DK165990B - Hypocholesterolaemic agent monacolin K - produced by culturing Monascus strain - Google Patents

Abstract

Monacoline K of formula (I) is new. It is produced by culturing a (I)-producing Moascus strain (esp. M. rubber 1005) in a suitable medium at 7-40 degrees C (esp. 20-35 degrees C) (I) inhibits cholesterol biosynethesis and is thus as a hypocholesterolaemic agent; it has higher activity than ML-236 (GB 1453425) and low toxicity.

Description

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DK 165990B

The present invention relates to a process for the preparation of the carboxylic acid of a novel cholesterol synthesis inhibitory compound called monacoline K having the formula: ΗΟγγΟ v ° k ο

A ^ AA

or salts thereof, characterized in that a strain of Monascus rubs is grown in a culture substrate containing 15 assimilable carbon or nitrogen sources, an alkali is added to the culture mixture to obtain alkali salts of the carboxylic acid of monacoline K, and then either is isolated the alkali salts from the alkaline culture mixture or extract the acid with an organic solvent, preferably 20 with ethyl acetate or chloroform, at an acidic pH.

High blood cholesterol levels are well known to be one of the major causes of cardiopathy, such as heart failure or arteriosclerosis. As a result, numerous scientific studies have been conducted to find physiologically acceptable compounds capable of inhibiting cholesterol biosynthesis and thereby lowering blood cholesterol levels.

One of these compounds is ML-236, which is disclosed in English Patent No. 1,453,425 and in Danish Patent No. 136,485. 30 ML-236 is prepared by culturing a microorganism of the genus

Penicillium.

It has now been found that strains of the species Monascus rubber, especially Monascus rubber strain 1005 (FERM 4822), produce a closely related compound with ML-236 and that by treatment with alkali it is possible to induce ring opening of this compound. lactone ring to form the corresponding salts, 2

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whereupon the carboxylic acid of the compound can be isolated at an acidic pH by extraction with a suitable organic solvent such as ethyl acetate or chloroform.

5 Monascus rubber strain 1005 (FERM 4822) is a recently isolated microorganism exhibiting the following microbiological properties. It was isolated from feed produced in Thailand and deposited on February 16, 1979 under the accession number FERM 4822 in the Fermentation Research Institute, Agency <5f Indu-10 strial Science and Technology, Ministry of International Trade and Industry, Japan, and under the accession number NRRL 12073 in Agricultural Research Service, Northern Regional Research Laboratory, USA.

15 1. GROWTH

Growth on a potato-glucose-agar substrate at 25 ° C is rapid and the diameter of the colonies reaches 6-6.5 centimeters 10 days after seeding. The colony is flat and a relatively 20 thin basal layer of hyphae develops. The development of air hyphae is poor; the air hyphae are white and most of them are woolly. Kleistotesia form on the basal layer of the hyphae and they turn reddish brown upon maturation. Both the surface and the back of the colony are brown to reddish brown in color.

25 The growth of Sabouraud's agar substrate at 25 ° C is very rapid and the diameter of the colonies reaches 6-6.5 centimeters 10 days after sowing. The surface of the colony is very flat and basal hyphae and aerial hyphae develop better than on the potato-glucose-30 agar substrate. Kleistotesia is very sparse. The surface of the colonies is reddish-yellow to reddish-brown in color, and the reverse is reddish-brown to dark brown.

The growth of oatmeal agar at 25 ° C is slow and the diameter of the colonies reaches 1.5-2 centimeters 10 days after sowing.

The colony is flat. The development of air hyphae and the formation of cleistothecia are very poor for both. Both the surface and the opposite side of the colony are dark red to reddish-brown in color.

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The growth of Czapek's agar substrate at 25 ° C is very slow and the diameter of the colonies reaches 1.6 - 1.8 centimeters 10 days after seeding.

5 The growth rates of each of the above substrates at 37 ° C are substantially equal to the growth rates at 25 ° C.

2. MORPHOLOGICAL PROPERTIES

10 Kleistotesia are spherical and 30 - 60 microns in diameter; their walls are white and membranous; their stems have partitions and each consists of a hypha with a diameter of 3.5 - 4.5 microns and a length of 15 - 80 microns. The spore sac consists of 8 spores and it is almost spherical and fades away quickly. The ascospores 15 are colorless and ovoid or ellipsoid; they have a size of 4 - 5 x 4 - 7 microns; and their surface is smooth. Conidia are colorless and spherical or bulbous; their size is 6-9x6-11 microns; their base is just cut off and their walls are relatively thick and smooth. Conidia are linked mid-point flying to each other as a kind of formation tissue arthrospore. The conidiophore is like a vegetative hyphae and is branched or unbranched; Conidia forms at the top. The mycelia are colorless and branched and have partitions; most of them have a diameter of 3 - 5 microns.

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Based on these observations of the properties, the microorganism was identified as a strain of Monascus ruber van Tieghem.

