GB2111989A - New compounds having cholesterol-reducing activity - Google Patents

Abstract

Dihydro- and tetrahydromonacolin L, of the general formula (I> <IMAGE> wherein X stands for-CH=CH- or -CH2-CH2- and derivatives thereof of formula <IMAGE> where Y is a metal cation or an alkyl group have cholesterol-reducing activity, making them useful as hyperlipemia treating agents. Dihydromonacolin L can be prepared by cultivation of a microorganism of the genus Monascus. The other compounds can be prepared by synthesis from dihydromonacolin L.

Description

SPECIFICATION New compounds having cholesterol-reducing activity The present invention relates to a novel physiologically active substance having a cholesterol-reducing activity. More particularly, the present invention relates to novel physiologically active substances, dihydro- and tetra - hydromonacolin L represented by the following general formula (I)

wherein A stands for-CH=CH- or-CHrCH=, having a cholesterol-reducing activity and metal carboxylate salt as well as alkyl carboxylate ester thereof represented by the following general formula (II),

wherein Y stands for M (in which M is a metallic element and n is an atomic valency of said metallic element), or a lower alkyl group having 1 to 6 carbons atoms;X has the same meaning as above, a process for preparing the same and a hyperlipemiatreating agent containing these substances as an active ingredient.

Hyperlipemia, particularly hyperchloesterolemia is known as one of the important factors of heart diseases such a myocardial infarct, and atherosclerosis. Therefore, the present inventor has made research investigations in order to find excellent novel physiologically active substances having a cholesterol-reducing activity from the microbial products. As a result, the present inventor have succeeded in obtaining an active substance dihydromonacolin L from the cultivated medium of a strain of the fungi.

It was found in an animal experiment using rats that the present substance is effective as a cholesterol-reducing agent. Further, the present substances were found to be novel substances from the examination of the physico-chemical properties of said substance.

Dihydromonacolin L is specified by the above said general formule (I) wherein X is-CH=CH- and represented by the following formula (Ill).

The present inventor has further conducted investigation and found thattetrahydromonacolin L represented by the following formula (IV)

which is specified by the above general formula (I) in which X is-CH2CHr has the similar effect and that metal carboxylate salt and alkyl carboxylate ester of dihydro- and tetra - hydromonacolin L are also excellent novel physiologically active substances having cholesterol-reducing activity like dihydromonacolin L, and thus the present invention has been accomplished.

An object of the present invention is to provide novel physiologically active substances having a cholesterol-reducing activity as well as a process for preparing the same.

Another object of the present invention is to provide a hyperlipemia-treating agent containing a novel phsiologically active substance having a cholesterol-reducing activity as an active ingredient.

Further object and advantages of the present invention will be clarified by the following description.

In the accompanying drawings, Fig. 1 is an infrared absorption spectrum (KBr) of dihydromonacolin Land Fig. 2 and Fig. 3 show the NMR spectrum (CDC13) and mass analysis spectrum of dihydromonacolin L, respectively.

Dihydromonacolin L provided according to the present invention and represented by the following formula (Ill)

can be obtained by cultivating microorganisms, a kind of fungi, particularly microorganisms belonging to genus Monascus and separating from the cultivated medium.

The microorganisms used in the present invention are dihydromonacolin L-producing strains belonging to genus Monascus and the strain which is considered by the present inventor as particularly effective is, for example, Monascus ruber No. 1005 which was isolated by the present inventor from foodstuffs produced in Thailand, and said strain was deposited on 16th February 1979 under the accession No. FERM-P 4822 at the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan and on 22nd November 1982 under the accession No. ATCC 20657 at the American Type Culture Collection, U.S.A.

It is needless to say that not only variant but also mutant strains of the microorganisms belonging to genus Monascus other than the above said one can be used if they have a dihydromonacolin L-producing ability.

The bacteriological properties of said dihydromonacolin L-preducing microorganism are as follows: 1. Growth The growth on a potato - glucose - agar medium at 25"C is fast and the diameter of the colony reaches 5-6 centimeters 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 colony are brown to reddish-brown in color.

The growth on Sabouraud's agar medium at 25"C is very fast amd the diameter of the colony reaches 6-6.5 centimeters 10 days after inoculation. The surface of the colony is 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 color and the reverse is reddish-brown to dark brown.

