JPH0333698B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the formula (In the formula, R ¹ is a group

[expression] or

[Formula] is shown. R ² and R ³ are the same or different and represent a lower alkyl group. ), a pharmacologically acceptable salt thereof, an ester thereof, or a closed ring lactone thereof. Conventionally, compounds in which R ² is a hydrogen atom and R ³ is a methyl group in the general formula () and derivatives thereof have been disclosed in JP-A-57-2240, JP-A-57-67575,
It is described in No. 57-108039 and No. 58-10572, and is known to exhibit a cholesterol synthesis inhibitory effect. The present inventors have discovered that a carboxylic acid having the general formula (), a pharmacologically acceptable salt thereof, an ester thereof, or a closed ring lactone thereof all exhibit a cholesterol synthesis inhibitory effect and are highly stable, The invention has been completed. In the general formula (), R ² and R ³ are the same or different and are methyl, ethyl, propyl,
Indicates a lower alkyl group such as isopropyl or butyl. Pharmaceutically acceptable salts of the compound having the general formula () of the present invention include, for example, metal salts, amino acid salts, and amine salts. Examples of metal salts include alkali metal salts such as sodium and potassium;
Examples include alkaline earth metal salts such as calcium and magnesium, and aluminum salts, iron salts, zinc salts, copper salts, nickel salts, and cobalt salts, among which alkali metal salts, alkaline earth metal salts, and aluminum salts are preferred, and the sodium, potassium, calcium and aluminum salts are most preferred. Examples of amino acid salts include arginine, lysine, histidine,
Basic amino acids such as α,γ-diaminobutyric acid and ornithine are preferred. Suitable examples of the amine salt include t-octylamine, dibenzylamine, dicyclohexylamine, morpholine, D-phenylglycine alkyl ester, and D-glucosamine. Examples of the ester of the compound having the general formula () include alkyl esters such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and pentyl. Methyl is preferred. The ring-closed lactone of the compound having the general formula () above refers to a compound in which the formula () is represented by the following ring-closed structural formula. In the general formula () of the present invention, the following geometric isomers exist depending on the arrangement of substituents. Furthermore, various optical isomers also exist due to the presence of asymmetric carbon atoms. In the general formula (),
All of these isomers and mixtures of these isomers are shown in a single formula. Therefore, in the present invention, the carboxylic acid having the general formula (), its pharmacologically acceptable salt, its ester or its ring-closed lactone, includes the carboxylic acid having the general formula () to (), its It includes not only pharmacologically acceptable salts, esters thereof, or ring-closed lactones thereof, but also mixtures of these isomers. The object compound of the present invention has an effect of lowering blood lipids by inhibiting cholesterol synthesis, and can be used in medicine, for example, as a therapeutic agent for hyperlipidemia or an agent for preventing arteriosclerosis. These compounds can be administered orally or parenterally, for example, in the form of capsules, tablets, injections, and the like. The dosage varies depending on age, symptoms, body weight, etc., but it is usually about 0.2 to 200 mg per day for adults.
is administered in 3 to 4 divided doses. However, larger amounts can be used if desired. The object compound of the present invention has the formula (wherein R ² and R ³ have the same meanings as defined above), a pharmacologically acceptable salt thereof, an ester thereof, or a ring-closed lactone thereof (raw material compound) is 3-hydroxylated. or 6
- Enzymatic hydroxylation by contacting with a hydroxyl converting bacterium or a cell-free extract thereof, and then optionally subjecting the resulting conversion reaction product to a hydrolysis reaction, salt formation reaction, esterification reaction or lactonization reaction. , obtained by collecting from the reaction solution. Examples of microorganisms that can convert the raw material compound into a carboxylic acid having the general formula (), a pharmacologically acceptable salt thereof, an ester thereof, or a closed ring lactone thereof include Nocardia genus belonging to Zygomycetes, Synthephalastrum ( Genus Syncephalastrum, Mucor
Genus, Rhizopus, Zygorynchus, Circinella
Genus, Actinomucor, Gongronella, Phycomyces, Absidia,
Examples include the genera Cunninghamella and Mortierella, and the genera Pychnoporus (formerly known as Trametes), Streptomyces, and Rhizoctonia. Among these microorganisms, especially Nocardia autotrophica (Nocardia
autotrophica) SANK62781
(Nocardia autotrophica subsp. nov.) Nocardia autotrophica subsp.