The microbiological properties of Monascus rubs are listed in the following literature: Takada, Transactions of the Micological

Society of Japan, 9, 125-130 (1969) [Materials for the Fungus Flora of Japan (7)], and van Tieghem, Bull, Soc.Botan.France, 31, 227 (1884). Ascospore formation of the species is discussed by Cole et al in the Canadian Journal of Botany, £ 6, 987 (1968), 35 "Conidium Ontogeny in hyphomyceres. The imperfect state of

Monascus ruber and its meristem arthrospores ".

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Other strains of the species Monascus rubs capable of producing monacoline K can be used in the process of the invention.

In cultivation, methods well known to those skilled in the art of cultivating fungi and other microorganisms are used. Eg. the monacoline K-producing microorganism can first be grown on a suitable substrate, and then the microorganisms produced can be collected and seeded and grown in another growth substrate 10 to produce the desired monacoline K; the culture substrate used for propagating the microorganisms and for the production of monacolin K may be the same or different.

Any culture substrate known to those skilled in the art of growing mushrooms may be used provided that, as is known, it contains the necessary nutrients, in particular a source of assimilable carbon and a source of assimilable nitrogen. Examples of suitable sources of assimilable carbon are glucose, maltose, dextrin, starch, lactose, sucrose and glycerol. Of these sources, glucose, glycerol and starch are particularly preferred for the production of monacoline K. Examples of suitable sources of assimilable nitrogen are peptone, meat extract> yeast, yeast extract, soybean meal, peanut flour, corn mold liquid, rice husks and inorganic nitrogen. Of these 25 sources of nitrogen, peptone is particularly preferred. When producing monacoline K, an inorganic salt and / or a metal salt may optionally be added to the culture substrate. Further, a smaller amount of heavy metal can also be added.

Preferably, the microorganism is cultured under aerobic conditions using cultivation methods well known to those of skill in the art, e.g. solid cultures, shaking cultures or cultures under aeration and stirring. The microorganisms grow within a wide temperature range, e.g. from 7 to 40 ° C, but 35 especially for the formation of monacoline K is the preferred culture temperature in the range 20 to 35 ° C.

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During the cultivation of the microorganisms, the production of monacoline K can be controlled by sampling the culture substrate and measuring the physiological activity of monacolin K in the culture substrate by experiments described below. Cultivation is continued until a substantial accumulation of monacoline K is obtained in the culture substrate, at which time monacoline K can be isolated and recovered from the culture broth by any combination of isolation methods selected for its physical and chemical properties. Eg. any or all of the following isolation techniques may be used: Extraction of liquid from the culture broth with a hydrophilic solvent (eg diethyl ether, ethyl acetate, chloroform or benzene); extracting the organism itself with a hydrophilic solvent (e.g., acetone or an alcohol); concentration; dissolving in a more polar solvent (e.g., acetone or an alcohol); removing impurities with a less polar solvent (e.g., petroleum ether or hexane); gel filtration through a column of a material of "Sephadex" 20 (trade name for a material sold by Pharmacia

Co., Ltd., USA); absorbent chromatography with active carbon or silica gel, etc. Using an appropriate combination of these methods, the subject monacoline K can be isolated from the culture broth as a pure compound.

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Monacolin K has been found to have the following properties: 1. Color and shape: - colorless crystals 2. Melting point: - 157-159 ° C (under decomposition) 30 3. Elemental analysis: C 71.56%; H, 8.85%; 019.59%.

4. Molecular weight: - 404 (by mass spectrometry): 5. Molecular formula - C24H36 ° 5 · 6. Ultraviolet absorption spectrum (methanol): 35 As shown in FIG. 1 there is etmaxdmum. at 232, 238 and 246 μπι.

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7. Infrared absorption spectrum (KBr):

The spectrum is as shown in FIG. 2nd

8. Nuclear Magnetic Resonance Spectrum (60 MHz proton): 5 The spectrum is shown in FIG. 3 in deuterated chloroform, using tetramethylsilane as the internal standard.

9. Nuclear Magnetic Resonance Spectrum (C) -:

The spectrum is shown in FIG. 4 in deuterated methanol.

10 10. Solubility:

Soluble in alcohols (eg methanol, ethanol and propanol), acetone, chloroform, ethyl acetate and benzene. Insoluble in petroleum ether and hexane.

11. Specific rotation: [cc] D = 307.6 (c = 1, methanol).

12. Thin-layer chromatography: Rf = 0.47 [No. 5715 "Kieselgel 60F254 silica gel (Merck & Co., Ltd.) elicited with a 4: 1 volume mixture of methylene chloride and acetone, visible as an ultraviolet radiation absorbent stain, 50 v / v sulfuric acid (a faint red and reddish brown color produced by heating ) or with iodine].

The compound is neutral and insoluble in neutral or acidic aqueous substrates. It is converted to an acidic compound by treatment with an alkali, and then it can be dissolved in water. The acidic compound can be extracted with ethyl acetate or chloroform at an acidic pH and it will be converted to 30 monacoline K by evaporation of the solvent.

The physiological effect of monacoline K salts can be investigated and quantitatively determined by the following experiments.

