 in the Merck Culture Collection was also placed for permanent storage under ATCC 20542. This latter organism provides better yields. Although the use of these microorganisms described herein is described in connection with the process of the invention ¹ , other Aspergillus organisms, including mutants, are also capable of producing novel compounds and can be used in the process of the invention.

The morphological characteristics of MF-4833 and MF-4845 are Aspergillus. Using the criteria set forth in the standard literature of the Aspergilli, Charles Thom and Kenneth B. Kasper, published by Williams at Wilkins Company, Baltimore, Lida, 1945, and compared to known species, both strains were found to be Aspergillus terreus.

The cultivation of these organisms for the production of the novel compounds is carried out in an aqueous medium such as those used for the production of other fermentation products. These media contain a source of carbon, nitrogen and inorganic salts assimilated by the microorganism.

Generally, sugars such as glucose, fructose, maltose, sucrose, xylose, mannitol, and the like, and starches such as grain starches such as oats, rice, corn, either alone or in combination with other assimilable carbon sources, may be used. . The exact amount of carbon source or sources used in the medium depends in part on the other components of the medium, generally the amount of carbohydrates is usually between about 1% and 6% by weight of the medium.

These carbon sources may be used individually or several may be mixed in the medium. In general, many proteinaceous materials can be used as nitrogen sources. Suitable nitrogen sources include, for example, yeast hydrolyzates, primary yeast, soy flour, cottonseed flour, casein hydrolyzates, corn leaches, distillation residues or tomato paste, and the like. Nitrogen sources may be used alone or in combination and are from about 0, 2% to 6% wt. aqueous medium.

Among the nutrient inorganic salts that may be added to the culture medium, salts capable of providing sodium, potassium, calcium ions, and ammonium, phosphate, sulfate, chloride, carbonate, etc. are also suitable. Trace metals such as cobalt, manganese, iron and magnesium.

It should be noted that the media described in the examples are merely examples of a variety of usable media and do not limit the present invention in any way. Namely, the carbon sources used in the nutrient medium for preparing the novel compounds of the invention are dextrose, dextrin, oatmeal, molasses, citrates, soybean oil, glycerol, malt extract, cod liver oil, starch, ethanol, figs, sodium ascorbate and lard. Peptoned milk, autolysed yeast, ribonucleic acids, yeast, tomato paste, casein, primary yeast, ground peanuts, distillation residues, corn extracts, soybean meal, cornmeal, NZ amine, beef extract, asparagine, cottonseed are used as nitrogen sources. seeds and ammonium sulfate. The main ionic components are calcium carbonate, KHgPO4, KgSO4. 7H ₂ O and NaCl and small amounts CaClg. 6H ₂ O and traces of iron, manganese, molybdenum, boron and copper.

The fermentation is carried out at a temperature in the range of 20 to 37 ° C, but for optimum results, it is preferable to carry out the fermentation at a temperature of from 22 to 30 ° C. The pH of the nutrient medium suitable for Aspergillus culture growth and for the production of novel compounds ranges from about 6.0 to 8.0.

Although the novel compounds are produced by both surface and submersible culture, it is preferred that the fermentation be carried out in the submersive state; The small fermentation is preferably carried out by seeding a suitable nutrient medium and carrying out the fermentation at a constant temperature of about 28 ° C for several days.

Fermentation is initiated in a sterile medium flask through one or more inoculation stages. the feed medium for the inoculation stages may comprise any suitable combination of nitrogen and carbon sources. The seed flasks are shaken for two days at a constant temperature at 28 ° C until growth is sufficient and a portion is used to inoculate the second seed stage or production medium. Intermediate flasks, if used, are incubated in the same manner, i.e. the number of flasks from the last inoculation stage is used to inoculate the production medium. The inoculated flasks were shaken for several days at constant temperature and, at the end of the incubation period, the contents of the flasks were centrifuged or filtered.

For large-scale production, it is advantageous to carry out the fermentation in suitable tanks with a stirrer and a device for aerating the fermentation medium. According to this method, the nutrient medium is processed in a tank and sterilized by heating to temperatures up to about 120 ° C. After cooling, the sterile medium is inoculated with a previously grown seed culture and the fermentation is carried out for a period of time, for example 3-5 days, while stirring and / or aerating the nutrient medium and maintaining the temperature at about 28 ° C. This method of producing the novel compounds is particularly suitable for the preparation of large quantities.

The compounds are preferably isolated from the fermentation medium in the form of lactones I and II. Alternatively, they may be isolated in the form of salts of compounds III and IV.

Compounds X and II can be hydrolyzed with bases such as NaOH to give sodium salts of compounds III and IV. Use of bases with other pharmaceutically acceptable cations provides salts of these cations. Careful acidification of the salts yields hydroxy acids III and IV. Hydroxy acids III and IV are converted to compounds I and II at acidic pH. Reaction of compounds I and II under acidic or basic conditions with methanol, ethanol, propanol or butanol or with phenyl, dinethylamino or acetylamino alkanols gives the corresponding esters of compounds III and IV, which are also part of the present invention.

The compounds III and IV and in particular III can advantageously be isolated without chromatography in the form of ammonium salts. This isolation is preferable to bulk chromatography.

Further, salts III and IV are more active than compounds I and II in vitro in inhibiting cholesterol biosynthesis and as a fungicidal agent. In fact, hydroxy acids (and their salts) are active forms. These salts are one of the particularly suitable dosage forms. In addition to the ammonium salts, preferred salts are the tetramethylammonium salt and salts with ethylenediamine, sodium, potassium, calcium, N-methylglucamine, lysine, arginine and ornithine.

The physicochemical properties of the compound of formula I (MSD-803) are as follows:

1. Melting point 170-171 ° C

2. Molecular weight (mass spectrum)

404

3. Formula (calculated by mass spectrometry)

404.2555

404.2563

221919 6

4. UV spectrum (in acetonitrile): maximum

230.5 nm with E% 505.7

237.5 nm with E * 576.6 246 nm with E96 395.2

5. ¹³ C NMR chemical shifts. The spectrum was recorded in a CDCl 3 solution (20.1 mg) in 0.35 mL) Chemical shifts are given relative to the internal standard, tetramethylsilane as zero ppm. Under experimental conditions, solvent (CDCl 3) exhibits a signal at 70.0 ppm. In accordance with the mass spectrum, spectral data for 24 carbons were found and their chemical shifts are as follows:

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 ppm.