subsp.canberrica subsp.nov) SANK62881 (Feikokuken Bibori No. 6182) Nocardia autotrophica subsp. Amethystina subsp.nov. (Nocardia autotrophica
subsp.amethystina subsp.nov.) SANK62981 (Feikoken Bacteria No. 6183)
autotrophica) IFO12743 Nocardia asteroides (Nocardia
asteroides) IFO3424 Nocardia falcinica (Nocardia
farcinica) ATCC3318 Nocardia coeriaca
coeliaca) ATCC17040 Syncephalastrum nigricans SANK42172 (FTE No. 6041) SANK42272 (FTE No. 6042) SANK42372 (FTE No. 6043) Syncephalastrum nigricans・Syncephalastrum racemosum IFO4814 IFO4828 Mucor hiemalis f.hiemalis IFO5834 IFO5303 IFO8567 IFO8449 IFO8448 IFO8565 CBS117.08 CBS109.19 CBS200.28 CBS242.35 CBS110.19 CBS201.65 Mucor Basiliformis
bacilliformis) NRRL2346 Mucor circinelloides f.
circinelloides) IFO4554 Same as IFO5775 Mucor hiemalis f.corticolus (Mucor hiemalis f.corticolus) SANK34572
(Feikoken Bibori No. 5913) Mucor Deimorphosporus (Mucor Deimorphosporus)
dimorphosporus) IFO4556 Mucor fragilis
CBS23635 Mucor genevensis
IFO4585 Mucor globosus
SANK35472 (February 5915) Mucor circinelloides f.
griseocyanus) IFO4563 Mucor heterosporus (Mucor
heterosporus) NRRL3154 Mucor Spinescens (Mucor
Rhizopus sinensis) IAM6071 Rhizopus sinensis
chinensis) IFO4772 Rhizopus cilcinans (Rhizopus
circinans) ATCC1225 Rhizopus arrhizus
ATCC11145 Zygorynchus moereri
moelleri) IFO4833 Circinella muscae
IFO4457 Circinella rigida
NRRL2341 Circinella amberata (Circinella
umbellata) IFO4452 IFO5842 NRRL1713 Actinomucor elegans ATCC6476 Gongronella
butleri) IFO8080 Phycomyces blakesleeanus (Phycomyces blakesleeanus) SANK45172 (Feikoku Kenkyori No. 5914) Absidia coerulea (Absidia coerulea)
IFO4423 Absidia glauca var.paradoxa IFO4431 Cunninghamella echinulata IFO4445 IFO4444 ATCC9244 Mortierella
isabellina) IFO6739 Pycnoporus coccineus (Pycnoporus
Streptomyces roseochromogenus
NRRL1233 IFO3363 IFO3411 Streptomyces halstedii IFO3199 Streptomyces platensis NRRL2364 Strep
-tomyces fulvissmus) NRRLB-1453 Rhizoctonia solani
SANK22972 (Feikoken Bibori No. 5917) is suitable. All microorganisms suitably used in the present invention have been deposited with the Institute of Fine Technology, or have been deposited with public storage institutions (IFO, CBS, ATCC, IAM, etc.).
