CA1129794A

CA1129794A - Antihypercholesteraemic agent, monacolin k, and its preparation

Info

Publication number: CA1129794A
Application number: CA345,983A
Authority: CA
Inventors: Akira Endo
Original assignee: Sankyo Co Ltd
Current assignee: Sankyo Co Ltd
Priority date: 1979-02-20
Filing date: 1980-02-19
Publication date: 1982-08-17
Also published as: IE800321L; PL222120A1; NL191540C; DK149095B; DE3006216C2; FI800506A; SU969702A1; NL191540B; FR2449685B1; SU1158048A3; NO153974C; PL124304B1; DD154494A5; MX6314E; KR830002030A; KR830002801B1; DE3051175C2; IT8067262A0; FI66427B; ES488796A0

Abstract

ABSTRACT

A new compound, which we refer to as Monacolin K, has the molecular formula C24H3605 and has been found to have valuable antihypercholesteraemic activity.
It can be produced by cultivating suitable micro-organisms from the genus Monascus, especially Monascus ruber strain 1005 (FERM 4822).

Description

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"ANTIHYPERCHOLESTERAEMIC AGENT, MONACOLIN K, AND ITS

PREPARATION"
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The present invention relates to a new compound having antihypercholesteraemic activity and which we 5 have named Monacolin K. Monacolin K can be produced by cultivating various microorganisms of the genus ! Monascus.

Thus the present invention consists in a compound, Monacolin K, having the formula:

H W
,. ~fO

The invention further consists in a process for . preparing an antihypercholesteraemic agent designated Monacolin K, which comprises cultivating a Monacolin K-producing microorganism of the genus Monascus in a culture medium therefor.

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7~4 The invention still further consists in a pharmaceutical composition comprising Monacolin K in admixture with a pharmaceutically acceptable carrier or diluent.

High blood cholesterol levels are recognized as being one of the main causes of cardiopathy, such as cardiac infarction or arteriosclerosis. As a result, considerable research has been undertaken with a view to discovering physiologically acceptable substances which are capable of inhibitlng cholesterol biosynthesis and thus reducing blood cholesterol levels. One such compound is ML-236, which forms the subject of our United Kingdom Patent Specification No. 1,453,425. ML-236 is produced by cultivating microorganisms of the genus Penicilllum.

' On investigating fungi of the genus Monascus, it was found that these, particularly Monascus ruher i strain 1~05 tFERM 4~22), produced an antihyper-I cholesteraemic agent having substantially better ¦ 20 activity than that of ML-236. This agent was named Monacolin K.

~' All microorganisms o~ the genus Monascus which are capable of producing Monacolin K may be employed ;

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in the process of the present invention. Especially useful are strains of Monascus ruber, particularly Monascus ruber strain 1005 ~fERM 4822).

Monascus ruber strain 1005 (FERM 4822) is a newly isolated microorganism having the following microbiological properties. It was isolated -From foodstuffs produced in Thailand and deposited on 16 February 1979 under the accession No. FERM 4822 with the Fermentation Research Institute, Agency of Industrial Science and TechnologyJ Ministry of International Trade and Industry, Japan and under ; the accession No, NRRL 12073 with the Agricultural Research Service, Northern Regional Research Laboratory, USA.

1. Growth ~ The growth on a potato-glucose-agar medium at i 25C is fast and the diameter of the colony reaches 6 - 6,5 centimetres 10 days after inoculation. The colony is flat and a relatively thin basal layer of hyphae develops. Development of aerial hyphae is poor; the aerial hyphae are white and most of them are woolly. Many cleistothecia are formed on the basal layer of hyphae and turn reddish-brown on maturity. Both the surface and the reverse of the , .

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colony are brown to reddish-brown in colour.

The growth on Sabouraud's agar medium at 25C is very fast and the diameter of the colony reaches 6 - 6.5 centimetres 10 days after inoculation.
The surface of the colony ls very flat, and basal hyphae and aerial hyphae develop better than on potato-glucose-agar medium. Cleistothecia counts are very few. The surface of the colony is reddish-yellow to reddish-brown in colour and the reverse 10 i9 reddish-brown to dark brown.