The growth on oatmeal agar at 25"C is slow and the diameter of the colony reaches 1.5-2 centimeters 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 color.

The growth of Czapek's agar medium at 25"C is very slow and the dimater of the colony reaches 1.6-1.8 centimeters 10 days after inoculation.

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

2. Morphological Properties The cleistothecia are spherical and 30-60 microns in diameter; their walls are thin and membranous; their stalks have septal walls and each consists of a hyphae of diameter 3.5-4.5 microns and length 15-80 microns. The ascus consists of 8 spores and is nearly spherical and evanescent. The ascospores are ovoid or ellipsoid; they have a size of 4-5 x 4-7 microns; and their surfaces are smooth. The conidia are colourless and spherical or pyriform; their size is 6-9 x6-11 microns; their bases are truncate and their walls are relatively thick and smooth. The conidia are linked basipetally. The conidiophore is like is vegetative hypha and is branched or unbranched, the conidia being formed at the top. The mycelia are branched and have septal walls; most of them have a diameter of 3-5 microns.

Based on the observation of its characteristics as mentioned above, this microorganism was identified as a strain of Monascus rubervan Tieghem.

Microbiological properties of Monascus ruber have been reported in the following literature: van Tieghem, Bull. Soc. Botan. France, 31,227 (1884); Cole et al, The Canadian Journal of Botany, 46,987 (1968), and Takada, Transactions of the Micological Society of Japan, 9,128 128(1969).

Dihydromonacolin L is produced by cultivating the dihydromonacolin L-producing strain aerobically in a nutrient medium in the known method as a cultivating method of fungi. For example, dihydromonacolin L-producing microorganism is cultivated serially on the medium containing 2% of soluble starch, 1 % of glucose, 2% of peptone and 2% of agar. For the purpose of producing dihydromonacolin L, the cell bodies grown on this agar medium are directly inoculated on the production medium and cultivated. Further, the cell bodies grown on the production medium are cultivated on a new production medium, wherein dihydromonacolin L can be produced.

The dihydromonacolin L-producing microorganism is grown at a temperature ranging from 7" to 40"C, but usually dihydromonacolin L is preferably produced at a temperature from 20 to 35"C. For cultivation of the dihydromonacolin L-producing microorganism belonging to genus Monascus, the nutrient sources known for cultivation of fungi and other microorganisms can all be employed. For example, glucose, maltose, dextrin, starch, lactose, saccharose, glycerin and the like can be used as a carbon source. Among these carbon sources, glucose and starch are favourable carbon sources for the production of dihydromonacolin L.

All of the nitrogen sources known for the growth of the microorganisms belonging to the genus Monasuc or others can be employed for the production of dihydromonacolin L. For example, peptone, meat extract, yeast extract, soybean meal, peanut meal, corn steepe liquor, rice bran and inorganic nitrogen source and the like can be used. In the production of dihydromonacolin L by the cultivation of the dihydromonacolin L-producing microorganism, an inorganic salt and a metallic salt are added, if required. A trace amount of a heavy metal can also be added, if required.

Dihydromonacolin L is obtained by cultivating the dihydromonacolin L-producing microorganism aerobically, and generally employed aerobic cultivation methods, for example, solid cultivation, shaking cultivation, and cultivation under aeration and agitation are employed. If antifoaming is required during the cultivation or sterilization of the medium, antifoaming agents such as, silicon oil, surface active agents and te like can be used. The temperature from 20 to 35 C is preferable for cultivation. The cultivation is continued until dihydromonacolin L is substantially accumulated and usually carried out for4to 17 hours.

Extraction of the present substance from the cultivated material can be carried out by a suitable combination of a variety of methods on the basis of the properties of the present substance which were disclosed by the present inventor. That is to say, these methods include extraction with a solvent such as ether, ethyl acetate, chloroform, benzene and the like; dissolution into a solvent having a large polarity such as acetone, alcohol, and the like; removal of impurities by the use of petroleum ether, hexane and the like having a small polarity; gel filtration by the use of Sephadex (RTM) column; an adsorption chromatography bythe use of activated carbon, silica gel and the like. The present substance is isolated from the cultivated medium as homogeneous substance by the use of an appropriate combination of these means.