6. 'H NMR Spectrum

The spectrum was scanned in a CDCl 3 solution and chemical shifts are shown in Figure 1 in ppm relative to the internal tetramethylsilane standard at zero ppm.

7. Infrared spectrum

The infrared spectrum was recorded in KBr microparticles. It is shown in Figure 2.

8. Optical rotation

The specific optical rotation [α] - 320.7 °, was determined in a solution of 5.30 mg / ml

CH 3 CN. This value was obtained when measuring the wavelength of the sodium D line.

Based on these and other data, the compound with a high degree of certainty corresponds to the stereochemical structure

The corresponding hydroxy acid III has the structure:

The absolute configuration of the centers of asymmetry in these molecules was determined by X-ray diffraction.

The physico-chemical properties of compound II (MSD-883) are summarized as follows: 1. Melting point 129-131 ° C

2, Molecular Weight 406

3. Formula ° 24 ^Η 38 ^θ 5 (not determined by mass spectrometry) 406,2706 calculated 406,2719

4. 1 H NMR spectrum

The spectrum was recorded in a CDCl 2 solution and the chemical shifts are shown in Figure 3 in ppm relative to the internal tetramethylsilane standard at zero ppm.

5. Infrared spectrum

The infrared spectrum was determined in KBr lozenges. It is shown in Figure 4,

6. Optical rotation

The specific optical rotation [α] = 148.6 °, was determined in a solution of 5.23 mg / ml CH 2 CN.

This value was obtained by measuring at the wavelength of the sodium D-line.

7. ³ C NMR chemical shifts in CDCl 3

11.8, 14.9, 16.5, 21.1, 23.1, 26.7 (20, 30.9, 31.3, 33.0, 35.7, 35.9, 37.4, 38.5, 38.6, 41.9 (20, 62.3, 70.1, 76.5, 131.0, 132.6, 171.2, 176.7.

Based on these and other data, it is assumed that the structure of the product with a high degree of certainty has a chemical structure

The corresponding hydroxy acid, compound IV, then has the structure:

The present invention is illustrated by the following examples.

Example 1

Preparation of compounds I and III

A. Fermentation

The lyophilized culture tube MF-4833 is opened under aseptic conditions and the contents are suspended in a 250 ml unobstructed Erlenmeyer flask (seed flask) containing about 20 ml of medium A. Medium A has the following composition:

and

Medium A: corn extracts 10 g tomato paste 80 g oatmeal 20 g glucose 20 g trace elements, mixture no. 2 20 g distilled water 000 ml pH 6.8 by addition of NaOH Trace Elements, Mix No. 2 FeSO ₄ . 7H ₂ O 1 000 mg MnS0 ₄ . 4H ₂ O 1 000 mg CuClg. 2H ₂ O 25 mg CaCl ₂ . 2HgO 100 ALIGN! mg h ₃ or ₃ 56 mg (NH ₄ ) ₆ Mo ₇ 0 ₂₄ . 4H ₂ O 19 Dec mg ZnS0 ₄ . 7H ₂ O 200 mg distilled deionized water 1 000 mg

The inoculated flask was incubated for 48 hours at 28 ° C on a 220 rpm shaker (5.1 cm shift). Two 2 liter septum flasks without baffles containing 500 ml of medium B are then inoculated with 10 ml per flask of the grown flask from the seed flask. Medium B has the following composition:

Medium B tomato paste 20 g primary yeast 10 g

CPC starch 20 g

CoCl ₂ . DHgO 5 mg distilled water 1000 ml pH 7.2 to 7.4 by addition of NaOH

The two inoculated flasks were incubated at 28 ° C for 96 hours. Incubate one flask without stirring. The second flask is incubated on a shaker at 150 rpm (5.1 cm shift). After 46 hours, the contents of each flask were separately processed and the product isolated.

B. Isolation of Compound I

The incubated medium is centrifuged for 20 to 30 minutes. The solids were extracted. The clear liquid (pH 6-8) was placed in a 950 mL flask and 150 mL of ZAD-2 resin was added. Using an automatic extractor operating according to a pre-set program, the mixture is stirred for 2 hours. The liquid is then drained, the resin is washed twice with 200 ml of deionized water and the wash solution is discarded. Then 300 ml of solvent: isopropanol, ethyl acetate dichloromethane 25:45:30 was added. The mixture was stirred for two hours. The solvent-resin suspension is filtered on a Buchner funnel or on a sintered glass funnel and the resin is discarded. The solids from the medium are mixed with 100 ml of acetone for 30 minutes. The mixture is then centrifuged and the supernatant is decanted. The combined filtrates and decanted solution are concentrated to 15 ml.

C. Test Compounds I

The filtrates were tested for inhibition of HMG-CoA reductase by the method described in Beg,

Stonik and Brewer (1977 FEBS Letters 80 123-129) using enzymes prepared according to the work

Kleinsek, Rangatham and Porter (1977 Proc. Hat. Acad. Sci. 74 1431-1435). A positive assay (more than 90% inhibition at a dose of 20 µg per ml - IC 50 of 2.3 µg per ml indicates the presence of a very potent HMG-CoA reductase steroid synthesis inhibitor.

Example 2

Preparation of compounds 1 β III

A. Fermentation

Test tube with freeze-dried culture Aspergillus sp. The MF-4833 is opened aseptically and the contents are suspended in a 250 ml Erlenmeyer flask (seed flask # 1) containing 40 ml of medium C. Medium C has the following composition:

Medium C corn extracts 5 tomato paste 40 oatmeal 10 glucose 10 trace elements mixture no. 2 10 Distilled water 1 000

pH 6.8 by addition of NaOH

The inoculated flask was incubated for 24 hours at 28 ° C on a rotary shaker at 220 rpm (5.1 cm shift). An additional 8,250 ml) of septum-free Erlenmeyer flasks, each containing 40 ml of medium C and 2 ml of grown culture from flask # 1, are then inoculated. at 220 rpm (5.1 cm shift). Twenty 2-liter septum flasks without baffles containing 500 ml of medium B are then seeded in 14 ml of pooled grown cultures from flasks # 2. These 20 flasks are incubated at 28 ° C without stirring for 11 days. After 11 days, the contents of the 20 flasks were combined.

