DD154494A5 - PROCESS FOR PREPARING MONACOLINE K - Google Patents

Abstract

The invention relates to a process for the preparation of monacolin K for use as a medicament for hypercholesterolemia. The aim of the invention is to provide a compound having an improved action for lowering the cholesterol content in the blood. According to the invention, a Monacolin K compound of the following formula is prepared by culturing microorganisms of the culture strain Monascus ruber, strain 1005, in a suitable culture medium at a temperature of from 7 to 40 ° C, especially from 20 to 35 ° C.

Description

Berlin, 3.9.1980

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Process for the preparation of monacolin K Field of application of the invention

The present invention relates to a new compound having hypercholesterolemic activity! It is the Monacolin K.

In particular, the invention relates to a process for the preparation of an agent against hypercholesterolemia under the name Monacolin K.

The present invention further relates to a pharmaceutical composition of monacolin K in association with a pharmaceutically acceptable carrier or excipient.

Characteristic of the known technical solutions

High cholesterol levels in the blood are considered to be a major cause of cardiopathy, such as a myocardial infarction or atherosclerosis. As a result, extensive research has been undertaken for the purpose of finding physiologically acceptable substances capable of delaying cholesterol biosynthesis and thus lowering cholesterol levels in the blood. One such compound is ML-236, which is the subject of British Patent No. 1,453,425. The substance ML-236 is prepared by growing microorganisms of the genus Penieilium.

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Object of the invention

The aim of the invention is to provide a composition with an improved action for lowering the cholesterol content in the blood.

Explanation of the essence of the invention

The invention has for its object to find new microorganisms from which substances with improved activity for lowering the cholesterol content in the blood can be bred.

In the study of fungi of the genus Monaseus, it was found that these, in particular the culture strain 1005 ^of .MonasCus ruber (FERN 4822), produced an agent with an activity against hypercholesterolemia which had a substantially better activity than the substance ML-236 , This product was called Monacolin K,

The compound monacolin K which has been cultured according to the invention corresponds to the following formula:

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All microorganisms of the genus MojTgg which are capable of producing the monacolin K can be used in the process according to the invention. Particularly useful are culture strains of Monascus ruber, of which especially the culture strain 1005 of Monascus ruber (FERM 4822),

The culture strain 1005 of Monascus ruber (FERM 4822) corresponds to a newly isolated microorganism having the following microbiological properties: It was isolated from foods produced in Thailand and purchased on February 16, 1979 under the number FERM 4822 as a new acquisition at the Fermentation Institute, Industrial Science and Technology, Ministry of International Trade and Industry, Oapan, and NRRL 12073 as a new acquisition from the Agricultural Research Service, Northern Regional Research Laboratory, USA.

The growth proceeds on a potato-glucose agar medium

at 25 ⁰ C rapidly and the diameter of the colony reaches 6 to 6.5 cm 10 days after inoculation · The colony is flat and a relatively thin base layer of fungal hyphae develops (hyphae) »The development of aerial fungal filaments is poor; the air mushroom threads are white and most of them are woolly. "Many corkeries are formed on the base layer of the 'fungal threads and assume a reddish-brown coloration during maturation." Both the surface and the underside of the colony have a brown to reddish-brown color.

The growth on a Sabouraud agar medium is very rapid at 25 G and the diameter of the colony reaches 6 to 6.5 cm 10 days after inoculation. The surface of the colony is very flat and the fungal filaments as well as the fungal filaments develop better than on the potato-glucose-agar medium. "The number of cisterns is very low. * The surface of the colony has a reddish-yellow to reddish-brown color and the underside is reddish-brown to dark brown ·

The growth on an oatmeal agar medium is slow at 25 G and the diameter of the colony reaches 1.5 to 2 cm 10 days after the successful inoculation * The colony is flat »The development of aerial fungal threads and the formation of Kleistotheken is always very bad. Both the surface and the underside of the colony have a dark red to reddish brown color.