More concretely, the present substances can be isolated from the cultivated medium by the combination of the following steps: Extraction with a solvent such as ethyl acetate and the like from the cultivated medium, evaporation to dryness of the extract, dissolving the extract into an organic solvent such as benzene and the like, extraction with an alkaline hydroxide aqueous solution, acidifying the aqueous solution by adding an acid and subjecting the solution to extraction with an organic solvent such as ethyl acetate, evaporation to dryness of the extract, dissolving the extract into benzene, removal of crystals formed by concentrating evaporation to dryness of the solution, dissolving the dried product into an organic solvent such as benzene, and finally purifying the crude product by means of an ordinary column chromatography to give a purified product.

The physico-chemical properties of dihydromonacolin L are described below.

Dihydromonacolin L represented by the above-mentioned general formula (III) is a white crystalline powder and soluble in the solvents such as lower alcohols, for example, methanol, ethanol, propanol and the like; acetone, chloroform, ethyl acetate, benzene and the like. It is insoluble in hexane, petroleum ether and the like. this substance is neutral and is not soluble in both neutral and acidic water. The lactone structure is ring-opened by the usual saponification with alkali and it is converted into a water-soluble acidic substance. This acidic substance is extracted with a solvent such as ethyl acetate, chloroform and the like in the acidic pH range and reconverted into dihydromonacolin L by evaporation to dryness.

The elementary analysis of dihydromonacolin L show the following values: carbon, 74.50%; hyd rogen,9.85%; and oxygen, 15.65%. It has a molecularweight of 306 and a molecular formula of C1gH3003. Figs. 1,2 and 3 show the infrared absorption spectrum, NMR spectrum and mass analysis spectrum of this substance, respectively.

Thin layer chromatography by the use of silica gel (manufactured by Merck & Co., No. 5715, Kiesel gel 60-F254) gives a single spot at a Rf value of 0.48 when hexane-acetone (1:1) is used as a developing solvent. Said spot can be detected by sulfuric acid spray (colored in reddish brown with weak heating) and iodine.

Tetrahydromonacolin L represented by the above-mentioned general formula (IV) is a white crystal and is soluble in lower alcohols such as methanol, ethanol, propanol and the like, and other solvents such as acetone, chloroform, ethyl acetate, benzene and the like, but insoluble in hexane, petroleum ether and the like. This substance is neutral and insoluble in water, but readily converted into a water-soluble acidic substance by ring-opening of the lactone structure with the usual alkali treatment.

Elementary analysis of tetrahydromonacolin L showed the following values: carbon,74.03%; hyd rogen, 10.39; oxygen, 15.58%. It has a molecular weight of 308 and the molecular formula thereof is C19lIO3.

Tetrahydromonacolin L can be obtained by dissolving monacolin L or dihydromonacolin L represented by the above said general formula (liy) in an organic solvent such as, for example, tetrahydrofuran, ethyl acetate and the like, and hydrogenating the dissolved compound in the presence of a catalyst such as, for example, platinum oxide, palladium-carbon and the like. Hydrogenation is carried out at a temperature from 1 0'C to 60"C, preferably 15 to 40"C and is usually completed in 10 to 24 hours. The amount of the catalyst is usually 1 to 60% by weight, preferably 2 to 30% by weight based on the weight of monacolin L or dihydromonacolin L.

Monacolin L used as a starting material is a known compound and can be obtained by isolating from the cultivated medium of Monascus ruber No. 1005 (FERM P-No. 4822), as disclosed in the Japanese Patent Application KOKAO (Laid-Open) No.57798/ 81.

Subsequently, a compound of the general formula (II) is explained.

As metal carboxylate salts of dihydro- and tetrahydromonacolin L, there are alkali metal salts such as sodium, potassium and the like; alkaline earth metals such as calcium, magnesium, and the like; aluminum salt; iron salt; zinc salt; copper salt; nickel salt; and cobalt salt or the like. Among them, particularly alkali metal salt, alkaline earth metal salt, and aluminum salt are preferred, and furthermore sodium salt, calcium salt and aluminum salt are most preferred.

As examples of alkyl carboxylate ester, there can be mentioned methyl ester, ethyl ester, n - propyl ester, isopropyl ester, n - butyl ester, n - hexyl - ester and the like.

The representatives of these compounds are shown below.