Available from NRRL). When carrying out the method of the present invention, the enzymatic hydroxylation method involves culturing the converting bacteria under culture conditions suitable for its growth, and adding the raw material compound to the medium during the cultivation of the converting bacteria. A method in which the converted bacteria are cultured and collected, and the resulting converted bacteria cells are brought into contact with a raw material compound, and a method in which a cell-free extract prepared from the converted bacteria cells is brought into contact with the raw material compound. It will be held in The converting bacteria can be cultured in a medium containing nutrients that can be used by microorganisms. As the nutrient source, any known nutrient source used for general microbial culture can be used. For example, glucose, sucrose, starch, glycerin, starch syrup, molasses, soybean oil, etc. can be used as carbon sources. Further, as the nitrogen source, soybean flour, wheat germ, meat extract, peptone, corn steep liquor, dried yeast, ammonium sulfate, etc. can be used. In addition to salt, potassium chloride, calcium carbonate, phosphate, etc., additives necessary to aid the growth of bacteria and promote the production of the enzyme having the hydroxylation ability may be used in appropriate combinations as necessary. As a culture method, culture methods generally used for microorganisms, such as liquid culture methods, are possible.
Industrially, the deep culture method is suitable. Cultivation is carried out under aerobic conditions, with a culture temperature of 20~20°C.
37°C, preferably 26-28°C. The method is carried out by adding a raw material compound to a medium in the middle of culturing the converted bacteria and culturing it. The timing of addition varies depending on the optimal culture conditions of the conversion bacteria used, especially the culture equipment, medium composition, medium temperature, etc.
The best time is when the hydroxylation ability of the converting bacteria begins to increase.
Usually, it is preferable to carry out the reaction 2 to 3 days after the start of culturing the converting bacteria. The amount of raw material compound added is based on the medium.
Selected from the range of 0.01 to 5.0%, but 0.05 to 0.1%
A range of is suitable. Cultivation after addition of the raw material compound is carried out under aerobic conditions at the above-mentioned culture temperature. The culture period is 3 to 5 days after addition of the raw material compound. The method involves culturing the converted bacteria using the method described above, and culturing the converted bacteria until the hydroxylation ability of the converted bacteria reaches its maximum. That is, the hydroxylation ability differs depending on the type of medium, temperature, etc., but usually reaches its maximum 4 to 5 days after the start of culture, so the culture is terminated at this point. Bacterial collection is performed by subjecting the culture to centrifugation, straining, etc. It is preferable that the collected converted bacterial cells be washed with normal saline, buffer solution, etc. before use. In order to bring the thus obtained converted bacterial cells into contact with the starting compound, it is usually carried out in an aqueous medium, e.g.
It is carried out in a phosphate buffer with a pH of 5-9. The reaction temperature is 20-45°C, preferably 25-30°C. The concentration of the raw material compound is usually selected from the range of 0.01 to 5.0%. The reaction time depends on the concentration of the raw material compound, the reaction temperature, etc., but is usually about 1 to 5 days. The cell-free extract in this method is obtained by applying physical or chemical means to the transformed bacterial cells obtained in the above method, such as by grinding, ultrasonication, etc. Obtained as a bacterial cell lysate by treatment with an activator or an enzyme. The method of contacting the cell-free extract thus obtained with the raw material compound is carried out in the same manner as the method of contacting the converted bacterial cells with the raw material compound described above. After the conversion reaction is completed, the target compound can be directly collected, separated, and purified from the product by known methods. For example, the product is filtered, the resulting liquid is extracted with an organic solvent that is not easily miscible with water such as ethyl acetate, the solvent is distilled off from the extracted liquid, and the resulting crude target compound is extracted with silica gel, alumina, etc. It can be separated and purified by subjecting it to column chromatography and eluting with an appropriate eluent. Furthermore, if desired, the obtained product can be converted into the target compound by subjecting it to a hydrolysis reaction, salt formation reaction, esterification reaction, or lactonization reaction according to conventional chemical methods, and can be easily collected. can. All of these methods are conventional methods, such as the following method. Carboxylic acids with formula () are used when the product of the conversion reaction is a carboxylate salt, and the resulting liquid