I The growth on oatmeal agar at 25C is slow ,~ and the diameter of the colony reaches 1.5 - 2 cænti-metres 10 days after inoculation. The colony is flat. Development of aerial hyphae and formation of cleistothecia are both very poor. Both the surface and the reverse of the colony are dark red to reddish-brown in colour.

The growth on Czapek's agar medium at 25C
J is very slow and the diameter of the colony reaches 20 1.6 - 1.8 centimetres 10 days after inoculation.

The rates of growth on each of the above media at 37C are substantially equal to tnoss at 25C.

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2. Mo~ l~ ical properties The cleistothecia are spherical and 30 - 60 microns in diameter~ their walls are thin and mem-branous, their stalks have septal walls and each consists of a hypha o-F diameter 3.5 - 4.5 microns and length 15 - ao microns. The ascus consists o~
8 spores and is nearly spherical and evanescent.
The ascospores are colourless and ovoid or ellipsoid~
they have a size of 4 - 5 x 4 - 7 micronsS and their sur-Faces are smooth. The conidia are colourless and spherical or pyriform; their size is 6 - 9 x 6 - 11 microns~ their bases are truncate and their walls ars relatively thick and smooth. The i conidia are linked basipetally as a type of meristem arthrospore. The conidiophoreis like a vegetative hypha and is branched or unbranched, the conidia being formed at the top. The mycelia are colourless ; and branched and have septal walls~ most of them have a diameter of 3 - 5 microns.

Z0 ~ased on the observations of its charactsristics as reported above this microorganism was identified as a strain of Monascus ruber van Tie~hem.

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Microbiological propsrties of Monascus ruber have been reported in the following literature:
Takada, Transactions of the Micological Society of Japan, 9, 125 - 13û (1969) [Materials for the Fungus Flora of Japan (7)]~ and van Tieghem, Bull, Soc. Botan. France, 31, 227 (1884). Ascospore generation of the strain has been reported by Cole et al in the Canadian Journal of Botany, 46, 987 (196B), "Conidium Ontogeny in hyphomycetes~ The imperfect state of Monascus ruber and its meristem arthrospores".

Although the use of Monascus ruber strain 1005 I is hereafter speci-Fically exemplified, it will be appreciated that any strains o-F the genus Monascus, including varieties and mutants, which are capable of producing Monacolin K can be used in the process - of the invention.

Monacolin K may be produced by cultivating the chosen microorganism in a culture broth under aerobic conditions, using the same techniques as are well known in the art for the cultivation of fungi and other microorganisms. For example, the Monacolin K- producing microorganism may first be 7.
cultivated on a suitable medium and then the produced microorganisms may be collected and inoculated into and cultivated on another culture medium -to produce the -desired Monacolin K; the culture media used for multiplication of the microorganism and for production of Monacolin K may be the same or differentO
~ ny culture medium well known in the art for the cultivation of fungi may be employed, provided that it contains, as is well known, the necessary nutrlent materials, especially an assimilable carbon source and an assimilable ni-trogen source. Examples of suitable sources of assimilable carbon are glucose, maltose, dextrin, starch, lactose, sucrose and glycerine. Of -these sources, glucose, glycerine and starch are particularly preferred for the production of Monacolin K. ~xamples of suitable sources of assimilable nitrogen are peptone, meat - extract, yeast, yeast extract, soybean meal, peanut meal, corn steep liquor, rice bran and inorganic nitrogen sources. Of these nitrogen sources, peptone is particularly preferred. When producing Monacolin K, an inorganic salt and/or a metal salt may, if necessary, be added to the culture mediu~
~urthermore, if necessary, a minor amount of a heavy metal may also be added.
~ he microorganism is preferably cultivated under aerobic conditions using cultivation methods .