1. Dihydromonacolin L sodium carboxylate salt 2. Tetrahydromonacolin L sodium carboxylate salt 3. Dihydromonacolin L calcium carboxylate salt 4. Tetrahydromonacolin L calcium carboxylate salt 5. Tetrahydromonacolin L aluminum carboxylate salt 6. Dihydromonacolin L methyl carboxylate ester 7. Tetrahydromonacolin L methyl carboxylate ester 8. Tetrahydromonacolin L ethyl carboxylate es ter These compounds are synthesized in the following manner.

Tetrahydromonacolin L alkyl carboxylate ester is represented by the following general formula (V)

wherein R stands for an alkyl group having 1 to 6 carbon atoms, and is obtained by reducing catalytically carboxylate ester of the corresponding monacolin L or dihydromonacolin L represented by the following general formula (VI)

wherein - stands for a double bond in the case of monacolin Land a single bond in the case of dihydromonacolin L; R has the same meaning as above, in an organic solvent such as, for example, alcohol such as methanol, ethanol and the like; tetrahydrofuran, ethyl acetate and the like in the presence of a catalyst such as platinum oxide, palladium-carbon and the like.Reduction is carried out at a temperature ranging from 10' to 60 C, preferably 15 to 49 C. The catalyst is used usually in an amount of 1 to 60% by weight, preferably 2 to 30% by weight based on the weight of carboxylate ester of monacolin L or dihydromonacolin L.

The carbo2cylate ester can also be obtained by reacting the tetrahydromonacolin L with an alcohol (RCH, wherein R has the same meaning as above) corresponding to the desired ester.

Alkyl carboxylate ester of dihydromonacolin L is obtained usually by reacting dihydromonacolin L with ,rr alcohol corresponding to the desired ester.

In the above reaction, there is employed as a catalyst an inroganic acid such as hydrochloric acid, sulfuric acid and the like; boron fluoride, acidic ion exchange resin and the like and, as a solvent, an alcohol of the same kind, of benzene, chloroform, ether and the like which does not take part in the reaction.

Monacolin L aklyl carboxylate ester and dihydromonacolin L alkyl carboxylate ester used as a starting material can be obtained by reacting monacolin Lordihydromonacolin Lwith an alcohol corresponding to the desired ester.

Metal carboxylate salts of dihydro- and tetrahydromonacolin L are represented by the general formula (Vll)

wherein each of X, M and n has the same meaning as above, and they are synthesized in the following manner.

That is to say, they are obtained by dissolving dihydro- or tetrahydromonacolin L represented by the general formula (I)

wherein X stands for-CH=CH- or-CHl=CIHr in a solvent such as methanol, ethanol, acetone and the like, and reacting said compound with a metal hydroxide represented by the general formula (VIII).

M(OH)n (Vlel) wherein M stands for alkali metal such as sodium, potassium and the like; alkaline earth metal such as calcium, magnesium and the like; or metal element such as aluminum, iron, zinc, copper, nickel, cobalt, and the like; n is an atomic valency of said metallic element. Said metal hydroxide is usually used as an aqueous solution.

The physiological activity of dihydromonacolin L, tetrahydromonacolin Land derivatives thereof according to the present invention can be assayed by the following method which detects the blood cholesterol reducing activity in rats. Triton (RTM) WR-1339 [4-(1, 1,3,3 -tetramethylbutyl) phenol polymer with formaldehyde and oxirane, a surface active agent, manufactured by Rohm & Haas Co., U.S.A.], having a blood cholesterol elevating activity) is given to a group of 5 rats intravenously in a dosage of 400 mg/kg and at the same time a certain amount of each compound is administered orally.

The rats are blooded to death after 14 hours and the blood cholesterol is determined by the conventional method (Treated group). On the other hand, the rats to which Triton WR-1 339 only was given intraveneously are treated in the similar manner to determine the blood chloesterol (Control group).

The effect of dihydromonacolin L can be determined quantatively by comparing the cholesterol levels in both groups.

The serum cholesterol reducing activity, effective dosage and toxicity are shown below.

(1) Serum cholesterol reducing activity.

(A) Triton WR-1339 was given to rats intravenously in an amount of 400 mg/kg and at the same time dihydromonacolin b, tetrahydromonacolin Lora derivativethereofwasadministered orally (in the form of an aqueous solution or aqueous suspension). The rats were blooded to death after 14 hours and the blood cholesterol was determined by the conventional method. The results are shown in Table 1.

