KR900008248B1 - Process for preparing physiologically active substance fa-5859 - Google Patents

Abstract

Cpds. of formula (I) (where R1 is H or acetyl) and their salts are new. The N-acetyl cpd. of (I), designated FA-5859, is prepd. by cultivating appropriate strains of the genera Emericella or Aspergillus. It can be hydrolyzed conventionally to the primary amine. (I) inhibit degradation of fatty acids so are useful as antidiabetic agents and as reagents for studying fatty acid metabolism.

Description

Preparation of bioactive substance FA-5859

1 is an infrared absorption spectrum of the bioactive material FA-5859 obtained in Example 2.

2 is an infrared absorption spectrum of the hydrochloride of the bioactive material FA-5859 obtained in Example 3.

The present invention relates to a physiologically active substance FA-5859, derivatives thereof, their preparation and preparation.

Conventional compounds having a fatty acid degradation inhibitory activity include 4-pentenoic acid (PCHolland et al., Biochemical Journal, Vol. 136, p-157 and p-173, 1973, HSASherratt, etc. , Biochemical, Biochemical Pharmacology, Vol. 25, p-743, 1976), Hypoglycin (HSASherratt et al., Biochemical Pharmacology, Vol. 25, p-743). , 1976], decanoyl-(+)-carnitine and 2-bromopalmitoyl. Coenzyme A (2-bromopalmitoyl CoA) [IBFritz et al., Proceedings of the National Academy. Proceedings of the National Academy of Sciences USA 54, p-1226, 1965], but are not used clinically due to their toxicity and effects. Methyl-2-tetradecyl glycidate and 2-tetradecyl glycidate (methyl-2-tetradecyl-glycidate, 2-tetradecylglycidate) [(GFTutwiler et al., Diabetes 28, p-242, 1979 And Methods. Enzymology, Vol. 72, p-533, 1981, are known to have a fatty acid degradation inhibitory activity and to have an oral hypoglycemic effect.

In recent years, new mechanisms of action against diabetes and its complications, which continue to increase gradually, and the development of more effective anti-diabetes drugs are expected. That is, in diabetes, the release of fatty acids in adipose tissue is promoted due to insulin deficiency, thereby increasing the supply of fatty acids to the liver, and at the same time, increasing the decomposition of fatty acids, thus increasing the production of ketone bodies, which is called ketonemia. In addition, in the tissues of the liver, ketone bodies produced by poor use of glucose are used as energy sources. Therefore, the inhibition of fatty acid degradation is expected to lead to the use of glucose in response to the reduction of ketone bodies and consequently to lower the blood sugar level. Therefore, it is considered that the specific inhibitor of fatty acid degradation can be used as a novel diabetic therapeutic agent by a new mechanism. In light of these circumstances, the present inventors have searched and isolated a substance that inhibits fatty acid degradation in cultures of microorganisms belonging to the genus Emerisella or genus Acepergillus. As a result, this substance was found to be a novel substance and had an excellent inhibitory effect on fatty acid degradation, and it was called the bioactive substance FA-5859.

The present inventors estimated that the chemical structure of the bioactive substance FA-5859 is as follows.

The present inventors then conducted various studies to obtain derivatives of the bioactive substance FA-5859. As a result, the deacetylate obtained by hydrolyzing the bioactive substance FA-5859 is a novel substance as well as a remarkable fatty acid degradation inhibitory effect. The physiologically active substance was found to be called deacetyl FA-5859.

The inventors also conducted various studies on the chemical synthesis of the bioactive substance deacetyl FA-5859 and the bioactive substance FA-5859.

(In formula, R <_2> represents protecting groups other than an acetyl group.) The compound represented by it or its salt is carried out by protecting group desorption reaction and acetylation reaction as needed, and is represented by Formula (3).

Compound (1) is advantageously compounded by trimethylation reaction of the salt or a salt thereof and hydrolysis reaction if necessary.

It was found that the compound represented by (wherein R ₁ represents hydrogen or an acetyl group) or a salt thereof can be produced.

The inventors of the present invention have continued to study the results to complete the present invention.

The present invention (1) general formula

(Wherein R ₁ represents hydrogen or an acetyl group) A compound or a salt thereof.

(2) A physiological activity characterized by culturing the bioactive substance FA-5859 producing bacteria belonging to the genus Emerisella. Or Aspergillus in a culture medium and producing and accumulating the bioactive substance FA-5859 in the culture. Preparation of the substance FA-5859.