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well known in -the art, for example solid culture, shaken culture or culture under aeration and agitation. ~he microorganism will grow over a wide temperature range, e.g. from 7 to 40 C, but, especially for -the pro-duction of Monacolin K, the more preferred cultivationtemperature is within the range from 20 to 35 C.
During the cultivation of the microorganism, the production of Monacolin ~ may be monitored by sampling the culture medium and measuring the physiological activity of the Monacolin K in the culture medium by the test described hereafter.
~ultivation may then be continued until a substantial accumulation of Monacolin K has been achieved in the culture medium, at which time the Monacolin K may be isola-ted and recovered from the culture broth by any suita~le combination of isolation -techniques ; chosen having regard to its physical and chemical properties. For example, any or all of the following ; isolation techniques may be employed extraction of ; 20 the liquor from the culture broth with a hydrophilic solvent (for example, diethyl ether, ethyl aceta-te, chloroform or benzene); extraction of the organism with a hydrophilic solvent (such as ace-tone or an alcohol); concentra-tion; dissolution into a more polar solvent (e.g. acetone or an alcohol); removal of impurities with a less polar solvent (such as petroleum ether or hexane~; gel filtration through 517~

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a column of a material such as Sephadex (a trade name for a material available from Pharmacia, Co., Ltd., U.S.A.); absorptive chroma-tography with acti~e - carbon or silica gel; and so onn By using a suitable combination of these techniques, the desired Monacolin K can be isolated from the culture broth as a pure ::
substance.

Monacolin K was ~ound to have the ~ollowing ~ :
properties~

1. Colour and form:
Colourless crystals.
2. Melting point:
157-159 C (with decomposition)-

3. ~lemental analysis:
C, 71.56%; H, 8~85%; 0, 19.59%.

4. Molecular weight:
. 404 (by mass spectrometry) . 5. Molecular formula:
C2L~H3605 .

, 20 6. Ultraviolet absorp-tion spectrum (methanol):
As shown in Figure 1 of the accompanying drawin~ss ha.ing maxima at 232, 238 and 246 m).

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7. Infrared absorption spectrum (KBr):
As shown in Figure 2 of the accompanying drawings.
8. Nuclear magnetic resonance spectrum (60 MHz proton):
As shown in ~igure 3 of the accompanying drawings in deuterated chloroform, using tetr~methylsilane as internal standard.
9. Nuclear magnetic resonance spectrum (13C):
As shown in Figure 4 of the accompanying drawings, in deuterated methanol.
10. Solubility:
Soluble in lower alcohols (e.g. me-thanol, ethanol and propanol), acetone, chloroform, ethyl acetate and benzene.
Insoluble in petroleum ether and hexaneO
11. Specific rotation:
[C<~2D5 = +~07O6 (c=1, methanol).
12. Thin layer chromatography:
Rf = 0.47[No. 5715 Kieselgel 60~25L~ silica gel ~Merck & Co., ~td.) developed by a 4:1 by volume mixture of methylene chloride and acetone, detectable as an ultraviole-t radiation-absorbing lump, 50% v/v sulphuric acid (a pale red to reddish-brown colour develops on heating) or with iodinel.

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The compound is neutral and is insoluble in neutral or acidic aqueous media. It is cDnverted to an acidic substance upon treatment with an alkali and can then be dissolved in water. This acidic substance can be extracted with ethyl acetate or chloroform at an acid pH value and will revert to Monacolin K on evaporation of the solvent.

The p~ysiological activity of Monacolin K
can be assayed and determined quantitatively by the following in vivo test.

In vivo tsst with rabbits In this test, the ability o-f Monacolin K to reduce cholesterol levels in rabbit blood is measured.
The animals employed should weigh from 2.5 to 3.0 kg.
Immediately prior to starting the test, blood is collected from the vein in an ear of each rabbit and ths cholesterol level in the blood serum is measured by a conventional method. A predetermined quantity of Monacolin K is then administered orally continuously for 1 to 5 days and the cholesterol level in the blood serum after administration is measured. The potency of the Monacolin K or MDnacolin K-containing culture medium can be determined quantitatively from the cholesterol values obtained prior to and after administration of Monacolin K.