Table 1. Serum cholesterol reducing activity in rats

Compound dosage Serum cholesterol mg/kg reducing percent age (%) Dihydromonacolin L 20 23.4 100 30.6 Tetrahydromonacolin L 5 21 20 29 Tetrahydromonacolin 5 19 L methyl ester 20 31 Tetrahydromonacolin 5 20 L sodium salt 20 29 (B) Triton WR-1399 was given to rats intravenously in an amount of 400 mg/kg and at the same time each of monacolin L, dihydromonacolin L, tetrahydromonacolin L, or derivatives thereof was administered orally in a dosage of 10 mg/kg (in the form of an aqueous solution or aqueous suspen sion) Further, each of the above compounds was administered orally in a dosage of 10 mg/kg after 8 hours (total dosage being 20 mg/kg). The rats were then blooded to death after 12 hours (20 hours after administration of Triton WR-1339 and the serum cholesterol was determined by the conventional method.The results are shown in Table 2. Table 2

Compounds Serum cholesterol reducing percent age (%) Monacolin L 18.9 Dihydromonacolin L sodium salt | 31.3 Dihydromonacolin L methyl | 34.1 ester Tetrahydromonacolin L sodium 34.2 salt Tetrahydromonacolin L 1 38 methyl ester Asis apparent from the above results, the com- pounds of the present invention have an excellent cholesterol-reducing activity and particularly dihyd rnmonacolin L methyl ester, tetrahydromonacolin L and derivatives thereof have an excellent effect.

(2) Acute toxicity.

Acute toxicity was determined by adminstering orally and intraperitoneally an aqueous solution or aqueous suspension of hydromonacolin L, tetrahydromonacolin L and derivatives thereof. The results are shown in Table .

Table 3. Acute toxicity

Compound Animal Administration LD50 (mg/kg)~ method Orally > 1,000 Mouse Dihydrcmonacolin L. Intraperitoneally > 500 orally > 1,000 Rat Intraperitoneally > 500 Orally > 2,000 Mouse Intraperitoneally > 500 Tetrahydrcmonacolin L Orally > 2,000 Rat Intraperitoneally > 500 Orally > 2,000 Tetrahydrcmonacolin L Mouse Introperitoneally > 500 |methyl ester Orally > 2,000 Rat Intraperitoneally > 500 Table 3 (Cont'd)

Orally > 2,000 House Tetrahydromonacolin L . Intraperitoneally > 500 sodium salt Orally > 2,000 Rat Intraperitoneally > 500 It was found from the above table that dihydromonacolin L, tetrahydromonacolin L and derivatives thereof have a very low toxicity.

As mentioned above, dihydromonacolin L, tetrahydromonacolin Land the derivatives thereof have a blood cholesterol-reducing activity and they can be used as pharmaceuticals, for example, as an anti-hyperliprnic agent and an anti-atheroslerotic agent.

These compounds are administered in such manner as intravenous injection or oral administration and the like. The dosage varies depending on the age, body weight, symptoms, adminstration route and the like, and usually to adults dihydromonacolin L is administered in a dosage of 10 to 2000 mg, preferably 50 to 300 mg, per day in 1 to 3 divided doses, and tetrahydromonacolin Las well as the derivatives thereof is administered in a dosage of 5 to 3,000 mg, preferably 10 to 300 mg, per day in 1 to 3 doses. However, further dosage may be administered, if required.

Dihydromonacolin L, tetrahydromonacolin Land the derivatives thereof can be prepared into the dosage form in the optional and customary manner by the analogy with other lipemia reducing agent, for example, Clofibrate or Simfibrate. Consequently, the present invention includes also a preparation composition containing at least one of the dihydromonacolin L, tetrahydromonacolin L, and the derivatives thereof, which are suitable as pharmaceuticals for human beings. Such compositions may be provided for use in the customary manner by the use of an optional and necessary pharmaceutical carrier or an excipient.

This composition is desirably offered in a form suitable for absorption from the gastrointestinal organs. Tablets and capsules for oral administration are unit dosage form and may contain customary excipients, for example, binders such as syrup, gum arabicum, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; excipients such as lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine; lubricants such as, for example, magnesium stearate, talc, polyethylene glycol, or silica; degrading agents such as, for example, potato starch or permissible wetting agents such as, for example, sodium laurylsulfate. Tablets may be coated by the conventional method known to the art.The liquid preparation of oral use may be in the form of an aqueous or oil suspension, solution, syrup, elixic and the like or may be a dry product ready to be re-dissolved with water or other suitable vehicles prior to use. These liquid preparations may contain usually employed additives, for example, suspending agents such a sorbitol syrup, methylcellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, or hydrogenated edible fat; emulsifiers such as lecithin, sorbitan monooleate, or gum arabicum; non-aqueous vehicles such as almond oil, fractionated coconut oil, oily ester, propylene glycol or ethyl alcohol; preservatives such as methyl p - hydroxybenzoate, propyl p - hydroxybenzoate, orsorbic acid.