B. Extraction

10.2 liters of combined incubated medium, pH 6.0 is mixed in a mixer to break heavy mycelial horses, centrifuged and the clear supernatant decanted. After filtration, 10 liters of the filtrate obtained are extracted with 3 liters of ethyl acetate to give 1820 ml of a clear extract. A second extraction with 3 liters of ethyl acetate yields 3,350 ml of clear extract. The solids from the medium were extracted by stirring with 2 liters of methanol for 1 hour and filtered to yield 20 ml of filtrate.

Aliquots of these extracts were dried and tested as in Example 1C. The following results were obtained:

Extract volume (ml) total fixed total units proportions (mg) activities 1 820 1 133 1 496 695 3 350 787 314 900 2 100 13.15 1,144,067

C. Gel filtration

The total solids obtained from the first two extracts in Example 2B were combined, dissolved in methanol and filtered to remove insoluble solids. 30 ml of the filtrate is loaded onto a gel filtration column (2.5 cm x, 200 cm, 980 ml) packed with Sephadex LH-20 and the fractions are separated by molecular weight using methanol as solvent. Using refractive index and UV detection, the best fractions are identified by bioassay for total solids (mg) of total activity unit

fraction 1 89 106 27, fraction 2 278 1 099 680 fraction 3 779 210 357

D. Division and cleaning

A sample from fraction 2 above was filtered on 1 g of Waters Bondapak C18 / Porasil B and eluted with five volumes of methanol. Concentrate the methanolic eluates to 0.5 ml. This sample was chromatographed in several runs on a Waters / IC18 column (3.9 mm x 30 cm) with a mixture of methanol and 0.05 M ammonium phosphate, pH 2.9 (75:25) as eluent. Fractions are monitored on a Beckmann spectrometer and those exhibiting absorption maxima at 236 nm with 229-45 nm inflexes are pooled and concentrated under reduced pressure to an aqueous solution. The pH of the concentrate was adjusted to 6.5 by the addition of 2M potassium hydroxide and the active ingredients were extracted with ethyl acetate. The organic phases are dried, concentrated to dryness and the residue is dissolved in 0.3 ml of methanol. The methanolic solution is chromatographed as above and recycled. The fractions containing the previously eluted component were combined, concentrated to an aqueous solution, and extracted with chloroform. The chloroform residue was dissolved in methanol and the solvent was evaporated under nitrogen. 3.5 ml of dry product was identified, which was identified as hydroxy acid (compound III). The fractions containing the second component were combined and extracted with chloroform as described above. It is obtained. 0.87 mg of dry product identified as lactone (Compound I).

Example 3

The best method of fermenting MF-4833 to produce compounds I and III

Test tube of lyophilized culture Aspergillus sp. The MF-4833 is opened aseptically and the contents are suspended in a 250 ml Erlenmeyer flask (seed flask) containing 40 ml of medium C). The inoculated flask was incubated at 28 ° C on a shaker at 220 rpm for 48 hours.

5,1 cm. Two 250 ml Erlenmeyer flasks, each containing 40 ml of medium D, are then inoculated with 2 ml of an inoculated flask culture. Medium D has the following composition:

Medium D lactose 20 g distillation liquor 1 5 g autolysed yeast 5 g distilled water 1000 ml pH 7.0 by addition of NaOH.

The two inoculated flasks were incubated for 96 hours at 28 ° C on a 1550 rpm shaker (5.1 cm shift). After 96 hours of incubation, the contents of the two flasks were extracted as described in Example 2B. Total production in these flasks is 1,450 to 2,000 units / ml.

Example 4

Preparation of compounds I and III

The Aspergillus MF 4845 freeze-dried tube is opened aseptically and the contents are suspended in a 250 ml septum-free Erlenmeyer flask (Seed flask δ. 1) containing medium C (40 ml). The inoculated flask is incubated for 24 to 48 hours at 28 ° C on a 220 rpm shaker (5.1 cm shift). A portion of this flask (about 0.5 ml) is then used to inoculate a slanted culture in a tube containing medium E. Medium E has the following composition:

Medium E yeast extract 4 g malt extract 10 g dextrose 4 g agar 20 g distilled water 1000 ml pH 7.0 by addition of NaOH.

The inoculated slant culture is incubated for 11 days at room temperature. It is then stored at -60 ° C for 3-4 months. a portion of the contents of this inclined culture is then suspended in a 250 ml non-septum Erlenmeyer flask (seed flask # 2) containing 40 ml of medium C. The inoculated flask is incubated for 24 hours at 28 ° C on a 220 rpm shaker (5 µl shift). cm). Six

250 ml Erlenmeyer flasks without baffles (flasks no. 3) containing po. The 40 ml of medium C is then inoculated with 2 ml of the grown culture from flask # 2. The six inoculated flasks are incubated for 48 hours at 28 ° C on a rotary shaker at 220 rpm (5.1 cm feed). Six 2-liter Erlenmeyer flasks without baffles containing 500 ml of medium F are then inoculated with the contents of inoculation flask No. 3. Medium F has the following composition:

Medium F corn extracts 1 5 g

CPC starch 20 g corn flour 1 g soya flour 4 g glucose 5 δ. soybean oil 2.5 g (NH ₄ ) ₂ SO ₄ 4 g

KH ₂ PO ₄ 0.3 g

CaCo ₃ 6 g distilled water 1000 ml pH 6.7 by addition of NaOH.

The inoculated flasks were incubated at 28 ° C for 11 days without stirring. After 11 days, extraction was performed as in Example 2B. Total production in these flasks is 1,231 units / ml.

Example 5

The best way to ferment MF-4845 to produce compounds I and III • The Aspergillus MF-4845 freeze-dried culture tube is opened aseptically and the contents are suspended in a 250 mL bulkhead Erlenmeyer (seed flask) containing 40 ml of medium C. The inoculated flask is incubated for 30 hours. at 28 ° C on a 220 rpm shaker (5.1 cm shift). 250 ml without septum flask containing 40 ml of medium G is seeded with 2 ml of the grown medium from the seed flask. Medium G has the following composition:

Medium G dextrose 45 g

Peptoned milk 24 g autolysed yeast 2.5 g polyglycol P2000 2.5 ml distilled water 1000 ml pH 7.0 by addition of NaOH.