(3) expression

Hydrolysis reaction of the compound or salt thereof represented by

The manufacturing method of the compound shown by these, or its salt.

(4) General formula (2)

(In formula, R <_2> represents protecting groups other than an acetyl group.) The compound (1) or its salt manufacturing method characterized by deprotection of a protecting group and acetylation reaction as needed.

(5) (3)

A compound (1) or a method for producing the salt thereof and (6) a compound for treating diabetes comprising (1), which is characterized by trimethylation of a compound or a salt thereof and a hydrolysis reaction if necessary.

In the present specification, a compound in which R ₁ is hydrogen in compound (1) is referred to as "bioactive substance deacetyl FA-5859", or only "deacetyl FA-5859", in compound (1), in which R ₁ is an acetyl group. Are sometimes referred to as "bioactive substances FA-5859", or simply "FA-5859", respectively.

Examples of protecting groups other than the acetyl group represented by R ₂ in the general formula include t-butyloxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl and the like. .

FA-5859 (glass type) of the present invention has the following physicochemical properties.

(a) Elemental analysis (%): (dried under reduced pressure for 10 hours at 60 DEG C over phosphorus oxide).

C 51.16 ± 2.0

H 9.06 ± 1.0

N 13.26 ± 1.0

(b) Molecular weight: 2.4 to 3.3 × 10 ² (H ₂ O)

(By VPO law)

-17.4

±3

(c=1, H₂O)(c) specific light intensity:

-17.4

± 3

(c = 1, H ₂ O)

(d) Ultraviolet absorption spectrum: No specific absorption.

(e) Infrared absorption spectrum: (main absorption (wavenumber) of absorption spectrum by potassium embrittlement tablets) 1660, 1590, 1485, 1400, 1325, 1292, 970, 945 (cm ^-1 ).

(f) Solubility in solvents: insoluble: petroleum ether, hexane, diethyl ether, benzene, ethyl acetate, chloroform, poorly soluble: pyridine, acetone, dimethyl sulfoxide, dimethylformamide, soluble: ethanol, methanol, use: water.

(g) Color reaction: positive: okdo, negative: glagg. Liback reaction, ninhydrin reagent, sagakuguchi reaction, Morishshu reagent, Erlich reagent.

(h) Basic, acidic, neutral three stars: positive substances.

(i) Color of substance: colorless

FA-5859 can be produced by culturing the bioactive substance FA-5859 producing bacteria belonging to the genus Emerisella. Or Aspergillus in a medium, and producing and accumulating the bioactive substance FA-5859 in the culture.

Microorganisms that can be used in this method include the genus Emericella.

Or any species belonging to the genus Aspergillus and producing the bioactive substance FA-5859. Examples include, for example, E. quadrilineata, E. meridrila. Needleland. E. nidulans var. Acristata, E. mericella. cleistominuta, E. nidulans var.nidulans, E. nidulans var.lata, E. medulla, Ruglossa (E. rugulosa), Emerisella.Enldulans, Emerisella.Savulata, E.sublata, Aspergillus.sp.No. For example, Emerisella.Quadrillinea IFO 5859, Emerisella.Quadrillaneta IFO 30911, Emerisella.Quadrillineata IFO 30912, Emerisella.Quadrillaneta IFO 30850, Emerisella Cudrillineta IFO 30851, Emerisella. Needlelan. Bar. Acrysta IFO 30063, Emerisella. Needleless. Bar. Acresta IFO 30844, Emé Selah Clythe Inuta IFO 30839, Emerisella.Nidlans.Bar.Nidrance IFO 30872, Emerisella.Nidlans.Bar.Lata IFO 30847, Emerisella.Ruglossa IFO 8626, Emerisella.Ruglossa IFO 8629, Emerisella.Lugglos IFO 30913, Emerisella.Rugloss IFO 30852, Emerisella.Ruggosa IFO 30853, Emerisella.Nidlans IFO 5719, Emerisella.Nidlans IFO 7077, Emerisella Needlelans IFO 30062, Emerisella.Sarlatta IFO 30906, Aspergillus.sp No.3704.

List of IFO 5859, IFO 30063, IFO 8626, IFO 8629, IFO 5719, IFO 7077 and IFO 30062 shares issued by the Foundation of Fermentation Research Institute (IFO). Cultures, 1978, Sixth Edition).

