DE3112566C2 - - Google Patents

Description

The invention relates to monacolin derivatives which are the Inhibiting cholesterol biosynthesis, a method to their Preparation and medicaments containing these derivatives.

Hyperlipemia, especially hypercholesterolemia, are known to be a major cause of heart disease, like myocardial infarction or atherosclerosis. It is therefore Significant efforts have been made, connections to discover the lipid and especially the cholesterol levels lower the blood. A group of such Compounds is described in US-PS 39 83 140. These Group of compounds becomes microorganisms of the genus Penicillium isolated and collectively referred to as ML-236.

ML-236A and ML-236B (two compounds of the type described in US Pat 39 83 140 described ML-236 complex) have the formulas II or III

The compounds of the invention differ from the compounds described in US Pat. No. 3,983,140, that they are in the 6'-position, ie in the partially hydrogenated Naphthalene nucleus, another methyl substituent. The prior art was no indication to see that the introduction of a methyl group in the naphthalene core a desirable modification of the known compound, because, due to the known Effect of compound ML-236 as an agent for competitive inhibition of 3-hydroxy-3-methylglutaryl  Coenzyme A at most was to be expected that the introduction a methyl group in the lactone ring is effective.

In DE-OS 30 06 215 and 30 06 216 is another such connection described as Monacolin K or MB-530B is and by breeding microorganisms of the Genus Monascus, in particular the trunks Monascus ruber, is obtained.

MB-530B (or Monacolin K) has the following formula IV:

A new set of compounds has now been isolated which contribute to their better absorption oral administration and their easier availability as a pro-drug (i.e., drug, after administration by chemical or biochemical Body reactions converted into an active or more active form will) show a better activity development.

The invention relates to monacolin derivatives of the general Formula I  

in which R² is a straight-chain or branched C₂ to C₂₀ alkanoyl group except the α- methylbutyryl group, a straight-chain or branched C₃ to C₂₀ alkenoyl group or the benzoyl group.

The invention also relates to medicaments containing at least one of the compounds of general formula I according to Claim 1 or 2 and pharmaceutically acceptable Carriers, excipients and / or diluents.

The compound of the invention in which R² is a hydrogen atom is, d. H. the compound of formula V

was designated as MB-530A and can be manufactured thereby be that one has a MB-530A-producing microorganism of the genus Monascus in a suitable culture medium  and the produced MB-530A from the culture medium wins.

The other compounds of the invention considered 8'-acylated derivatives of MB-530A can be made by acylating Manufacture MB-530A. For clarification These acylated derivatives are as follows as the ester of compound MB-530A from which they are obtained by acylation were.

In the definition of the compounds of the invention is the following numbering is applied:

Preferred aliphatic acyl groups are straight chain or branched alkanoyl with 2 to 6 carbon atoms and straight or branched  Alkenoyl of 3 to 6 carbon atoms.

Examples of saturated aliphatic acyl groups are Acetyl, propionyl, butyryl, isobutyryl, valeryl, Isovaleryl, hexanoyl, 2-methylvaleryl, 3-methylvaleryl, 4-methylvaleryl, 2-ethylbutyryl, heptanoyl, octanoyl, 2-ethylhexanoyl, nonanoyl, isononanoyl, decanoyl, undecanoyl, Dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, Palmitoyl, stearoyl, isostearoyl, nonadecanoyl, Eicosanoyl and pivaloyl.

Examples of suitable unsaturated aliphatic acyl groups are acryloyl, 3-butenoyl, methacryloyl, 2-pentenoyl, 4-pentenoyl, 2-undecenoyl, 4-undecenoyl, 2-tridecenoyl, 2-tetradecenoyl, 2-hexadecenoyl, linolenoyl, linoleyl, Arachidonoyl, propioloyl, crotonoyl, tigloyl, angeloyl, Senecionoyl, 2-heptenoyl, 2-octenoyl and 2-nonenoyl.  

