DK151273B - PROCEDURE FOR THE PREPARATION OF THE CHOLESTEROL BIOSYNTHESE INHIBITOR MB-530A. - Google Patents

Description

in 151273

The present invention relates to a process for the preparation of the compound termed MB-530A with inhibitory effect on the biosynthesis of cholesterol. MB-530A has the formula (V): 50 (V) 10 L in ch 3

O ^ J

Hyperlipidemia, especially hypercholesterolemia, is known to be one of the major causes of cardiopathy, such as heart attack or arterial sclerosis. As a result, considerable research has been carried out to find compounds capable of reducing the amount of lipid, and especially the cholesterol 2Q sterol in the blood. A group of compounds of this type is disclosed in U.S. Patent No. 3,983,140, which compounds are isolated from microorganisms of the genus Penicillium.

This group of compounds is collectively referred to as ML-236.

25 ML-236A and NL-236B (two of the compounds of the ML-236 complex disclosed in U.S. Patent No. 3,983,140) have formulas (II) and (III), respectively:

so to'rV ΗΟγ Y

γ Y

x \

OH 1 9 T

CH, I I (ID CH I i (III)

35 YyS Y yS

XXX X XX

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It has now been found that the compound MB-530A can be prepared by growing a strain of the Monascus rubber species, and the method according to the invention is characterized by cultivating a strain of the Monascus 5 rubber species in a culture medium therefor, and that the MB-530A compound formed is separated from the culture medium.

MB-530A is preferably produced by cultivating the strain Monascus ruber SANK 15177. This strain was deposited on the 27th.

April 10, 1979 under the accession number FERM 4956 at The Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry,

Japan, and on January 25, 1980 under accession number NRRL 12081 at The Agricultural Research Service Culture Collection, 15 Northern Regional Research Laboratory, Peoria, Illinois, U.S.A.

From the specification to Danish patent application No. 731/80, which corresponds to the present disclosure no. 148.807, it must be considered known to produce monacoline K of formula (IV): 20

HS V

Y o 25 i i ^ 0 CH3 \ Yv> ^ k (IV) ^ Ύ 'CH-30 3 by cultivating strains of the species Monacus ruber, i.a. Monacus 35 ruber SANK 15.177.

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From the description to the Danish patent application no. 461/81, it must be considered known to produce, inter alia, MB-530A by a chemical process.

The compound MB-530A can be prepared by culturing the selected microorganism in a culture substrate under aerobic conditions using the same technique well known for growing fungi and other microorganisms. For example, the selected strain of Monacus rubber can first be grown IQ on a suitable medium and the microorganism produced can then be collected and seeded and grown on another culture medium to prepare the desired MB-530A compound.

The culture medium used for the propagation of the microorganism and the culture medium used for the production of MB-530A may be the same or different.

Any well-known culture medium for growing mushrooms can be used provided that, as is well known, it contains the necessary nutrients, in particular an assimilable carbon source and an assimilable nitrogen source. Examples of such sources of assimilable carbon include glucose, maltose, dextrin, starch, lactose, sucrose and glycerol. Of these sources, glucose, glycerol and starch are particularly preferred for the preparation of MB-530A. Examples of suitable sources for assimilable nitrogen are peptone, meat extract, yeast, yeast extract, soybean meal, peanut flour, corn mold liquid, rice bran and inorganic nitrogen sources. Of these nitrogen sources, corn molding liquid and peptone are particularly preferred. In preparing MB-530A, an inorganic salt and / or a metal salt can be added to the culture medium if necessary. Furthermore, if necessary, a smaller amount of a heavy metal can also be added.

The microorganism is preferably grown under aerobic conditions using cultivation methods well known in the art, e.g. solid culture, shaking or aeration and stirring. The microorganism will grow over a wide range of temperatures, e.g. 7 to 40 ° C, but for the preparation of MB-530A, the more preferred culture temperature is within the range of 20 to 30 ° C.