The inoculated flask was incubated for 120 hours at 28 ° C on a shaker at 220 rpm and a 5.1 cm shift. After 120 hours of incubation, the contents of the flasks were extracted as in Example 2B. The total production in this flask was 21,500 units / ml.

Example

A. Large-scale fermentation of MF-4833 to give compounds 1 and III

The medium used for each fermentation step contained:

corn extract 5 ε tomato paste 40 ε oatmeal 10 ε glucose. 10 ε trace element solution 10 ml distilled water 1 000 ml adjusted to pH 6.8 with sodium hydroxide. The trace element solution contained: FeSO ₄ . 7H ₂ O 1 ε MnSO ₄ . 4H ₂ O 1 ε CuC1 ₂ . ŽH / 25 mg CaClg 100 ALIGN! mg ^H 3 ^B ° 3 56 mg (NH ₄ ) ₆ Mo _? 0 ₂₄ . 4H ₂ O 19 Dec mg ZnSO ₄ . 7H ₂ O 200 mg Distilled water 1 liter

All media were tested for sterility prior to inoculation with the microorganism.

A 250 ml Erlenmeyer flask without septum was charged with 40 ml of medium and the contents of one tube of lyophilized MF 4833. The flask was then shaken on a rotary shaker at 220 rpm at 28 ° C for 24 hours. Fill the new flasks with 40 ml of medium, add 1 ml of the contents of 1 flask, and shake at 28 ° C for 24 hours. 400 ml of medium and 10 ml of the second-stage fermentation broth are then placed in a 2-liter flask and shaken at 28 ° C for 24 hours.

A 910 liter stainless steel fermentation tank is filled with 501 liters of medium containing:

lactose 2% w / v

soluble distillation residues 1.5% w / v

autolysed yeast 0.5% w / v

Polyglycol P2000 0.25% w / v

whose pH is adjusted to 7.0. The medium is sterilized for 15 minutes at 121 ° C. 1 liter of the third inoculation stage is added to the medium and the mixture is incubated for 96 hours at 28 ° C, 130 rpm and an air flow rate of 0.28 m 2 / min.

B. Isolation of Compound I

About 17 kg of silica filter material is added to 501 liters of the medium obtained by culturing MF-4833 described above and the mixture is filtered through a 46 cm filter press. The clear filtrate (pH 6.6) was adjusted to pH 4.0 by carefully adding 450 mL of concentrated hydrochloric acid and extracted by stirring with about one third of the volume (about 164 liters) of ethyl acetate. After separation, the upper solvent phase was separated and the aqueous phase was re-extracted with ethyl acetate (173 L). After separation, the two extracts were combined and washed with about 54 liters of water. After separation, the ethyl acetate solution is concentrated under vacuum at a temperature below 30 ° C first in a stirred boiler and finally in a vacuum rotary evaporator to a volume of less than 4.5 liters.

Approximately 3800 ml of the ethyl acetate concentrate from the previous extraction was further concentrated on a rotary evaporator (40 ° C bath, 1.3 kPa) to a syrup, which was then evaporated twice after addition of about 1 liter of methylene chloride in two portions. A polar solvent-free syrup is obtained. About 300 mL of residual oil containing about 250 g solids, depending on dry weight, was dissolved in about 750 mL of ethyl acetate and methylene chloride (30/70; v / v) and a slurry was prepared by adding 200 g of silica gel. x 36 cm containing 2.5 kg of silica gel in about 7.5 L of the same solvent mixture. Developing with the same solvent is carried out until 3 liters of the front are collected.

Elution was then started with a mixture of ethyl acetate and methylene chloride (50/50, v / v) and 800 ml fractions were collected. 12 fractions were collected and then eluted with 100% ethyl acetate, and after a further seven fractions the elution was started with 100% acetone. Fractions 4 to 24 determine the biological activity of HMG-CoA reductase inhibition by the assay described in Example 1. Considerable activity was found in fractions 7-11. The main activity was found in fraction 8. This was concentrated to an oil for further purification. Determined dry weight of solids 9.0 g.

Fraction 8 from the silica gel column was triturated in 50 mL of methylene chloride, filtered. and the filter cake weighs 4.9 g. The filtrate is applied to a 1 m long column of diameter

5.1 cm packed with Sephadex LH20 dextrated gel (Pharmacia) wetted and equilibrated in methylene chloride and the column eluted with methylene chloride at a rate of 15 ml / min. Compound I eluted between 0.64 and 0.81 column volumes. The solvent was removed from the strip to give a slightly brown residue of about 0.290 g. This residue (213 mg) was dissolved in 1.5 mL of a 65/35 mixture of dichloromethane and acetonitrile and loaded onto a pre-filled and equilibrated silica gel column ( EM LOBAK size B) and eluted with a mixture of dichloromethane and acetonitrile. (65/35) at a rate of 5 ml / min. Evaporation of the solvent from the peak eluted between 235 and 360 ml of solvent gave 121 mg of crystalline product, m.p. 155-160 ° C. In high pressure liquid chromatography, this material was run on an EM RP 18 reverse phase analytical column. E. Merck Hlbar II, Cat. No. 906046) using 0.05 M sodium phosphate pH 3.0 and acetonitrile (45/55) as eluent at a rate of 2 ml / min shows a peak with a characteristic UV absorption at 11 min.

mg of this material was recrystallized with 0.6 ml of absolute ethanol and then again from 0.4 ml of the same solvent and after drying overnight in a desiccator over Pg®5 ^with 40 mg of white feathery crystals. Analytical high pressure liquid chromatography in the system described above gives one sharp peak with an elution time of 11 minutes. After further recrystallization, a melting point of 170-171 ° C was obtained.

The product was identified by spectra et al. As Compound I. This material in an in vitro HMG-CoA reductase assay (Example 1) gave an LC 40 of 0.01 micrograms per milliliter.

Example 7

Direct isolation of the ammonium salt of compound III

The fermentation broth obtained in Example 20A (454 liters) was acidified with phosphoric acid to pH 5. - Ethyl acetate (318 liters) was added and the mixture was stirred vigorously.