In addition, IFO 30911, IFO 30912, IFO 30850, IFO 30851, IFO 30844, IFO 30839, IFO 30872, IFO 30847, IFO 30913, IFO 30852, IFO 30853 and IFO 30906 are published by the Fermentation Research Institute. 1981 (Institute For Fermentation, Osaka, Research Communications, No. 10, 1981).

The bacteriological properties of emerisella and kudrillineata in the microorganisms are described in Japanese Journal of Mycology, Vol. 20 No. 4, p-481 (1979), Transactions.of.the.economics, sociati.of. J.Pen (Transactions of The Mycological Society of Japan) vol.20, No.4, 481 (1979). (50 years old AD 1975), report. Of the the. In the report (The Report of The Tottori Mycological Institute) No. 12, 171 (1975), Emerisella. In the Transactions of The British Mycological Society vol 52, No. 2, 33l (1969), the mycelial characteristics of Emerisella, Nidrance, Var. Microbiology (Korean Journal of Microbiology) vol. 18, No. 2 104 (1980), the bacteriological properties of Emerisella. Raper, BIFennell, The Genus of Aspergillus p-500, The Williams & Wilkins Company, Baltimore, 1965, Emercellula. Journal of Korean Society for Publications, Vol. 20, No. 4, p-481 (1979), Transactions of the Mycological Society of Japan Vol.20, No.4 481 (1979), the bacteriological properties of Emerisella.Nidlans are described in KBRaper, BIFennell, The Genus Aspergillus p-145, The Williams & Willkins Company, Baltimore, 1965. The bacteriological statues of Emerisella and Sablita are described in the Japanese Society for Mycology, Vol. 20, No. 4, p-481 (1979) .Transactions of the Mikologic, Society, of Japan. Mycological Society of Japan) vol. 20, No. 4,481 (1979) are the same as the bacteriological properties described in each.

Aspergillus sp.No.3704 is a fungus isolated from the foot soil of Kagonishi, Yamato-cho, Hyogo-go, Japan.

Growth:

1) ZAbeck Agar Badge

Growth was slow and colony size was 1.4-2.0 cm after 24 ° C for 2 weeks. It is a swelling fungus with a slight bulge in the center, and the periphery is irregular and deeply dug under the agar. Seedling growth showed that the mycelia were reticular. It has a yellowish tinge pale green color, and becomes light brown as it is torn off. The number of conidia plums is small. The back side color is light brown to brown. When torn off yields a light brown soluble pigment.

2) wort agar medium

The growth was vigorous and the size of the clonie after 24 ° C and 2 sections was 4.0-5.0 cm. It is flat and the surroundings are thin and slightly swollen. The mycelia are distracting. The formation of conidia plums is very good and has a grayish yellowish green color. The back side is light brown to yellow brown. There is no formation of soluble pigment.

shape :

Conidia plums: The size is not constant, but the length is 75-100μ, the diameter is 20-40μ, and it is a radial but roughly cylindrical shape.

Conidia: 40-60μ in length, 2.0-4.5μ in diameter, wall surface smooth, colorless, smooth and curved.

Seminal vesicles: Flask type, flat at the top, 4.5-7.5μ in diameter

Metor: Cylindrical, 4.5-6.2 × 2.4-3.4μ

Pooiride: Club type, 4.5-6.5 × 2.0-3.0μ

Conidia: Real to oval, dark green, 2.5 to 3.5μ in diameter

When the above formulation is combined with the low "fungal illustration" of Sunichi, Koichi (Godansha, 1978), the composition of Aspergillus fungus, based on p-1006, is a strain of Aspergillus. Belonging to is obvious.

The Aspergillus sp No 3704 strain has been deposited in the fermentation laboratory of the Foundation as of November 6, 56 Digestion No. IFO 31171. In addition, this microorganism has been deposited with the FRI (Industrial Institute of Technology) Microorganism Industry and Technology Research Institute (FRI) as accession number FERM P-6224 from November 18, 2016, which was converted into a deposit by the Budapest Treaty, and accession number FERM BP It is kept at the FRI as -185 and deposited at KAIST with the number KAIST 830426-07810.

The genus Emerisella is a group of taxonomic genus Aspergillus, which is a full generation, and thus it is possible to use any strain capable of producing FA-5859 regardless of the sexual or asexual generation of the strain. none.