An injectable composition is provided in the form of a unit dosage ampule or a container for multiple doses together with an added preservative. The composition may be in the form of a suspension, solution, an emulsion in an oily or aqueous vehicle, and may contain a prescribed agent such as a suspending agent, a stabilizer and/or a dispersing agent. On the other hand, the active ingredient may be a powder ready to be re-dissolved in a suitable vehicle, for example, pyrogen-free and sterilized water, prior to use.

The present invention will be illustrated in further details with reference to Examples and Preparation Examples, but the present invention is not limited thereto.

Example 1 Monascus ruber No. 1005 (FERM-P 4822, ATCC 20657) was inoculated into a liquid medium containing 3% of glucose, 1% of peptone, 3% of defatted soybean powder, 7% of glycerol, 0.2% of sodium nitrate, and 0.1% of magnesium sulfate.haptahydrate (MgSO4.7H2O) and cultivated at 28"C aerobically for 10 days. 6N Hydrochloric acid was added to the resulting cultivated filtrate (50 I) to make a pH of 3 and then the filtrate was extracted with an equal amount of ethyl acetate. The extract was concentrated to dryness and the residue was dissolved in 11 of benzene and the insoluble material was removed by filtration.The filtrate was washed twice with 500 ml of a 5% aqueous solution of sodium bicarbonate. Subsequently, to the benzene solution was added 11 of a 0.2 N sodium hydroxide solution and the resulting mixture was stirred at room temperature. An aqueous layer was separated after disappearance of dihydromonacolin Lfrom the benzene layer was confirmed by the thin layer chromatography. This aqueous layer was made to pH 3.0 with 6 N hydrochloric acid and extracted twice with 11 of ethyl acetate. The extract was concentrated to dryness to obtain 3.1 g of an oily material. This oil material was dissolved in benzene and subjected to crystallization to precipitate monacolin Kwhich was produced simultaneously. The mother liquor from which crystals were removed was dried and then dissolved in benzene and adsorbed on a column packed with 80 g of silica gel (Wako gel C-200). The column was developed with 400 ml of dichlor omethane, 51 of a mixture of dichloromethane and ethyl acetate (9:1), and 7 1 of a mixture of dichloromethane and ethyl acetate (8:2), successively. A fraction containing dihydromonacolin L [a fraction giving a brown spot by spraying sulfuric acid and by heating at 1200C at the Rf value of 0.45 developed by thin-layer chromatography using Silica gel No. 5715, Kiesel gel 60 F254 (manufactured by Merck & Co.), solvent: CH2Cl2/acetone = 9:1] was dissolved in benzene and adsorbed on a column packed with 40 g of silica gel (Wako gel C-200) and developed by 250 ml of hexane and 41 of a mixture of hexane and acetone (9:1) successively.A fraction containing dihydromonacolin L was dried and dissolved in 60 ml of benzene and washed twice with 30 ml of a 5 % sodium bicarbonate solution. The benzene solution was then dehydrated and concentrated to dryness to obtain 30 mg of a white powder. This white powder was subjected to high speed liquid chromatography (silica-gel-ODS type column, developed with a solvent consisting of acetonitrile - 0.'. % phosphoric acid solution, 1:1) to obtain a dihydromonacolin L fraction. After distilling off the solvent, there was obtained 18 mg of dihydromonacolin L in the form of a crystalline white powder.

Elementary analysis Calculated C: 74.50%; H,9.85% Found C: 74.63% H, 9.79% Molecular weight: 306 (C19H3003) Example 2 100 mg of monacolin Lwas dissolved in 5 ml of dried tetrahydrofuran and to this solution was added 5 mg of platinum oxide and the resulting solution was subjected to hydrogenation with hydrogen while stirring at room temperature for 15 hours. The catalyst was removed by filtration and the resulting product was crystallized from a mixture of ethyl acetate and hexane to obtain 63 mg of a white crystal oftetrahydromonacolin L.