The mycelium residues are filtered off and the filter cake is washed with a small amount of ethyl acetate which is combined with the main extract The organic phase is separated and mixed with 22.7 liters of 0.2N sodium hydroxide solution. The mixture was stirred vigorously and then allowed to settle. The aqueous phase was separated and the pH was adjusted from 9 to 5 'by the addition of phosphoric acid. It is then extracted first

9.1 liters of hexane-ethyl acetate and then 4.5 liters of the same mixture. The combined organic extracts were combined and dried over anhydrous magnesium sulfate. The drying agent is then filtered off and the filter cake is washed with one liter of the same hexane-ethyl acetate solution and the wash solution is combined with the filtrate. This filter solution, after further dilution with 2 liters of acetone, was stirred and ammonia gas was introduced. The gas is absorbed and a crystalline precipitate is formed. Once the ammonia is no longer absorbed and darkening is observed in the precipitate, it is discontinued by introducing ammonia and the mixture is allowed to stand for several hours, then filtered. The precipitated ammonium salt filter cake was washed with acetone until a colorless wash solution flowed through and then air dried.

The crude ammonium salt can be recrystallized by dissolving in 7.8 liters of a mixture of chloroform and methanol and concentrated aqueous ammonium hydroxide (80: 80: 2) and filtering off the undissolved material. The filtrate solution was then diluted with an equal volume of 1: 1 concrete-ether and allowed to stand overnight. Filtration gave a crystalline straw-colored ammonium salt (96.5 g).

Further purification can be accomplished by dissolving 70 g of the above recrystallized material in 2 liters of boiling isopropanol-concentrated aqueous ammonium hydroxide (95: 5). 10 g of activated carbon are added and the hot solution is filtered off. The filtrate was allowed to cool to room temperature and allowed to stand overnight at -20 ° C. After filtration, the filter cake is washed with cold (-20 ° C) isopropanol, cold acetone and then ether at room temperature. The product was dried under a nitrogen atmosphere to give about 60 g of white crystalline ammonium salt.

Example8

Salts of compound III

To a solution of 40 mg of the product of Example 6 in 2 mL of ethanol was added 1 mL of aqueous NaOH solution (10 ^-4 mol, 1 equivalent). After 1 hour at room temperature, the mixture was evaporated to dryness in vacuo to give the sodium salt of compound III.

In the same way, using one equivalent of potassium hydroxide, the potassium salt is prepared.

Example 9

L-lysine salt of compound III

A solution of 146 mg of L-lysine in 1.5 ml of 65% ethanol was added to a solution of 440 mg of ammonium salt of compound III in 11.5 ml of 85% ethanol. The solvents were distilled off under vacuum. The residue is triturated with 10 ml of hot ethanol, cooled and filtered. A white solid was obtained containing the salt of compound III with L-lysine, m.p. 178-180 ° C (dec.).

Analysis for ^C 30 ^H 52 ^N 2 ° ^8: calculated: 63.35% C 9.22% H 4.93% N Found: 62.80% C 9.13% H 4.83% N

He did

Salt of compound III with L-arginine

A solution of 174 mg of L-arginine was reacted with a solution of 440 mg of the ammonium salt of compound III as described in Example 9. The solvent was evaporated in vacuo and the residue was triturated with hot ethanol, cooled and filtered. Drying gives the L-arginine salt of compound III.

They did

Salt of compound III with L-ornithine

As described in Example 9, a solution of 132 mg of L-ornithine free base and a solution of 440 mg of the ammonium salt of compound III were mixed. The solvent was evaporated in vacuo and the residue was triturated in hot ethanol, cooled, filtered and dried to give the L-ornithine salt of compound III.

Example 12

Compound III salt with N-methylglucamine

A solution of 195 mg of N-methylglucamine in 1.5 ml of water and 440 mg of ammonium salt of compound III in 11.5 ml of 85% ethanol was mixed as described in Example 9. The solvent was evaporated in vacuo to give the salt of compound III with N-methylglucamine.

Example 13 *

The ethylenedlammonium salt of compound III g of compound I is dissolved in 180 ml of hot isopropanol and treated with 90 ml of 0.5 M aqueous sodium hydroxide solution, stirred for 1 hour and diluted with 180 ml of water, evaporated in vacuo to remove isopropanol and cooled in vacuo. ice bath. 90 ml of 0.5 M HCl are slowly added and the mixture is extracted with 2 x 150 ml of ethyl acetate, which is washed again with 100 ml of water and dried over magnesium sulfate. The solvent was removed in vacuo at low temperature and the residue was dissolved in 150 ml of ethanol. Add 3 ml of ethylenediamine, and the solvent was evaporated in vacuo and the residue triturated with boiling ethyl acetate, cooled, filtered and recrystallized from 30 ml of isopropanol and dried in vacuo over ^P 2 ° 5 * IEK ^with 13.1 g of white crystals, m.p.

Mp 152-153.5 ° C.

Analysis for (^ 24 ^ 7 ^ 2 * ^ 2 ^ 0 ^ 2 ^ 5 : calculated: 66.35% C, 9.35% H, 3.09% N, started up: 66.08% C, 9.49% H, 3.01% N.

Example 14

Calcium salt of compound III

87.9 mg of the ammonium salt of compound III is dissolved in 3 ml of HgO with stirring and heating. 7.4 mg of analytical Ca (OH) _{2 are} then added and the mixture is stirred and heated until no more ammonia evaporates and only a small haze remains which is removed by centrifugation. The colorless clear supernatant was lyophilized and samples of dry material were crystallized from various solvents and solvent mixtures. The product crystallizes in the needles when the hot, concentrated solution in anhydrous isopropanol is allowed to cool to room temperature.