It can be obtained by adjusting the pH to 4 or less, preferably 3 to 4. The acid used is not limited to organic acids or mineral acids as long as it does not affect the target compound; for example, trifluoroacetic acid, hydrochloric acid, sulfuric acid, etc. are preferably used. The carboxylic acid thus obtained is extracted,
After processing such as washing and dehydration, it can be used in the following reaction. A metal salt of a carboxylic acid having the formula () can be obtained by contacting a hydroxide, carbonate, etc. of the metal with the above carboxylic acid in an aqueous solvent. Examples of aqueous solvents used include water; methanol;
Alcohols such as ethanol, acetone, n
- A mixed solvent of water and an organic solvent such as hexane or ethyl acetate is suitable. Particularly suitable is a mixed solvent of a hydrophilic organic solvent and water. The reaction is usually suitably carried out at around room temperature, but may be carried out under heating if necessary. Amine salts of carboxylic acids having formula () are obtained by contacting amines with the above carboxylic acids in an aqueous solvent. Examples of the aqueous solvent used include water; alcohols such as methanol and ethanol; ethers such as tetrahydrofuran; and mixed solvents of water and nitriles such as acetonitrile; preferred is aqueous acetone. The reaction is usually suitably carried out at a pH of 7 to 8.5 and below room temperature, particularly at 5 to 10°C. The reaction is completed instantly. Alternatively, the carboxylic acid metal salt obtained above can be dissolved in an aqueous solvent, and then a mineral acid salt (eg, hydrochloride) of the desired amine can be added under the above conditions to obtain the salt exchange reaction. Amino acid salts of carboxylic acids having the formula () are:
It is obtained by contacting an amino acid with the above carboxylic acid in an aqueous solvent. Examples of the aqueous solvent used include water; a mixed solvent of water and alcohols such as methanol and ethanol; and ethers such as tetrahydrofuran. The reaction is usually carried out under heating, preferably around 50 to 60°C. The alkyl ester of a carboxylic acid having the formula () can be obtained by contacting the carboxylic acid obtained above with an alcohol. In this case, a mineral acid such as hydrochloric acid or sulfuric acid, boron fluoride, an acidic ion exchange resin, or the like is used as a catalyst, and as a solvent, the same alcohol or one that does not participate in the reaction, such as benzene, chloroform, or ether, is used. Alternatively, it can be obtained by contacting the carboxylic acid obtained above with a diazoalkane. The reaction is usually carried out by contacting with an ethereal solution of the diazoalkane. or,
It is obtained by contacting the metal salt of carboxylic acid obtained above with an alkyl halide. Suitable solvents to be used include, for example, dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and acetone. All reactions are preferably carried out at around room temperature, but depending on the type of reaction system, they may be carried out under heating if necessary. The lactone form of carboxylic acid having the formula () is
It is obtained by contacting the carboxylic acid obtained above with a catalytic amount of acid. The acid used is preferably an organic or mineral acid such as trifluoroacetic acid, hydrochloric acid, or sulfuric acid. The reaction is usually suitably carried out at around room temperature. Furthermore, the target compound thus obtained can be used as a raw material and converted into other target compounds according to the above-mentioned conventional chemical methods. The target compound thus obtained can be collected, separated, and purified by appropriately combining various methods. For example, this may be carried out by adsorption using various carriers such as activated carbon or silica gel, ion exchange chromatography, gel filtration using a Sephadex column, or extraction using an organic solvent such as ether, ethyl acetate, or chloroform. In particular, separation of isomers can be carried out by the above-mentioned separation and purification means at an appropriate time after the completion of the conversion reaction or after the completion of the desired step. The carboxylic acid having the general formula (), its pharmacologically acceptable salt, its ester, or its ring-closed lactone, which is the raw material compound of the present invention, is a new compound, for example, ML-236A
136885) according to the following steps to obtain its ring-closed lactone form. (In the formula, R ² and R ³ are the same as described above, and X represents a halogen atom.) The first step is a step of producing a compound having the above formula (), and having the above formula (). ML−
It is obtained by contacting 236A with t-butyldimethylsilyl halide in an inert solvent in the presence of a base. The second step is a step of producing a compound having the above general formula (), and includes contacting the compound having the above formula () with a 2,2-dialkylbutyryl halide in an inert solvent in the presence of a catalyst. obtained by. The third step is a step of producing a compound having the general formula (), which is obtained by contacting the compound having the general formula () with an acid or a base to remove the hydroxyl protecting group. The target compound in each step is collected by appropriately combining various methods as necessary.