The growth on a Czapek agar medium is very slow and the diameter of the colony reaches 10 days after the inoculation SUC-GTEN 1.6 to 1.8 cm x with 25 ^0C

The growth rates on each of the above-mentioned nutrients were 25

Nutrient media are essentially equal to 37 ° C

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2. Morphological properties

The cisterns are spherical and have diameters of 30 to 60 / um; their walls are thin and membranous; their stems have Septalwände and each consist of a mushroom thread with a diameter of 3.5 to 4.5 / um and a length of 15 to 80 , ma. The ascus consists of 8 spores and is almost spherical and disappearingly small. The ascorbic spores are colorless and ovate or ellipsoidal; they have a size of 4 to 5x4 to 7 / um; and their surfaces are smooth. "The conidia are colorless and spherical or pear-shaped; their size is 6 to 9x6 to 11 , um; their base surfaces are dull and their walls are relatively thick and smooth. The conidia are connected basipetally (directed toward the base) in the sense of a dividing arthrospore. "The conidiophor corresponds to a vegetative fungal thread and is branched or unbranched; the conidia is formed on the top · The mycelia are colorless and branched and have septal walls; most of them have a diameter of 3 to 5 / um *

Based on the observations of their characteristics given above, these microorganisms were identified as a culture strain of Mona sous ruber van Tieghem.

The microbiological properties of Monaseus ruber have been reported in the following literature: Takada, Transactions of the Micological Society of Japan, 9, 125-130 (1969) / "" Materials on the fungal flora of Japan (7) "7, and van Tieghem , Bull * Soc. Offered. Prance, 3J., 227 (1884), About the emergence of ascospores of the culture strain is from CoIe and others in the journal Canadian Journal of Botany ,. 46, 987 (1968): "Conidia ontogenesis in Hyphomycetes. (Moniliales) $ The imperfect state of Mnajgcujg r_u_ber and the existing divisible arthospore."

Although the use of the culture strain 1005 of Monas ^ cjj ^ s

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It will be understood more fully below that any of the strains of the genus Monaseus, including the varieties and mutants capable of producing the substance monacolin K, can be used in the process of the invention.

The substance Monacolin K can be obtained in the way

The selected microorganisms are grown in a culture liquid under aerobic conditions, using the same technical method ^7, as are well known in the art for the growth of fungi and other microorganisms. For example, the microorganisms which produce the substance monacolin K may first be cultivated on a suitable nutrient medium and subsequently the microorganisms formed may be collected and inoculated into another culture fluid and cultured on this other culture fluid in order to obtain the desired substance monacolin K; the breeding ground for the multiplication of microorganisms and for the formation and development of monacolin K may be the same or different from each other.

Each culture liquid that is well known in the art for the cultivation of mushrooms can _s be used provided, however, that, as is well known, contains the necessary nutrients, especially an assimilable carbon source and an assimilable nitrogen source * Examples Suitable sources of assimilable carbon include glucose, maltose, dextrin, starch, lactose, sucrose and glycerine. Of these carbon sources, glucose, glycerine and starch are particularly preferable for producing the substance Monacolin K. Examples of suitable sources of assimilable nitrogen are peptone, meat extract, yeast, yeast extract, soybean meal, peanut cake flour, corn steep liquor, rice bran and inorganic nitrogen sources Nitrogen sources, the peptone is particularly preferred. When extracting the substance Monacolin K can, if necessary,

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an inorganic salt and / or a metal salt are added to the culture liquid. In addition, if necessary, a smaller amount of a heavy metal may also be added.

The microorganism is preferably cultured under aerobic conditions using culturing methods well known in the art, for example by means of a culture on solid medium, a shaking culture or a culture with aeration and stirring. The microorganism is grown in a wide temperature range, for example from 7 C to 40 C, aber.besonders for obtaining the substance Monacolin K is the preferred growth temperature within the range of 20 ⁰ C to 35 ⁰ C.