Example 15

The tetramethylammonium salt of compound III mg of compound I in 1 ml of CH 2 Cl 2 was treated with 0.04 ml of 24% tetramethylammonium hydroxide in methanol. The product is precipitated with ether in partially crystalline form, centrifuged and the precipitate is first washed with ether and then recrystallized as hexagonal plates from 1 ml of isopropanol by adding 5 ml of ether and about 5 ml of low boiling petroleum ether. This gave 27 mg (65% yield). 1 H NMR spectrum of the tetramethylammonium salt of compound III

(6 mg / 0 , 35 ml at 25 ° C in CDCl 3 300 MHz) 0.83 t <3H, J = 6,5) 0.84 d (3 H, J = 7) 1, 02 d (32, J = 7) 1, 05 d (3 H, J = 7) 1.24 m (-> 1H); ί 1.30 to 1.80 wide multiplet

1, 88 ddd OH, J = 2.8, 15) 1.98 dd OH, 3, 15) 2.16 dd (1H, J = 8.5, 15.5) 2.23 m (1H, hidden) 2.32 m OH, hidden) 2.37 dd (1H, J = 3.15.5) 2.40 m (~ 1g, hidden) 3.42 with (12H, MeN ⁺ ) 3.79 m OH, symmetric multiplet) 4.06 m OH, symmetric multiplet) 5.32 dt OH, J * 3) 5.50 br. s (1H) 5.79 dd OH, J = 6.10) 5.98 d OH, J = 10) Chemical shifts are in ppm down from Constants in brackets are in Hz. Abbreviations: s = singlet, d = doublet, t í k 1 and d 16

Ammonium salt of compound III

Following the procedure described in Example 13, compound I was converted to the hydroxy acid, compound III was extracted into ethyl acetate, dried over magnesium sulfate, filtered and treated with anhydrous ammonia with stirring and cooling. The ammonium salt precipitates.

Example 17

Preparation of hydroxy acid of compound III

The sodium salt prepared in Example 7 was redissolved in 2 ml of ethanol-water (1: 1) and added to 10 ml of 0.1 N hydrochloric acid and the liberated hydroxy acid was extracted with ethyl acetate. The solvent was washed once with water, dried and evaporated in vacuo at a bath temperature not exceeding 30 ° C. On standing, the hydroxy acid slowly changes to the lactone.

Example 18

Compound III

453 mg of ethylenediammonium salt of compound III is dissolved in 6 ml of 80% ethanol, cooled in an ice bath, 1 ml of 1M HCl is added, evaporated in vacuo to remove ethanol, 3 ml of water are added, extracted into 2 x 5 ml of ethyl acetate and backwashed with water, keeping all solvents in an ice bath. The extract was dried over magnesium sulfate and concentrated to dryness in vacuo. The hydroxy acid is obtained as a colorless oil.

^{The 13} C-NMR spectrum in CDCl 3 (190 mg / ml) shows the chemical shifts shown for the first six carbons of the hydroxy acid-containing portion of the molecule. On standing, the hydroxy acid of the pomel is converted to the lactone.

• 17

Table

C-NMR spectrum, ppm down to tetramethylsilane

Hydroxy acid, compound III

174.8

42.4, 41.6

HIM

68.8

72.3

34.9

The spectrum of the remainder of the molecule is only slightly altered by cyclization.

Example: d 19

Ethyl ester of compound III

A suspension of 500 mg (1.24 mmol) of compound I (MSD-803) in 20 mL of ethanol was stirred at room temperature under a nitrogen atmosphere. Add a small piece of sodium (about 1 mg) After 15 minutes, add a second small piece. After a total of 30 minutes, the homogeneous reaction mixture was diluted with ether, washed with water and saturated sodium chloride solution and dried over magnesium sulfate. Evaporation of the solvent gave a waxy solid. Analysis by high pressure liquid chromatography on a Whatman Partasil PAC column (4.6 mm x 25 cm) with 10% isopropanol in hexane pumped at 6 mL / min, was found to be a mixture of ethyl ester and MSD-803 (77:23).

This mixture was separated by medium pressure liquid chromatography on silica gel 230-400 mesh eluting with 3% ethanol / methylene chloride. The ester containing fractions were combined and evaporated to give 358 mg (66%) of an off-white solid, mp 67 ° C.

some of this material was recrystallized from hexane to give white needles, mp 66.5-68.5 ° C.

Analysis for C ₂₆ H ₄₂ O _6:

calculated: 69.30% C, 9.40% H, found: 69.22% C, 9.58% H.

In the same manner, the corresponding esters are prepared using equivalent amounts of methanol, propanol, butanol, isobutanol, t-butanol, amyl alcohol, isoemyl alcohol, 2-dimethylaminoethanol, benzyl alcohol, phenethanol, 2-acetamidoethanol and the like. .

Example 20

Preparation of compounds II and IV

A. Fermentation

The tube of lyophilized culture MF-4845 is opened aseptically and the contents are suspended in a 250 ml septum-free Erlenmeyer flask (seed flask) containing about 10 ml of the medium of the following composition:

Medium: corn extracts 5 g tomato paste 40 g oatmeal 10 g glucose 10 g trace element solution 10 g distilled water pH 6.8 by addition of NaOH. 1000 ml Trace element solution FeSO ₄ . ZH ₂ O 1000 mg MnSO ₄ . 4H ₂ O 1000 mg CuC1 ₂ . 2H ₂ O 25 mg CaCl ₂ . 2H ₂ O 100 mg ^h 3BO ₃ 56 mg (NH _{4) 6} Mo ₇ O _24th 4H ₂ O 19 mg ZnS0 ₄ . 7H ₂ 0 200 mg distilled deionized 1000 ml

water

The inoculated flask was incubated for 24 hours at 28 ° C on a 220 rpm shaker (5.1 µl shift). A 2 liter Erlenmeyer flask containing 500 ml of medium is then inoculated with 10 ml of the seed mixture of the mature culture from the first stage. The flask was also shaken at 28 ° C for 24 hours.

The 910 liter fermentation tank is then filled with 485 liters of medium containing:

cerellose peptoned milk autolysed yeast polyglycol P2000

4.5% w / v

2.5% w / v 0.25% w / v 0.25% w / v whose pH is adjusted to 7.0. The medium is sterilized for 15 minutes at 121 ° C. One liter of the second inoculation stage prepared above is then added to the tank and the mixture is incubated for 12 hours at 85 rpm and then at 130 rpm under an air flow rate of 0.14 m 2 / min for 12 hours and then 84 hours at flow rate. 0.28 n / min.

B. Insulation

1. Extraction

The two batches from the 454 liter tanks were combined, acidified with stirring to pH 4.1 by carefully adding 800 mL of concentrated hydrochloric acid, and then extracted by adding 340 L of ethyl acetate and stirring for two hours.