E. coli and Aspergillus are general properties of microorganisms and can cause mutations either naturally or by mutagenic agents. For example, many mutant strains obtained by mutating by irradiation of X-rays, γ-rays, ultraviolet rays or the like, further separation of monospores, treatment with various agents or culture on a medium containing the agents, or other means, or Naturally obtained mutant strains and the like, which have the property of producing FA-5859, can be used in the production method of FA-5859.

The medium used for the cultivation of FA-5859 may be either liquid or solid as long as the strain used contains nutrients that can be used. However, liquid medium is more suitable for processing large quantities. The medium contains an assimilable carbon source, a digestible nitrogen source, an inorganic substance, and a micronutrient. Examples of carbon sources include glucose, lactose, sucrose, maltose, dextrin, starch, glycerin, mannitol, sorbitol, fats and oils (e.g. soybean oil, olive oil, strong oil, sesame oil, lard oil, chicken oil, etc.), various fatty acids (e.g. Urinic acid, myristic acid, palmitic acid, stearic acid, oleic acid, etc.), for example, broth extract, yeast extract, dry yeast, soybean meal, corn steak liquor, peptone, cottonseed flour, fructose, urea, ammonium Salts (eg ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.) and others are used. Salts containing sodium, potassium, calcium, magnesium and the like, metal salts such as iron, manganese, zinc, cobalt and nickel, salts such as phosphoric acid and boric acid, and salts of organic acids such as acetic acid and propionic acid are suitably used. Other amino acids (e.g. glutamic acid, aspartic acid, alanine, lysine, valine, methionine, proline, etc.), peptides (e.g. dipeptides, tripeptides, etc.), vitamins (e.g., B ₁ , B ₂ , nicotinic acid, B ₁₂ , C, and the like), nucleic acids (eg, purine, pyrimidine and derivatives thereof), and the like. Of course, an inorganic or organic acid, an alkali, a buffer, or the like is added for the purpose of adjusting the pH of the medium, or a spine such as an oil-based surfactant is added for the purpose of consumption.

Tens of thousands of cultures may be political culture, or a means such as shaking culture or aeration stirring culture may be used. Needless to say, it is preferable to use so-called deep aeration agitation culture for a large amount of treatment. Naturally, the conditions of the culture are not constant depending on the condition of the medium, the composition, the type of strain, the means of the cultivation, and the like. In particular, the medium temperature is preferably about 23 ° C to 32 ° C, and the initial PH is about 4 to 6 conditions. The period of incubation is not constant depending on the condition of the former, but it is better to incubate until the concentration of this bioactive substance is maximized. The time required for this is usually about 1 to 80 seconds in the case of shaking culture using a liquid medium or aeration stirring culture.

Since FA-5859 produced is mainly present in the culture filtrate, it is advantageous to separate the culture into the supernatant and the cells by centrifugation or filtration and to purify from the supernatant. However, it can also be purified directly from the culture.

In addition, in collecting this FA-5859, a means commonly used in collecting a metabolite that produces microorganisms can be applied. For example, after removing the cells by centrifugation, a method of separating, collecting and purifying the active substance from the filtrate is generally used.

That is, the difference in solubility and solubility in a suitable solvent, the difference in precipitation method and precipitation rate from a solution, the difference in various adsorption affinity, ion exchange chromatography by ion exchanger, or concentration under reduced pressure, freeze drying, crystallization, Means such as recrystallization and drying are used alone or in combination in any order or repeatedly.