During the growth of the microorganisms, the recovery of monacolin K may be monitored by sampling the culture fluid and measuring the physiological activity of the monacolin K substance in the culture fluid by the experiment described below. Cultivation may then be continued until substantial enrichment of monacolin K in the culture fluid has been achieved. At this time, the substance Monacolin K can be isolated and recovered from the culture fluid by any suitable combination of purification methods. These purification methods are selected with respect to the physical and chemical properties of the above substance. For example, any or all of the following purification methods may be used: extraction of the liquid from the culture fluid

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a hydrophilic solvent (for example with diethyl ether, ethyl acetate, chloroform or benzene); Extraction of the organism with a hydrophilic solvent (such as acetone or an alcohol); Enrichment; Dissolving in a more polar solvent (for example in acetone or an alcohol); Removal of impurities with a less polar solvent (such as petroleum ether or hexane); Gel filtration through a column of material containing Sephadex (a trade name); Absorption chromatography with activated charcoal or silica gel, etc. By using a suitable combination of these purification methods, the desired substance Monacolin K can be isolated from the culture liquid as a pure substance.

It has been found that the monacolin K has the following properties:

1 * Color and shape: colorless crystals «

2nd melting point: 157 to 159 ° C (with decomposition).

3. Elemental analysis: C - 71.56 %; H = 8.85 %; 0 = 19.59 %,

4. State of the art biomass: 404 (using mass spectrometry.

5. Molecular formula: 0 _{ο / 1} Η, ._ 0_.

6 UVJ WIPER RANGE ( methanol ): According to Figure 1 of the accompanying drawings, there are maxima at 232, 238 and 246 m ^ i.

7. Infra redabsor pti on ^ sp ^ ej ^ t rum (KB r): See Figure 2 of the accompanying drawings.

8. Mag η et Isc h e s Ke r nre 8 ο η a η ζ ep ek t_r u m_ (60JjHz ₁ Prot onι): See Figure 3 of the accompanying drawings in deuterated chloroform using tetramethylsilane as the internal standard.

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iil ^ .i- ^ iRxJlS.n ^ Il (. '. ", p.) ^: See here

Figure 4 of the accompanying drawings in deuteride methanol.

ir ^ äiAS, ™ ^? -? ^Solubility: soluble in lower alcohols (for example methanol, ethanol and propanol), acetone, chloroform, ethyl acetate and benzene; insoluble in petroleum ether and hexane "

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11. Specific turning ability; / ⁰ L / τ) = +307.6 (c = 1, methanol)

12. Thin layer chromatographic graph: R _f = 0.47

/ "" 2Tr. 5715 Kieselgel 60P ₂ C / silica gel (Merck & Co., Ltd. »), evolvable by a mixture of methylene chloride and acetone in a volume ratio of 4t-1» detectable as a UV radiation absorbing mass, using 50% sulfuric acid (v / v) (a light red to reddish brown color develops when heated) or with

The compound 'is neutral and is insoluble in neutral or acidic aqueous media.' Conversion to an acidic substance is carried out by treatment with an alkali and then the dissolution in water can be carried out. This acidic substance can be extracted with ethyl acetate or chloroform at an acidic pH and the reconversion into the substance monacolin K will take place upon evaporation of the solvent *

The physiological activity of the substance Monacolin K can be determined and quantified by the following experiment on a living object *

Attempt on living object with rabbit

In this experiment, the ability of the substance Monacolin K is measured to reduce the cholesterol content in the blood of rabbits * The animals used for this purpose must have a weight in the order of 2.5 kg to 3.0 kg.- Immediately before the start of the experiment Blood is taken from the vein in one ear of each rabbit and the cholesterol content in the blood serum is measured by a known method. A predetermined amount of monacolin K is then orally administered continuously over a period of 1 day to 5 days, and the cholesterol content in the blood serum is measured after the administration has taken place. The mode of action of the substance Monacolin K or de-r

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Monacolin K containing culture Fluseigke.it can then quantitatively from the. Cholesterol levels are determined before and after administration; the Substans Monasolin K were obtained »

We have demonstrated the ability of Monacolin K as a substance to reduce cholesterol levels in the blood and liver by various experiments on the living object *

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The animals used for these experiments were rats of the Wistar strain Imamichi with a particular body weight of about 300 g ₀ The experiments were conducted on groups of rats, each group would consist of 5 animals each animal (intravenously 400 mg / kg Triton WR-1339 a trade name for a material known to increase blood cholesterol content); while intraperitoneally administered 10 rag / kg Monacolin K "14 hours after intraperitoneal administration, the rats were killed by bloodletting and. the blood was collected and its cholesterol content by known means determined * As a result of this experiment that the Blutcholersteringehalte had been reduced was found ₅ compared with a Kontieollgruppe of animals ,; the Triton WR - 1339 alone was administered. "The reduction made 23 _? 9% off «.