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We have demonstrated the ability of Monacolin K
to lower the blood and liver cholesterol levsls by various in vivo tests Reduction of blood cholesterol levels in rats .
The animals used were rats of the Wistar Imamichi strain, each having a body weight of about 300 g. The rests were conducted on groups of rats, sach group con-sisting of 5 animals. Each animal was intravenusly injected with 400 mg/kg of Triton WR-1339 ta trade ~ff-~e for a material known to increase the blood cholesterol level) whilst simultaneously administering intraperitoneally 10 mg/kg of Monacolin K. 1~ hours after intraperitoneal administration, tlle rats were sacrificed by bleeding and ~ the blood was collected and its cholesterol level was s 15 determined by conventional means. As a result, it was established that blood cholesterol levels had been reduced, as compared with a control group of animals to which :f Triton WR-1339 alone had been administered, by 23.9%.
f Reduction o-f blood cholesterol levels in rabbits ff 20 The test animals used were rabbits having a body weight of from 2.7 kg to 2.9 kg. Each rabbit was given j orally 1 mg/kg of Monacolin K twice each day ~morning and evening) continuously for 5 days. Prior to administration and at 3 and 5 days after administration, blood was :, .. . .. . . ,, . _ .

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collected from a vein in the ear and the cholesterol levels in the blood serum were determined. As a result it was found that the cholesteroi levels at 3 and 5 days after administration of Monacolin K were 15~ and 29~j respectivelyJ lower than the level prior to administrati~n of Monacolin K, ~n addition to its valuable inhibitory effect on the biosynthesis of cholesterol, Monacolin K has a very low toxicity. Thus, the acute oral toxicity (LD50) of Monacolin K in the mouss is 1 g/kg body weight ; or more.

The ~onacolin K may be administered orally or parenterally in the form of a capsuleJ tabletJ
injectable preparation or any other known formulationJ
although we normally prefer to administer it orally.
The dose will vary, depending upon the ags and body weight of the patient and the severity of the condition, but,in general, the daily dose -for an adult would be from 0.5 to 50 mg, either as a single dose or in ' 20 2 or 3 divided doses. However, in view of the low ; toxicity of the compound, higher doses may be employed if required.
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The invention is further illustrated by the .
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folowing non-limiting Example.

EXAMPLE

Monascus ruber 1005 strain was inoculated onto a liquid culture medium containing 6% w/v glucose, 2.5% w/v peptone, 0.5% w/v corn steep li4uor and 0.5% w/v ammonium chloride. Cultivation was continued under aerobic conditions at a temperature of 28C for 10 days.
The resulting filtrate ~5 litrss) of the culture broth ; was adjusted to a pH value of 3 by the addition oF 6N
hydrochloric acid and then extracted with an equal volume of ethyl acetats. The solvent was evaporated under reduced pressure from the extract and the resulting residue was dissolved in 100 ml of benzene. Insolubles were filtered off.
The filtrate was washed twice, each time with 100 mI
~ of a 5% w/v aqueous solution of sodium bicarbonate.
;~ 100 ml of a 0.2N aqueous solution of sodium hydroxide ~ere then added to the washed -Filtrate and the mixture was stirred at room temperature. After confirming ~' ~0 the disappearance of Monacolin K from the benzene layer by thin layer chromatographyJ the aqueous layer was separated off. The pH value of the aqueous layer was then adjusted to 3 by addition of 6N hydrochloric acid and the resulting solution was extracted twice, each time ~; , 2~

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with 100 ml of ethyl acetate. The extract was 2vaporated -to dryness under reduced pressure, giving 260 mg of an oil. This oil was dissolved in benzene and allowed to crystallize and then recrystallized from an aqueous acetone solution to give 87 mg of cnlourless needles of Monacolin K having the properties : heretofor described.

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Claims

The embodiments of the invention in which an exclusive property or privilge is claimed are defined as follows:

1. A process for preparing Monacolin K, which process comprises cultivating a Monacolin K-producing microorganism of the genus Monascus in a culture medium therefor.

2. A process as claimed in Claim 1, wherein said microorganism is a strain of Monascus ruber.

3. A process as claimed in Claim 2, wherein said strain is Monascus ruber strain 1005.

4. A process as claimed in any one of Claims 1, 2 and 3, wherein the cultivation is carried out at a temperature of from 7 to 40°C.

5. A process as claimed in any one of Claims 1, 2 and 3, wherein the cultivation is carried out at a temperature of from 20 to 35°C.

6. Monacolin K when produced by a process which comprises cultivating a Monacolin K-producing micro-organism of the genus Monascus in a culture medium therefor and separating said Monacolin K from the culture medium.

7. Monacolin K when produced by cultivating Monascus ruber strain 1005 in a culture medium therefor and separating the Monacolin K from the culture medium.