Examples of compounds according to the invention are:
MB-530A-8'-acetate,
MB-530A-8'-propionate,
MB-530A-8'-butyrate,
MB-530A-8'-isobutyrate,
MB-530A-8'-valerate,
MB-530A-8 'isovalerate,
MB-530A-8 'pivalate,
MB-530A-8'-hexanoate,
MB-530A-8'-heptanoate,
MB-530A-8 '- (2-methylvalerate)
MB-530A-8 '- (3-methylvalerate)
MB-530A-8 '- (2-ethyl butyrate),
MB-530A-8'-octanoate,
MB-530A-8 '- (2-ethylhexanoate),
MB-530A-8 'nonanoate,
MB-530A-8 'isononaoat,
MB-530A-8'-undecanoate,
MB-530A-8 'tetradecanoate,
MB-530A-8 'pentadecanoate,
MB-530A-8 'palmitate,
MB-530A-8'-stearate,
MB-530A-8 'isostearate,
MB-530A-8 'nonadecanoat,
MB-530A-8 'eicosanoate,
MB-530A-8'-acrylate,
MB-530A-8 'propiolate,
MB-530A-8 'crotonate,
MB-530A-8 '- (3-butenoate)
MB-530A-8'-methacrylate,
MB-530A-8 '- (2-pentenoate)
MB-530A-8 '- (4-pentenoate)
MB-530A-8 'tiglat,
MB-530A-8 'Angelat,
MB-530A-8 'senecionat,
MB-530A-8 '- (2-heptenoate),
MB-530A-8 '- (2-octenoate),
MB-530A-8 '- (2-nonenoate),
MB-530A-8 'linoleate,
MB-530A-8'-oleate,
MB-530A-8'-linolenate,
MB-530A-8'-benzoate.

MB-530A, one of the compounds of the invention by growing a MB-530A producing microorganism of the Genus Monascus, preferably a tribe of Monascus ruber and in particular Monascus ruber SANK 15 177. This strain was found on 27.04.1979 under the No. FERM 4956 at the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan, and on 25.01.1980 under the entry no. NRRL 12 081 at Agricultural Research Service Culture Collection, Northern Regional Research Laboratory, Peoria, Illinois, USA. The The morphology and physiology of this strain are detailed described in DE-OS 30 06 215.

The desired MB-530A can be obtained by breeding the chosen Microorganism under aerobic conditions in a culture broth according to known methods of culturing fungi and other microorganisms. For example one can first select the chosen Monascus strain in grow a suitable medium, then the cultured microorganisms collect and vaccinate in another culture medium and continue breeding there to the desired MB-530A to obtain. That for the multiplication of the microorganism and the culture medium used for the production of MB-530A can be the same or different. It can Any culture media known for the cultivation of fungi be applied, provided that it has the known necessary nutrients contains, in particular assimilable carbon and Nitrogen sources. Examples of suitable assimilable Carbon sources are glucose, maltose, dextrin, starch, Lactose, sucrose and glycerin. These are for production of MB-530A, glucose, glycerin and starch are particularly preferred. Examples of suitable assimilable nitrogen sources are peptone, meat extract, yeast, yeast extract, soy flour, Peanut flour, corn steep liquor, rice bran and inorganic Nitrogen sources. Of these, corn steep liquor and  Peptone particularly preferred. For the production of MB-530A If desired, inorganic salts may be added to the culture medium and / or metal salts are added. Further, if necessary small amounts of heavy metals are added.

The microorganism is preferably under aerobic conditions bred using known culture methods, z. B. as a solid culture, shake culture or aerated stirring culture. The microorganisms grow over a wide temperature range, z. B. about 7 to 40 ° C, but is for the Production of MB-530A a breeding temperature of 20 to 30 ° C particularly preferred.

During the breeding of the microorganism, the production can MB-530A by sampling from the culture medium and measuring the physiological activity of the medium be monitored with known methods. The culture can then continue until sufficient in the culture medium MB-530A has accumulated, after which the MB-530A by a suitable combination of isolation techniques, with regard to on its physical and chemical properties selected from the culture medium and the tissues (Mycelium) of the microorganism isolated and wins. For example can use one or more of the following isolation methods to be applied:

Extraction of the liquid from the culture broth with a hydrophilic solvents (eg diethyl ether, ethyl acetate, Chloroform or benzene); Extraction of the organism with a hydrophilic solvents (eg, acetone or an alcohol); Concentrate, z. B. partial or total evaporation of the Solvent under reduced pressure; Dissolve in one more polar solvent (eg, acetone or an alcohol); Separate the impurities with a less polar one Solvent (eg, petroleum ether or hexane); gel filtration, z. With Sephadex®;  Absorption chromatography with activated charcoal or silica gel; and similar methods. By using a suitable combination These methods can be the desired MB-530A from the culture broth as a pure substance.