During the cultivation of the microorganism, the production of MB-530A 5 can be measured by sampling culture medium and measuring the physiological activity of the medium by well known experiments. Cultivation can then be continued until a significant accumulation of MB-530A has been obtained in the culture medium, at which time the MB-530A compound can then be isolated and recovered from the culture medium by any suitable combination of isolation technique selected taking into account its physical and chemical properties. For example, any or all of the isolation methods listed below may be used: Extraction of the liquid from the culture substrate with a hydrophilic solvent (such as diethyl ether, ethyl acetate, chloroform or benzene), concentration, e.g. by evaporation of some or all of the solvent under reduced pressure, dissolving in a more polar solvent (such as acetone 2q or an alcohol), removing impurities with a less polar solvent (such as petroleum ether or hexane), gel filtration through a column of a material , such as "Sephadex" (a trade name for a material obtained from Pharmacia Co. Limited, USA), activated carbon 25 or silica gel absorption chromatography, and other similar methods. When used.

by a suitable combination of these methods, the desired MB-530A compound can be isolated from the culture substrate as a pure substance. 1 2 3 4 5 6

The compound of the invention has been found to have a specific inhibitory effect against 3-hydroxy-3- 3 methylglutaryl coenzyme A reductase (HMG-CoA reductase), which 4 is the rate-controlling enzyme in the biosynthesis of 5 cholesterol.

6

The preparation of compound MB-530A is further illustrated in the following example.

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Example

Preparation of MB-530A

5,300 l of a culture medium with a pH of 5.5 before sterilization containing 5% w / v glucose, 0.5% w / v maize casting liquid and 2% w / v peptone (Kyokuto make, available from Kyokuto Seiyaku KK, Japan) and 0.5% ammonium chloride were charged to a 600 l fermentation vessel and seeded with a culture of Monascus ruber SANK 15177 (FERM 4956, NRRL 12081).

Culture of the microorganism was continued for 120 hours at 27 ° C with an aeration rate of 300 l / min and stirring at 190 rpm. minute.

At the end of this time, the culture substrate was filtered into a filter press to obtain a filtrate and a filter cake comprising wet cells of the microorganism.

The filtrate was adjusted to a pH of 3.0 by the addition of 6N hydrochloric acid and then extracted with 400 L of ethyl acetate.

The extract (about 400 L) was concentrated by evaporation under reduced pressure and then dewatered over anhydrous sodium sulfate, then evaporated to dryness to give ca. 60 g of an oily product. This oily product was washed with ethyl cyclohexane and with hexane, and the residue (20 g) was separated by chromatography using a high volume liquid chromatography apparatus (System 500 liquid chromatography, prepared by

Waters Co., USA), eluting. with 60% v / v aqueous methanol. Fractions with chromatographic retention time of 6 minutes were collected and concentrated by evaporation under reduced pressure to give 100 mg of the desired MB-530A compound as an oily product. This oily MB-530A compound was recrystallized from a mixture of acetone and diethyl ether to give 57 mg of the desired product in the form of colorless needles having the following properties:

Claims (2)

151273 A process for preparing the compound MB-530A of formula (V): 30 ° C) 35. OH ch3 ~ C ^ X ^ ~ - (V) 151273 characterized in that a strain of the species Monascus rubs in a culture medium therefor and that the MB-530A compound formed is separated from the culture medium. 1. Melting point: 92 - 93 ° C. 2. Elemental analysis: Calculated for c19 H28 ° 4: c 69/76%; H 8.68%. Found: C71.22%, H 8.81%. 3. Molecular weight: 320 (by mass analysis). 4. Molecular formula: C 28 H 28 ° 4 5. Ultraviolet absorption spectrum: As shown in Figure 1 of the drawing. 6. Infrared absorption spectrum: As shown in FIG. 2 of the drawing. 7. Nuclear Magnetic Resonance Spectrum; As shown in FIG. 3 of the drawing. 8. Solubility: Easily soluble in methanol, ethanol, acetone, ethyl acetate: Soluble in benzene: Insoluble in hexane and petroleum ether. 9. Color reaction: A pink color is seen when a thin layer chromatogram of the compound on silica gel is elicited with 50% volume / volume sulfuric acid. 10. Inhibitory effect on the biosynthesis of cholesterol: 20 A 50% inhibition of cholesterol synthesis in rat liver is observed at a concentration of 0.04 µg / ml. Claims. 25 --------------------- Method according to claim 1, characterized in that the microorganism is Monascus ruber SANK 15177 (FERM 4956, NRRL 12081).