One example is as follows. That is, the filtrate obtained by filtering the culture solution after the end of the culture is subjected to a strong acid cation exchange resin, for example, a column made of Amberlite IR-20 (H ⁺ ) [Rom & Haas (USA)]. When passed through, FA-5859 is adsorbed onto the resin. For elution from the resin, an aqueous solution of ammonia water, alkaline water or mineral or inorganic salt (for example, salt, ammonium chloride, sodium sulfate, ammonium sulfate) can be used. For desalination of the eluate of the active substance obtained here, for example, an adsorbent such as activated carbon is used to adsorb the active substance thereto and then eluted using a hydrophilic organic solvent system. As a hydrophilic organic solvent, lower ketones such as acetone, methyl ethyl ketone and methyl isobutyl ketone, lower alcohols such as methanol, ethanol, isopropanol, propanol and butanol isobutanol, or a mixed solution of water and a mixed solution Can be mentioned. In addition, when the water-soluble high molecular material is mixed, it is good to use a conventional molecular sieve method. That is, since FA-5859 is a low molecule, for example, Sephatex G-10 (manufactured by Pharmacia Fine Chemicals, Sweden) can be used to remove the water-soluble polymer material. In order to purify the crude material thus obtained again, column chromatography using a cation exchange resin buffered using the product as a positive substance can be advantageously used. That is, as a carrier, a strong acid cation exchange resin, for example, Amberlite IR-120, Dow X 50 (X ₂ ) (Dow Chemical Co., Ltd.) Dion SK1A (Mitsubishi Kasei (Japan)), etc., may be used. For example, buffered with a pH 4 buffer is used. For elution after adsorbing to the resin by passing the solution of the crude material, the elution is carried out using a buffer of pH higher than the pH value at the time of adsorption. Adsorption to re-use a strongly acidic cation exchange resin, the active segment, the demineralized not after the river for the purpose of removing impurities that are mixed again basic performed desorption ion exchange resin, for 1 (OH ^-) g Dowex Dow Chemical Co. A hygroscopic crystal of FA-5859 is obtained from a syrup-like substance obtained by passing through a column of (USA), concentrated under reduced pressure, and freeze drying.

FA-5859 can also be used as a pharmacologically acceptable salt by conventional means. As an acid in the case of forming this salt, hydrochloric acid, sulfuric acid, nitric acid, aquatic acid, acetic acid, succinic acid, citric acid, fumaric acid, etc. are mentioned, for example.

The physicochemical properties of FA-5859 (the vitreous) obtained in Example 2 below will be described below.

(a) Elemental analysis (%) (60 ° C on phosphorus pentoxide, dried under reduced pressure for 10 hours): C: 52.48%, H: 9.04%, N: 13.25%

(b) Molecular weight: 2.4 to 3.3 x 10 ² (H ₂ O) (by VPO method)

(c) Estimated molecular formula: C ₉ H ₁₈ N ₂ O ₃

-17.4℃(C=1, H₂O)(d) Specific light intensity:

-17.4 ° C (C = 1, H ₂ O)

(e) Ultraviolet absorption spectrum: No specific absorption above 210nm.

(f) Infrared absorption spectrum: The main absorption (wavelength) is

3420 (s), 3260 (sh), 3080 (m), 1660 (s), 1590 (s), 1485 (s), 1400 (s), 1325 (m), 1295 (m), 1145 (w), 1105 (w), 1060 (w), 970 (m), 945 (m), (cm ^-1 )

w: weak, m: medium, s: steel, sh: shoulder

See FIG. 1 (Potassium Embrittlement Tablet).

(g) Solubility in Solvents:

Insoluble: Petroleum ether, hexane, diethyl ether, benzene, ethyl acetate, chloroform

Sparingly: pyridine, acetone, dimethyl sulfoxide, dimethylformamide

Soluble: Ethanol, Methanol

Use: water

(h) color reaction:

Positive: Okdo reaction

Negative: grey. Lieback reaction, ninhydrin reagent, sagakuguchi reaction, morisch reagent, erlich reagent

(i) Distinguishing Basic, Acidic and Neutral: Positive Substances

(j) Color of substance: colorless

(k) appearance of crystals; Colorless hygroscopic crystalline material

(l) NMR spectrum: (CD ₃ OD 100 MHz) 1.98 (3H, s), 2.42 (2H, d), 3.19 (9H, s), 3.56 (2H, d), 4.7 (1H, m).

s: single line, d: double line, m: multiple line

(m) Stability: The aqueous solution of pH 3-pH 9 is stable at 100 degreeC and 10-minute heating.

The physicochemical properties of FA-5859 hydrochloride obtained in Example 3 below are described below.

(a) Elemental analysis (%): (dried under reduced pressure for 10 hours at 60 ° C on phosphorus pentoxide)

C: 45.29%, H: 8.18%, N: 11.24%, Cl: 14.36%

(b) Estimated molecular formula: C ₉ H ₁₈ N ₂ O ₃ HCl

(c) Melting point: 215 ° C (decomposition)

-20.5°(c=1, H₂O)(d) Specific light intensity:

-20.5 ° (c = 1, H ₂ O)

(e) Ultraviolet absorption spectrum: It shows no specific absorption above 210nm.

(f) Infrared absorption spectrum The main absorptions (wave numbers) are as follows.