Elementary analysis Calculated: C, 74.03%; H, 10.38% Found: C,74. 13%;H, 10,42% Molecular weight: 308 (C19H32O3) Ultraviolet absorption spectrum (MeOH): Terminal absorption Infrared absorption spectrum (KBr): 3500, 1730 cm- Melting point: 101 to 105 C Example 3 60 mg of dihydromonacolin L obtained by the method of Example 1 was dissolved in 5 ml of dried tetrahydrofuran and 5 mg of platinum oxide was added to this resulting solution. Thereafter, the procedure of Example 2 was repeated to obtain 33 mg of a white crystal of tetrahydromonacolin L.

Elementary analysis: Found: C, 74.11%; H, 10.44% Molecular weight: 308 Ultraviolet absorption spectrum: Terminal absorption Infrared absorption spectrum (KBr): 3,500, 1730 cm- Example 4 200 mg of monacolin L methyl carboxylate ester was dissolved in 30 ml of methanol and 10 mg of platinum oxide was added thereto as a catalyst and the resulting solution was reduced catalystically according to the conventional manner. After removing the catalyst, the concentrated residue was purified by silica gel chromatography (benzene-ethyl acetate, 9:1 and 8:2 mixture) to obtain 195 mg of tetrahydromonacolin L methyl ester as an oily substance.

Elementary analysis Calculated: C70.59%; H,10.59% Found: C, 70.60%; H, 10.61% Molecular weight 340 (C20H3 ;O4) Ultraviolet absorption spectrum (methanol): Terminal absorption Infrare absorption spectrum: -3200, 1740, 1730 cm- Example 5 120 mg of tetrahydromonacolin Lwas dissolved in 5 ml of benzene and 0,4 ml of a 0.1 N sodium hydroxide solution was added thereto and the resulting mixture was stirred at 40 C for 2 hours. An aqueous layer was separated from the mixture and washed three times with 2 ml of benzene and then freeze-dried to obtain 104 mg of tetrahydromonacolin L sodium salt as a white powder.

Elementary analysis Calculated: C, 65.52%; I^l,9.48% Found: C, 65.98%; H, 9.61% Ultraviolet absorption spectrum (H2O): Terminal absorption Infrared absorption spectrum (KBr): 3420,2930, 1720, 1650 cm By repeating the procedure of this Example, while using dihydromonacolin L instead of tetrahydromonacolin L, dihydromonacolin Lsodium salt was obtained.

Elementary analysis Calculated: C, 65.87%; H, 9.02% Found: C, 66.16%; H,9.14% Ultraviolet absorption spectrum (H2O): Terminal absorption Infrared absorption spectrum (KBr): 3420,2930, 1720 and 1650 cm~1 By using calcium hydroxide or magnesium hydroxide instead of sodium hydroxide in the above example, there was obtained calcium salt and magnesium salt, respectively.

Example 6 Synthesis of dihydromonacolin L methyl carboxylate ester: 90 mg Of dihydromonacolin Lwas dissolved in 10 ml of methanol and, to the resulting solution, was added 200 mg of an acidic ion exchange resin (Dowex (RTM) 50, H type) and the mixture was stirred at 60 to 700C for 3 hours. After completion of the reaction, the resin was removed and the mixture by filtration and the filtrate was subjected to evaporation under reduced pressure to remove the solvent, and the residue obtained was purified by means of a column chromatography using 10 g of a silica gel (developing solvent: benzene-ethyl acetate, 9:1 and 8:2 mixture) to obtain 63 mg of dihydromonacolin L methyl carboxylate ester an an oily substance.

Elementary analysis Calculated: C, 70.97%; H, 10.12% Found: C, 71.37%; H, 10.83% Ultraviolet absorption spectrum (H2O): Terminal absorption Infrared absorption spectrum (KBr): 3100, 1740, and 1725cm~1 Preparation Example 1 Capsules for oral administration: Tetrahydromonoacolin L 250 mg Lactose 75 mg Magnesium stearate 15 mg Totai 340 mg The powder of the above recipe was admixed and allowed to pass through a sieve of 60 mesh and then 340 mg of the powder was placed in a No. 1 gelatin capsule to prepare the capsule preparation.