About 11.3 kg of silica filter mass is added and the total suspension is pumped through a 61 cm filter press. An additional 340 liters of ethyl acetate was used to wash the filter cake, and extraction was carried out four times by repeated pumping back through the filter cake. All solvent washes are then removed from the filter cake and combined with the first filtrate. The biphasic filtrate was allowed to settle and the aqueous phase was separated. The ethyl acetate phase is washed with 45 liters of deionized water, the phases are allowed to separate and the ethyl acetate extracts are concentrated in vacuo to a residue of about 45 liters.

2. Lactonization (The following azeotropic treatment is performed to cyclize compound IV in extract to compound II).

Ethyl acetate extracts from an additional 1,365 liters of fermented medium were added to the extract prepared above and the volume reduced by vacuum distillation to about 136.4 liters. Ai 227 liters of toluene is added and the batch is concentrated to about 145 liters under vacuum. This step is repeated, sufficient toluene is added to reach a volume of 340 liters. Without vacuum, the batch is heated to boiling above 106 ° C for two hours.

The solution is then concentrated in vacuo to a small volume, which is then further concentrated in a large rotary evaporator under vacuum to an oily residue.

3. Silica gel chromatography

The extract obtained above was freed of other solvents by adding 9 liters of methylene chloride and concentrating to an oil.

The oily residue is dissolved in about 22.7 liters of a mixture of ethyl acetate and methylene chloride (30/70 v / v) and a suspension is prepared by adding 2.8 kg of silica gel.

The suspension is then applied to the top of a silica gel column (30 x 127 cm) packed in the same solvent mixture.

Elution was performed with a mixture of ethyl acetate and methylene chloride (40/60; v / v) at a rate of 800 ml / min. First a 45 liter front is collected and then a 18 liter fraction.

Fractions 6 to 10 inclusive were concentrated in vacuo to an oily residue which was dissolved in hot ethyl acetate, treated with charcoal, filtered hot and cooled. The MSD 803 crystals (compound I) are filtered off and the mother liquors are concentrated in vacuo to an oily residue which is chromatographed further.

4. Repeat chromatography on silica gel

The mother liquors from similar extractions of the fermented medium, equivalent to 2727 liters, were combined with the methylene chloride solution prepared above. One half of this solution is taken up for longer chromatography on silica gel. According to a small aliquot, the total solids content was 325 g. The solution was treated with 40 g of activated carbon, filtered and the filter cake was washed with methylene chloride. The combined filtrates and washings were concentrated in vacuo to an oily residue. This was redissolved in 800 ml of a mixture of ethyl acetate and methylene chloride (30/70; v / v) and mixed with 225 &lt; &gt; &gt; silica gel. Load the slurry onto a 14 x 36 cm silica gel column packed with the same solvent mixture. The column is developed with a mixture of ethyl acetate and methylene chloride (40/60; v / v). First, a front of three liters was collected and then a 800 ml fraction.

5. Concentrate the reversed phase ml chromatography from the chromatography fraction to a 500 mg oil residue and redissolve it in 5 ml of acetonitrile. This acetonitrile solution was applied to a 1.5 cm 30.5 cm diameter stainless steel column packed with reverse phase C18 / Porasil B preparative liquid chromatography material (Water Associates, Inc., Milford, Mass. 01 757). The column was eluted with a mixture of 55% acetonitrile and 45% 0.05 M ammonium phosphate pH 3 (v / v). The volume of solvents eluted between 1360 ml and 1700 ml was combined, as detected by refractive index.

^Of organic solvent was evaporated in vacuo and the residual aqueous solution was extracted with ethyl acetate. The ethyl acetate was evaporated in vacuo to give 120 mg of the title compound which crystallized from a concentrated acetonitrile solution. MSD 883 (compound XI) mp 129-131 ° C was obtained.

Example 21

Alternative isolation of compounds II and IV

The crude ammonium salt, isolated as described in Example 7 from a 500 liter fermentation, was lactonized by dissolving in water, acidified to pH 3 with concentrated hydrochloric acid, extraction into toluenes, and heating to boiling for 2 hours according to Example 20-B-2. Concentration in vacuo to a small volume yields crystals of crude I with a small amount of II. These are filtered off and dried.

The materials 12 of such batches are mixed and recrystallized from 46 kg of ethanol, filtered off and washed with ethanol. The combined mother liquors are washed and concentrated in vacuo to about 13.6 liters and a second crop of crystals of Compound I is filtered off. The mother liquors from this filtration are treated as follows:

About 0.5 L of these mother liquors are prepared for reverse phase chromatography by concentration in vacuo to remove ethanol, dissolving in acetonitrile and filtering off traces of insoluble material. The solution in 100 ml of acetonitrile is chromatographed on a 200 ml column of C-18 phase feed and the column is washed with a further 1 liter of acetonitrile. The combined filtrates were concentrated and dissolved in a total of 360 ml of solvent (60 parts acetonitrile and 40 parts water, v / v) and traces of insoluble matter were filtered off. According to the aliquot analysis, the solids content is 15 g.

The batch of 160 ml of this mixture is chromatographed on a Waters Prep 500 system using two 5 x 30 cm C18 reverse phase columns (Waters Assoc. Bound octadecyl silica gel coating) with acetonitrile-water (60/40 v / v) as the eluent, at teplotě30 ml / min at room temperature and refractive index detection. The impurities eluting in the first 3'900 ml are discarded. Compound I is obtained from the fractions eluted between 3900 to 5850 mL. The fractions obtained from 5850 ml to 6500 ml are left for rechromatography. The purified compound II is obtained from the last band eluted between 6 500 ml and 8 450 ml. The collected fractions, upon concentration, yielded 3 g of an oily residue.

One half (about 1.5 g) of this concentrate of compound II is prepared for ammonium salt of compound IV for chromatography by preparing a solution in 6 ml of clear methanol and immediately adding sufficient sodium hydroxide N while stirring. The pH was maintained between 10.5-1.1. For this purpose 3.5 to 3.8 ml of sodium hydroxide solution are required. After standing for half an hour, traces of insoluble matter were removed from the solution by filtration and the filtrate was pumped through a column of 2.54 x 86 cm of 200 to 325 mesh XAD-2 resin (styrene-divinylbenzene copolymer) and eluted with 23% acetonitrile / 77% % 0.1N ammonium hydroxide at a rate of 2 ml / min at 40 ° C. Collect 20 ml fractions. Fractions 1 to 44 contain impurities and a salt band of compound III. Fractions 45 to 61 were combined, concentrated in vacuo to a small aqueous residue, which afforded the ammonium salt of compound IV (0.72 g) by freeze-drying.