Can be purified. For example, it is carried out by an extraction method, a chromatography method, a gel filtration method, or the like. The ring-closed lactone having the general formula () thus obtained is converted into a carboxylic acid having the general formula (), a pharmacologically acceptable salt thereof, or an ester thereof by a conventional method, and is used as the raw material of the present invention. Used as a compound. Next, production examples and examples of the raw material compounds of the present invention will be shown, but the present invention is not limited thereto. Production example of raw material compound 1 Production of compound having the above formula () After dissolving 6.12g (0.02 mol) of ML-236A in 50ml of dry dimethylformamide, 2.0g of imidazole was added.
(0.03 mol) and 3.6 g (0.024 mol) of t-butyldimethylsilyl chloride were added, and the mixture was stirred at room temperature for 90 minutes. After the reaction is complete, add ethyl acetate to the reaction mixture.
500 ml was added thereto, and the mixture was washed successively with water, 1N hydrochloric acid, and saturated saline, and dried over sodium sulfate. After drying, the solvent is distilled off from the reaction mixture to form an oily substance.
8.2g was obtained. The resulting oil is fused with silica gel.
Chromatography was performed using 160 g of the product, and eluting with n-hexane-ethyl acetate (1:1) gave 7.6 g (yield: 90%) of the title compound as a white powder. (1) Thin layer chromatography: Rf value 0.37 Adsorbent: Merck silica gel Art5715 Developing solvent: n-hexane: ethyl acetate = 1:1 (2) Elemental analysis value C ₂₄ H ₄₀ O ₄ Si Calculated value C , 68.53; H, 9.58 Actual value C, 68.27; H, 9.45 2 Compound having the above formula () (R ² and R ³
is a methyl group) 8.4 g (0.02 mol) of the compound having the above formula ()
was dissolved in 50 ml of dry pyridine, 5.36 g (0.04 mol) of 2,2-dimethylbutyryl chloride and catalytic amounts of dimethylaminopyridine were added, and the
Heated at ~80°C for 3 hours. Next, 2.68 g (0.02 mol) of 2,2-dimethylbutyryl chloride was added and the mixture was further heated for 1 hour. After the reaction was completed, the reaction mixture was poured into 500 ml of ice water, and then 6N hydrochloric acid was added to make it acidic. The precipitate was extracted with ethyl acetate, and the extract was washed with saturated brine and dried over sodium sulfate. After drying, the solvent was distilled off from the extract to obtain 12.7 g of an oily substance. The obtained oil was chromatographed using 260 g of silica gel, and n-
Elution with a hexane-ethyl acetate (9:2) system gave 3.6 g (yield 34.7%) of the title compound as a pale green-yellow oil. (1) Thin layer chromatography: Rf value 0.28 Adsorbent: Merck silica gel Art5715 Developing solvent: n-hexane-ethyl acetate = 9:2 (2) Elemental analysis value C ₃₀ H ₅₀ O ₅ Si Calculated value C , 69.46; H, 9.71 Actual value C, 69.34; H, 9.68 (3) Nuclear magnetic resonance spectrum (δ: ppm) Measured at 60 MHz in deuterated chloroform using TMS as an internal standard. 1.06 (6H, singlet) 4.20 (1H, multiplet) 4.60 (1H, multiplet) 5.30 (1H, multiplet) 5.45 (1H, multiplet) 5.75 (1H, quartet) 5.95 (1H, doublet) ) 3 Compounds having the above formula () (R ² and R ³