The acylated derivatives according to the invention can be obtained from MB-530A according to one of the following procedures getting produced.

process 1

MB-530A comes with a suitable Acid chloride or acid anhydride to the desired Implemented acyl derivative. The reaction is preferably carried out in Presence of a base (which binds the acid), preferably an organic amine, such as pyridine, triethylamine, N, N-di- methylaminopyridine, N-methylpyrrolidone or N-methylmorpholine. The reaction is preferably in the presence of a solvent whose selection is not critical, as long as it does not affect the reaction negatively. suitable Solvents are z. As chloroform, methylene chloride and dimethyl ether. In some cases it is possible the reaction using an excess of one of the reactants or to carry out the base as a solvent. The reaction takes place over a wide temperature range, normally However, it is preferred for the reaction control, a relative apply low temperature, for. From -20 ° C to room temperature, especially -20 to 0 ° C. If desired, can However, higher temperatures are used.  

process 2

A carboxylic acid is treated with a chloroformate or a sulphonyl chloride in the presence of a base, e.g. B. one the above-mentioned organic amines, to a mixed Acid anhydride reacted, which then with MB-530A converts. The reaction takes place preferably in the presence of a solvent, its selection not critical, as long as the reaction is not negative affected. Suitable solvents are, for. B. diethyl ether, Benzene, chloroform and methylene chloride. The implementation occurs within a wide temperature range, eg. B. -20 ° C to room temperature, especially -20 to 0 ° C.

process 3

MB-530A is made with a carboxylic acid and a diazoalkyldicarboxylate in the presence of z. B. dicyclohexylcarbodiimide, triphenylphosphine or dimethylphosphonic acid amide implemented. The reaction is preferably carried out in the presence of a solvent, its selection not critical, as long as the reaction is not negative affected. Suitable solvents are, for. Chloroform, Methylene chloride, benzene and diethyl ether.

The above reactions are usually within 30 minutes to 5 hours complete, however, each depends required reaction time of the reactants and the Reaction temperature from. After the implementation is the desired product in the usual way from the reaction mixture separated, z. B. by evaporation of the solvent a solution of the desired product (this solution can be simple be the reaction mixture or a solution by Extraction of the reaction mixture with an organic solvent obtained), the solution optionally previously washed and dried. This can be the product  be cleaned in the usual way, for. By column chromatography, Thin layer chromatography and / or recrystallization.

Since the starting material for the acylation reaction two hydroxyl groups contains, the above reactions, the 3-monoester, the 3,8'-diester, the 8'-monoester or mixtures thereof. The type of product obtained can be determined by the quantities the reactants, the reaction temperature and other reaction conditions to be influenced.

The amount of the acylating agent, preferably the acid halide, is preferably about 1 equivalent per Equivalent MB-530A.

The compounds of the invention have a specific Inhibitory activity against 2-hydroxy-3-methylglytaryl-coenzyme A- Reductase (HMG-CoA reductase), which is the rate-limiting Represents enzyme in the biosynthesis of cholesterol. The inhibitory effect of some compounds of the invention Cholestrin biosynthesis is shown in the following table their I₅₀ values (concentration in μm / ml, the one 50% inhibition of cholesterol biosynthesis causes) shown. The measurement is carried out by the method of Knaus et al., J. Biol. Chem. Vol. 234, p. 2835 (1959).

R I₅₀ (μg / ml) butyryl 0,021 isovaleryl 0.026

Example 1 Production of MB-530A

300 liters of a culture medium before sterilization has a pH of 5.5 and 5% w / v glucose, 0.5% w / v corn steep liquor, 2% w / v peptone and 0.5% ammonium chloride placed in a 600 liter fermenter and with a Culture of Monascus ruber SANK 15 177 (FERM 4956, NRRL 12 081) inoculated. The microorganism is 120 hours at 27 ° C with aeration rate of 300 liters / min with stirring Bred 190 rpm.

Subsequently, the culture broth is passed through a filter press filtered to give a filtrate and a filter cake which consists of moist microorganism cells. The Filtrate is brought to pH by addition of 6 N hydrochloric acid of 3.0 and then extracted with 400 liters of ethyl acetate. The extract (about 400 liters) is under reduced Pressed, dried over sodium sulfate and then evaporated to dryness, with about 60 g of an oily Products are obtained. This is mixed with ethylcyclohexane and washed with hexane, whereupon the residue (20 g) chromatographically using a liquid chromatograph for large volume samples and elute with Separates 60% v / v aqueous methanol. The fractions with a chromatographic retention time of 6 minutes collected and evaporated under reduced pressure, Obtained 100 mg of the desired MB-530A as an oily product become. The oily MB-530A is made from acetone / diethyl ether recrystallized to give 57 mg of the desired Product in the form of colorless needles with the following Properties:

• 1st melting point: 92-93 ° C.
• 2. Elemental analysis for C₁₉H₂₈O₄:
  calc .:
  C 69.76%; H 8.68%;
  Found .:
  C 71.22%; H 8.81%.
• 3. Molecular weight: 320 (mass analysis).
• 4. molecular formula: C₁₉H₂₈O₄.
• 5. UV absorption spectrum: see FIG. 1.
• 6. IR absorption spectrum: see FIG. 2.
• 7. NMR spectrum: see FIG. 3.
• 8. Solubility: Easily soluble in methanol, ethanol, Acetone, ethyl acetate, chloroform and carbon tetrachloride; soluble in benzene; insoluble in hexane and Petroleum ether.
• 9. Staining reaction: in thin-layer chromatography on silica gel occurs at development with 50% V / V Sulfuric acid on a pink color.
• 10. Inhibitory effect on cholestrin biosynthesis: In one Concentration of 0.04 μm / ml becomes a 50% inhibition observed cholesterol synthesis in the rat liver.

example 2 MB-530A-8'-butyrate

309 mg MB-530A and 0.36 ml pyridine are added in 10 ml Dissolved methylene chloride. To the cooled to 0 ° C solution 0.16 ml of butyryl chloride is added dropwise. After completion of the implementation the reaction mixture is treated with water, the Separated methylene chloride layer, washed with water and dried over sodium sulfate. The by evaporation of the Methylene chloride is purified by column chromatography on silica gel (10 g). By recrystallization Diethyl ether gives 154 mg of the desired Product.

Elemental analysis for C₂₃H₃₄O₅:
calc .:
C 70.77%; H 8,72;
Found .:
C 70.58; H 8,61.
NMR spectrum (CDCl₃) δ ppm: 0.90 (3H, t); 4.17 (1H, m); 5.22 (1H, m).
IR spectrum (film) ν _max cm ^-1 : 3450, 1730.

example 3 MB-530A-8 'isovalerate

According to Example 2, 109 mg of the desired product made from 308 mg MB-530A and 0.12 ml isovaleryl chloride.

Elemental analysis for C₂₄H₃₆O₅:
calc .:
C 71.29%; H, 8.91%;
Found .:
C 71,17; H 8,69.
Nuclear Magnetic Resonance Spectrum (CDCl₃) δ ppm: 0.88 (6H, d); 4.15 (1H, m); 5.22 (1H, m).
IR spectrum (film) ν _max cm ^-1 : 3420, 1730.

example 4 MB-530A-8 'hexanoate

According to Example 2, 87 mg of the desired product made from 308 mg MB-530A and 0.15 ml hexanoyl chloride.

Elemental analysis for C₂₅H₃₈O₅:
calc .:
C 71.77%; H 9.09%;
Found .:
71.95%; H 9.17%.
NMR spectrum (CDCl₃) δ ppm: 0.90 (3H, t); 4.33 (1H, m), 5.33 (1H, m).
IR spectrum (film) ν _max cm ^-1 : 3440, 1735.

example 5 MB-530A-8'-octanoate

According to Example 2, 96 mg of the desired product made from 304 mg MB-530A and 0.11 ml octanoyl chloride.

Elemental analysis for C₂₇H₄₂O₅:
calc .:
C 72.65%; H 9.42%;
Found .:
C 72.52%; H 9.50%.
Nuclear Magnetic Resonance Spectrum (CDCl₃) δ ppm: 0.85 (3H, br t); 1.22 (12H, br s); 4.19 (1H, m); 5.23 (1H, m).
IR spectrum (film) ν _max cm ^-1 : 3400, 1735.

Claims (5)

1. Monacolin derivatives of the general formula I. in which R² is a straight-chain or branched C₂ to C₂₀ alkanoyl group except the α- methylbutyryl group, a straight-chain or branched C₃ to C₂₀ alkenoyl group or the benzoyl group. 2. Compounds according to claim 1, characterized in that R² is a straight-chain or branched C₂- to C₆-alkanoyl group, except the α- methylbutyryl group or a straight-chain or branched C₃- to C₆-alkenoyl group. 3. Compound M-530A of the formula 4. Process for the preparation of the compounds of the general Formula I according to claim 1, characterized in that the compound (V) according to claim 3 with an acylating agent acylated. 5. Medicaments containing at least one of the compounds of the general formula I according to claim 1 or 2 as well as pharmaceutically acceptable carriers, excipients and / or diluents.