Example 22

Salts of Compound IV

To a solution of 40 g of the product of Example 20 in 2 ml of ethanol was added 1 ml of an aqueous solution

NaOH (10 ^-4 mol, 1 equivalent). After one hour at room temperature, the mixture was evaporated to dryness in vacuo to give the sodium salt of compound IV.

In the same way, a potassium salt is prepared using one equivalent of potassium salt β calcium salt using one-half CaO equivalent.

Using the procedures described in Examples 9-12 and 14, but substituting the ammonium salt of compound III in each case for an equivalent amount of the ammonium salt of compound IV, the corresponding salts of compound IV with L-lysine, L-arginine, L-ornithine, N-methylglucamine and calcium are formed. .

Using the procedures described in Examples 13, 15 and 16. but substitution of compound I in each case for an equivalent amount of compound II produces the corresponding ethylenediammonium, tetramethylammonium and ammonium salts of compound IV.

Example 23

Preparation of hydroxy acid, compound IV

The sodium salt prepared in Example 22 was redissolved in 2 mL of ethanol-water (1: 1) and 10 mL of 0.1 N hydrochloric acid was added to liberate the hydroxy acid and extracted with ethyl acetate. The solvent was washed with water, dried and evaporated in vacuo. vacuum at a temperature not exceeding 30 ° C. The hydroxy acid on standing slowly changes to the lactone.

Example 24

Preparation of hydroxy acid, compound IV

221 mg of the ammonium salt of compound IV is dissolved in 4.5 ml of 65% ethanol. Cool with ice, acidify to pH 3 with about 0.5 ml of 1M hydrochloric acid and evaporate at low temperature on a rotary evaporator to a volume of about 2 ml. An additional 2 mL of water was added and extraction was performed with 2 x 3 mL of ethyl acetate. Wash the extract with 1 ml of water, keeping all solutions cold in an ice bath. The extract was dried (MgSO4) and evaporated in vacuo to give the hydroxy acid as a colorless oil.

The C-NMR spectrum in CDCl3 shows chemical shifts for the first six carbon atoms of the hydroxy acid-containing portion of the molecule as shown in the table. On standing, this hydroxy acid slowly changes to the lactone.

Table

hydroxy acid compound IV

HIM.

175.0

COOH

OH

42.2, 41.7

68.8

72.5

35.0

The spectrum of the remaining part of the molecule is only slightly changed by cyclization.

Example 25

Ethyl ester of compound IV

Using the procedure described in Example 19, but substituting 1 for 1.24 mmol with an equivalent molar amount of XI of Example 20 to give IV.

In a similar manner, methyl, propyl, butyl, isobutyl, t-butyl, amyl, isoamyl, 2-dimethylaminoethyl, benzyl, phenethyl and 2-acetamidoethyl esters are prepared.

Example 26

A. In vitro Inhibition of HMG Coenzyme A Reductase

The slightly modified method described by Beg et al. FEBS Letters 80, 123 (1977) is used. Incubation of the enzyme with the inhibitor is performed 5 minutes before starting the reaction with the substrate. With potent inhibitors such as the novel compounds of the invention, the standard procedure, performed simply by adding the enzyme to the inhibitor / substrate mixture, did not provide linear kinetics.

Using a modified procedure, the sodium salt of Compound IV provides IC 50 for inhibition of HMG-CoA reductase of 2.7 x 10 6 µM compared to 5.4 x 10 6 for ML 236 B.

B. In vivo inhibition of cholesterol synthesis (compound II)

Male Holzman rat groups were administered either 5% emulsion in sodium chloride solution or test compound in emulsion. After one hour, 80 µCi ¹ C acetate per kg was administered intraperitoneally. After 50 minutes, the rats are bled and the steroid synthesis is determined by cholesterol determination.

Dose, mg / kg% inhibition

0,15 38

0,6 51

1.2

Example 27

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

The procedure of AW Albertse and J. Biol. Chem. 249, 5241 (1974), measuring the amount of ¹ ^ C steroid biosynthesis from ¹ ^ C-acetate in mouse LM cells in culture, was used with slight modification. The test compound in 10 µL of DMSO is added to the monolayer culture along with 5> C ¹¹ -C ¹⁴ -acetate. After 3 hours of incubation, the cells are saponified and the &lt; 1 &gt; C sterol is extracted and isolated by thin layer chromatography on silica gel using petroleum ether-diethyl ether / acetic acid (75: 25: 1). The area of the ¹⁴ C steroid plate is identified by standing with I, A A and the C content is determined by liquid scintillation counting.

Using a modified procedure, compound II provides IC 50 for inhibiting HMG-CoA reductase at 17 nM compared to 22 nM for compound I and 46 nM for ML-236B.

Claims (5)

A process for the preparation of a mixture of hypocholesteremic compounds comprising compounds of structural formulas 1, II, III and IV, characterized in that the nutrient medium containing assimilable sources of carbon, nitrogen and nutrient inorganic salt is incubated at 20-37 ° C and pH 6.0-8, With microorganisms of the genus Aspergillus terreus deposited with the American Type Culture Collection under Nos. 20541 or 20542 and the products isolated. 2. A process for the preparation of a mixture of hypocholesteremic compounds comprising compounds of structural formulas I and III according to claim 1, characterized in that the nutrient medium is incubated with microorganisms of the genus Aspergillus terreus deposited with the American Type Culture Collection under Nos. 20541 or 20542 and recovered. 3. Process according to claim 2, characterized in that the isolation is carried out by solvent extraction of the fermentation mixture and subsequent chromatography. 4. A process for preparing a mixture of hypocholesteremic compounds comprising compounds of formulas II and IV (IV) according to claim 1, characterized in that the nutrient medium is incubated with microorganisms of the genus Aspergillus terreus deposited with the American Type Culture Collection under Nos. 20541 or 20542 and recovered. 5. A process according to claim 4, wherein the isolation is carried out by solvent extraction of the fermentation mixture and subsequent chromatography.