The experimental animals were rabbits weighing 2 _V 7 kg to 2 _S 9 kg. "Each rabbit received 1 mg / kg Monacolin K orally twice daily (morning and evening). For 5 days before administration and on 3 and 5 * days after the administration, blood was collected from a vein in the ear and collected, and the cholesterol contents in the blood serum were determined. As a result of these experiments, it was found that the cholesterol levels were 3 "and 5 * day after the administration of the substance

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Monacolin K were 15% and 29% below the levels prior to administration of Mona Colin K.

In addition to its valuable retarding effect on the biosynthesis of cholesterol, monacolin K has a very low toxicity. Thus, the acute oral toxicity (LD) of monacolin K in the mouse corresponds to 1 g / kg of body weight or more.

The Monacolin K can be administered orally or parenterally in the form of a capsule, tablet, injectable or any other known preparation, although it is usually preferred to administer the substance orally. The dose will vary, depending on the age and body weight of the patient, as well as the severity of the condition conditions, but generally the daily dose for an adult would be 0.5 to 50 mg, either as a single dose or in the form of 2 or 3 divided cans. However, given the low toxicity of the compound, higher doses may be used if necessary.

Exemplary Example

The present invention will be further illustrated by the following non-limiting example

Example .

The culture strain 1005 of MojTascus £ UJDe £ was made up of a fluid of 6 % massAlumen glucose, 2.5 % mass / volume peptone, 0.5% mass / volume corn steep liquor and

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0.5 / ο mass / volume of ammonium chloride inoculated "The cultivation was continued under aerobic conditions at a temperature of 28 C for 10 days. The resulting filtrate (5 L) of culture broth was adjusted to pH 3 by adding 6N hydrochloric acid. Then, ethyl acetate was extracted with an equal volume. The solvent was removed from the extract by evaporation under reduced pressure and

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the resulting residue was dissolved in 100 ml of benzene. Insoluble substances were filtered off.

The filtrate was washed twice, each time with 100 ml of a 5% (mass / volume) aqueous solution of sodium bicarbonate. 100 ml of one. 0.2 N aqueous solution of sodium hydroxide was then added to the washed filtrate and the mixture was stirred at room temperature. After confirming the disappearance of Monacolin K from the benzene layer by thin layer chromatography, the aqueous layer was separated. The pH of the aqueous layer was then adjusted to 3 by adding 6N hydrochloric acid. The resulting solution was extracted twice. each time with 100 ml of ethyl acetate. The extract was evaporated to dryness under reduced pressure to give 260 mg of an oil. This oil was dissolved in benzene and allowed to crystallize. Thereafter, a recrystallization was carried out from an acetone aqueous solution. mg Monacolin K were obtained in the form of colorless needles with the properties described above.

Claims (5)

1 »Process for the preparation of Monacolin K ₁ characterized in that Monacolin K-forming microorganisms are grown in a corresponding culture medium, 2 * Method according to item 1, characterized in that the microorganisms correspond to a culture strain of Mojiajscjjjs ^ ujser. 2 r ο ι 5 * »-» " ^{AP C ί?} - ^{D / 219} Ai ^ iOO - 1 & ~ 56 959/12 Erfindunqsanspruch 3, method according to item 2, characterized in that the culture strain is the strain 1005 of MoQg _- Scu_s .rujbejl. 09/03/1980 4.- Method according to any one of the preceding points, characterized in that the cultivation of the culture strain at
leads. Strain at a temperature of 7 ⁰ C to 40 ⁰ C be · 5 * Method according to item 4, characterized in that the temperature corresponds to 20 ⁰ C to 35 ⁰ C. ^ P? ££ *? !! // ζ ^ η inulin ν «° * ι ρ