Reference Example Synethesis of monacolin L methyl carboxylate ester: 1 g Of monacolin Lwas dissolved in 15 ml of methanol and, to this solution, was added 2 g of an acidic ion exchange resin (Dowex 50 W, H+ type, dried form) and the resulting mixture was heated and stirred at 60 to 70"C for 3 hours. After completion of the reaction, the resin was removed from the mixture by filtration, the filtrate was subjected to evaporation under reduced pressure to remove the solvent, and the residue obtained was purified by means of a column chromatography using 10 g of a silica gel (developing solvent: benzene-ethyl acetate 9:1 and 8:2 mixture) to obtain 650 mg of monacolin L methyl carboxylate ester as an oily substance.

Elementary analysis Calculated: C, 71.39%; H, 9.59% Found: C,71.44%; H,9.68% Ultraviolet absorption spectrum (H2O): Terminal absorption Infrared absorption spectrum: 3400,3100, 1710 cm-l

Claims (17)

1. Dihydro- and tetrahydromonacolin L of the general formula (I)

wherein X stands for-CH=CH- or-CHTCHT; and metal carboxylate salts and alkyl carboxylate esters of dihydro- and tetrahydromonacolin Loathe general formula (II)

wherein Y stands for 1 M (in which M is a metallic n element and n is the atomic valency of said metallic element) or an alkyl group having 1 to 6 carbon atoms; and X has the same meaning as above.

2. Dihydromonacolin L according to Claim 1, wherein X has the general formula (I) is -CH=CH-.

3. Tetrahydromonacolin L according to Claim 1 wherein X in general formula (I) is -CHTCHT.

4. A metal carboxylate salt of dihydro- and tetrahydromonacolin L according to Claim 1, wherein the metallic element is sodium, calcium, or aluminium.

5. A carboxylate ester of dihydro- and tetrahydromonacolin L according to Claim 1 wherein Y is methyl.

6. A process for preparing dihydromonacolin L of the formula (III):

which comprises cultivating a dihydromonacolin L-producing microorganism belonging to genus Monascus on a nutrient medium and isolating the resulting dihydromonacolin L.

7. A process according to Claim 6, wherein the dihydromonacolin L-producing microorganism belonging to genus Monascus is Monascus ruber.

8. A process according to Claim 7, wherein the Monascus ruber is Monascus ruber No. 1.005 (FERM-P 4822, ATCC 20657).

9. A process for producing dihydromonacolin L according to claim 6 substantially as hereinbefore described with reference to any one of the Examples.

10. A process for preparing tetrahydromonacolin Lofthe formula (IV) of an alkyl carboxylate ester thereof of the general formula (V)

wherein R represents an alkyl group having 1 to 6 carbon atoms, which comprises catalytically reducing monacolin L, or dihydromonacolin Loran alkyl carboxylate ester thereof in an organic solvent.

11. A process for producing tetrahydromonacolin L according to claim 10 substantially as hereinbefore described with reference to any one of the Examples.

12. A process for preparing a metal catboxylate salt or an alkyl carboxylate ester of dihydro- or tetrahydromonacolin L of the general formula (VII)

wherein Ystands for 1/n M (in which M is a metallic element and n is the atomic valency of said metallic element) or an alkyl group having 1 to 6 carbon atoms; and X represents-CH=CH- or-CH=CHr, which comprises dissolving dihydro- or tetrahydromonacolin L of the general formula (I)

wherein X is as defined above in an organic solvent and reacting said dissolved compound with an aliphatic alcohol having 1 to 6 carbon atoms or a metal hydroxide of the general formula (VIII) M(OH)n (Vlil) wherein M and n are as defined above.

13. A process for producing a metal carboxylate salt or an alkyl carboxylate ester of dihydro- or tetrahydromonacolin L according to claim 12 substantially as herein before described with reference to any one of the Examples.

14. A pharmaceutical composition comprising a compound according to any one of claims 1-5 together with a pharmaceutically acceptable diluent or carrier.

15. Acomposition accordingtoclaim 14substantially as hereinbefore described with reference to the Example.

16. A compound according to any one of claims 1-5 or a composition according to claim 14 or 15 for use in a method of treatment of the human or animal body by surgery or therapy or of diagnosis practised on the human or animal body.

17. A compound according to any one of claims 1-5 or a composition according to claim 14 or 16 for use in a method of treating hyperlipemia in humans.