35 1. Inhibition of cholesterol biosynthesis

Like monacoline K itself, monacoline K salts can specifically reverse the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which is the rate-determining enzyme in the biosynthesis of cholesterol. Table 1 lists the concentrations (in ng / ml) 7

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of the subject compounds which inhibit the activity of 3-hydroxy-3-methylglutaryl (HMG) -CoA reductase by 50 per cent. (measured by the method described in Analytical Biochemistry, 383 (1969), and the concentrations (in ng / ml) of the subject compound 5 inhibiting cholesterol biosynthesis by 50 per cent [measured by the method described in Journal of Biological Chemistry, 247, 4914 ( 1972)].

The corresponding results are also given for the known compound ML-236B (there is a compound known to have the same type of activity produced by growing microorganisms of the genus Penicillium disclosed in English Patent Specification No. 1,453 425). The concentration of monacoline K itself that inhibits cholesterol biosynthesis by 50 per cent is also indicated. It will be seen that while the concentration of 15 ML-236B required to inhibit cholesterol biosynthesis by 50 per cent is 10.0 ng / ml, the corresponding concentration of the sodium salt of monacoline K is approximately 0.14 ng / mg ( i.e. about seventy times better). The effects of the salts of monacoline K are comparable to or better than the mona-choline K itself.

Table 1 Compound Concentration (ng / ml) needed to inhibit by 50 per cent.

HMG-CoA cholesterol _ reductase_ biosynthesis sodium salt 2.0 0.14 3Q calcium salt 12.0 1.8 monacolin K - 2.0 ML-236B _10.0_10.0 2. Reduction of blood cholesterol levels

The experimental animals used were rats of the Wistar Imamichi strain, each weighing approximately 300 g. The experiments were performed on groups of rats, each consisting of five animals. Every animal

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8 S) was injected intravenously with 400 mg / kg of "Triton '**" WR-1339 (trade name for a substance known to increase blood cholesterol level) while simultaneously administering one of the compounds shown in Table 2 orally in the amount 5 are listed in the table. Twenty hours after oral administration of the animals, the animals were alive by bleeding, and blood and liver were examined and their cholesterol levels determined by conventional methods. The results are given in Table 2, which indicates the reduction in blood and liver cholesterol levels compared to a control group of rats given only "Triton®" WR-1339 alone.

For comparison, the corresponding results are given for monacoline K itself and for ML-236B, but these compounds were administered at doses substantially greater than the doses used by the compounds of this invention to achieve a similar reduction in cholesterol levels.

Table 2 20

Reduction of Cholesterol Compound Dose levels (%) in mg / kg of blood liver sodium salt 2 27.5 18/9 25 calcium salt 5 21.4 17.1 monacoline K 10 22.4 16.7 ML-236B 40 24.6 20 , 9 30

The reduction in blood and liver cholesterol levels is given by the following formula: 1 ", ft) x loo A - B 35

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wherein A = level in group treated only with "Tritor®" WR-1339 9 B = level in untreated control group 5 C = level in experimental group 3. Acute toxicity

The compounds studied were the sodium and calcium salts 10 of monacoline K. It was found that it was 50 per cent. lethal dose (LD 2) of each of the compounds was at least 2,000 mg / kg by intraperitoneal administration. Thus, the compounds have very low toxicity.

The results show that the compounds of this invention inhibit the biosynthesis of cholesterol and, as a result, lower blood cholesterol levels. Thus, they are valuable drugs in the treatment of hepatic lipaemia and arteriosclerosis.

The compounds of the invention are administered orally or parenterally in the form of capsules, tablets, injectable preparations or in other well-known ways, although it is usually preferred to administer them orally. The dose depends on the age and weight and condition of the patient, but usually a 25 daily dose is preferred for an adult of 0.1-100 mg, preferably 0.1-100 mg in the case of monacoline K salts.

The invention is further illustrated in the following example.

EXAMPLE

The Monascus rubber 1005 strain was seeded on a liquid liquid substrate containing 6% w / v. glucose, 2.5% w / v. peptone, 0.5% w / v. corn molding liquid and 0.5% w / v. ammonium chloride. Cultivation was continued under aerobic conditions at a temperature of 28 ° C for 10 days. The resulting filtrate (5 L) from the culture broth was adjusted to a pH of 3 by the addition of 6N hydrochloric acid, then extracted with an equal volume of ethyl acetate. The solvent is evaporated under reduced pressure from the extract and the resultant

Claims (4)

A process for the preparation of the carboxylic acid of a cholesterol synthesis inhibitory compound called monacoline K and of the formula: 3 or VO, or alkali salts thereof, characterized in that a strain of Monascus rubs are grown in a culture medium containing assimilable carbon and nitrogen sources, add an alkali to the culture mixture to give alkali salts of the carboxylic acid of monacoline K, and then either isolate the alkali salts from the alkaline culture mixture or extract the acid with an organic solvent, ethyl acetate or chloroform, at an acidic pH. Method according to claim 1, characterized in that the strain is Monascus ruber strain 1005 (FERM 4822, NRRL 12073). Process according to claim 1 or 2, characterized in that the cultivation is carried out at a temperature of 7 to 40 ° C. Process according to claim 3, characterized in that the temperature is from 20 to 35 ° C. 20 25 30 35