is a methyl group) After dissolving 3.1 g (6 mmol) of the compound having the above formula () (R ² and R ³ are methyl groups) in 20 ml of dry tetrahydrofuran, 2.2 ml (36 mmol) of acetic acid and tetrabutylammonium fluoride were added. 18 ml (18 mmol) of a tetrahydrofuran solution (1 mmol/ml concentration) of ((n-C ₄ H ₉ ) ₄ NF) was added thereto, and the mixture was stirred at 40 to 50°C for 60 minutes. After the reaction is complete,
The solvent was distilled off from the reaction mixture, and the resulting residue was dissolved in 300 ml of ethyl acetate, washed five times with saturated brine, and dried over sodium sulfate. When the solvent was distilled off from the reaction mixture, 4.0 g of an oily substance was obtained. The obtained oil was chromatographed using 100 g of silica gel and eluted with a benzene-ethyl acetate (3:2) system to obtain 1.4 g (yield 57.8%) of the title compound as a white foam. . (1) Thin layer chromatography: Rf value 0.20 Adsorbent: Merck silica gel Art5715 Developing solvent: Benzene: Ethyl acetate = 3:2 (2) Elemental analysis value C ₂₄ H ₃₆ O ₅ Calculated value C, 71.25; H, 8.97 Actual value C, 71.04; H, 8.85 (3) Nuclear magnetic resonance spectrum (δ; ppm) Measured at 60 MHz in deuterated chloroform using TMS as an internal standard. 1.06 (6H, singlet) 4.35 (1H, multiplet) 4.70 (1H, multiplet) 5.28 (1H, multiplet) 5.55 (1H, multiplet) 5.85 (1H, quartet) 6.05 (1H, doublet) ) (4) Mass spectrum M/Z: 404 Example 1 Ring-closed lactone of formula () (R ² and
(R ³ is a methyl group) Ten 500 ml Erlenmeyer flasks containing 100 ml of a medium with the following composition were inoculated with Mucor hymalis IFO 5834 at 26°C and 220 r.pm.
After 4 days, a ring-closed lactone of formula () (R ² and R ³ are methyl groups) was added to a final concentration of 0.05.
% and further incubated at 26℃ and 220r for 6 days.
Culture at pm. Medium composition: Glucol 1.0% Peptone 0.2 Meat extract 0.1 Yeast extract 0.1 Corn steep liquor 0.3 Tap water Remaining (PH not corrected) After completion of the culture, the conversion reaction solution was filtered and the pH of the solution was adjusted to 3 with trifluoroacetic acid. Then, by extracting 1 three times with ethyl acetate, a fraction containing the carboxylic acid of formula () (R ² and R ³ are methyl groups) is obtained. Carboxylic acid of formula () (R ² and R ³ are methyl groups) was converted to Rf by thin layer chromatography (TLC) (plate; Merck silica gel Art5715 solvent; benzene:acetone:acetic acid = 50:50:3).
Indicates a value of 0.50. Conversion rate 40%. The above extract was washed with a saturated sodium chloride solution, then dehydrated with sodium sulfate, and then lactonized by adding a catalytic amount of trifluoroacetic acid. Next, the above extract was washed with a 5% aqueous sodium bicarbonate solution, dehydrated with sodium sulfate, and then concentrated to dryness. The obtained residue was applied to a Rover column (Si60 manufactured by Merck & Co., size A) and purified using a benzene:acetone=1:1 system to obtain 70 mg of the title compound. Physical property value 1 Nuclear magnetic resonance spectrum (Fig. 1) (δ: ppm) Measured at 90 MHz in deuterated chloroform using TMS as an internal standard. 2 Ultraviolet absorption spectrum

Claims (1)

[Claims] 1 formula (wherein R ¹ represents a group [formula] or [formula]; R ² and R ³ are the same or different and represent a lower alkyl group), a pharmacologically acceptable salt thereof, and an ester thereof. or a ML-236B derivative consisting of a closed ring lactone thereof.