3400 (m), 3250 (s), 3190 (sh), 3045 (s), 2600-2400 (w), 1730 (s), 1660 (s), 1530 (m), 1480 (s), 1420 (m ), 1405 (s), 1375 (m), 1290 (m), 1205 (m), 1160 (s), 1140 (sh), 1135 (s), 1040 (w), 960 (w), 935 (m ), 915 (m), 865 (w), 800 (m), 665 (m), 625 (w), 600 (s), 560 (w) (cm ^-1 )

(w: weak, m: medium, s: strong)

See Figure 2 (Potassium Embrittlement Tablet)

(g) Solubility in solvents: insoluble: petroleum ether, hexane, diethyl ether, benzene, ethyl acetate, chloroform.

Poorly soluble: pyridine, acetone, dimethyl sulfoxide, dimethilformamide.

Soluble: Ethanol, Methanol

Use: water

(h) color reaction:

Positive: Okdo reaction

Negative: grey. Lieback reaction, ninhydrin reagent, sagaguchi reaction, morisch reagent, erlich reagent

(i) Color of substance: colorless

(j) Appearance of the crystal: colorless needle crystal

(k) Stability: The aqueous solution of pH 3-9 is stable by heating at 100 degreeC for 10 minutes.

)의 존재가 인정된다. 또 이 2조의 메틴 프로톤은 δ4.7ppm의 메틴 프로톤과 각각 커플링하고 있음이 데커플링에 의하여 명백해져서 -CH₂

의 부분식의 존재가 추정된다. 이 분자식으로부터 카르본산의 존재가 추정되며 이는 유리체로서 1590cm^-1에, 염산염으로 1730cm^-1에 c=0 흡수가 인징되는 점에 의하여도 분명하다 따라서 FA-5859의 평면 구조식으로서From the signal of δ 3.19 ppm (9H, s), which is recognized by the molecular formula and NMR spectrum of FA-5859, the presence of trimethyl ammonium in this molecule is estimated, and at δ 1.98 ppm (3H, s), methine proton of acetyl (CH ₃ CO-) 2.42 ppm (2H, d), 3.56 ppm (2H, sd) in two sets of methine protons (-CH ₂ -x 2), δ 4.7 ppm (1H, m)

) Existence is recognized. In addition, it is evident by decoupling that these two sets of methine protons are coupled to δ4.7 ppm methine protons, respectively, and are -CH _2.

The presence of subexpression of is estimated. To 1590cm ^-1 is believed that the presence of a carboxylic acid as a glass body, which from the molecular formula, the hydrochloride is also evident by a point that is to 1730cm ^-1 c = 0 absorption injing therefore a plane structural formula of FA-5859

Is estimated and FA-5859 is considered to be a novel compound.

Next, the measurement of fatty acid degradation inhibitory activity of the bioactive substance FA-5859 was carried out using a rat rat soy homogenate, and the metabolism of lipids, p-75 (1975, Doochem Chem. It was performed by the method described below according to the method of the same). In other words, SD rats (7-year-old, male) were fasted for 2 days, and then slaughtered by bleeding, and immediately harvested soy sauce to mix 10-fold (w / v) 5 mM Toris-HCl buffer (PH 7 5). The solution is added and homogenized with a tufted rod homogenizer. Fermented by centrifugation at 600 xg for 20 minutes and 30.000 xg for 30 minutes was suspended in a concentration of 0.29 / 0.5 ml of soy-moisture weight solution in the above-mentioned sucrose solution, and 0.5 ml was used as the enzyme solution for the reaction. have.

30 μmol potassium phosphate buffer (pH 7.5), 300 μmol KCl, 3 μmol ATP, 3 μmol MgCl ₂ , 120 μmol per book, ^1-14 C palmitic acid (0.1 μCl bovine serum albumin added to molar ratio 1: 5) pH 7.5 ) 0.6 μmol, L-cami-tin 0.6 μmol, Coenzyme A (0.6 μmol), 0.2 μmol oxalacetic acid, 0.1 ml of aqueous solution containing water or inhibitor and 0.5 ml of enzyme solution 3.0 ml of the roasted reaction mixture was placed in a test tube suspended in immersed in amine hydroxide 10-X (manufactured by Pakasa) and shaken at 37 ° C. for 20 minutes. 0.4 ml of 7% perchloric acid was added to stop the reaction, and the produced ¹⁴ CO ₂ was measured to measure the enzyme activity. The inhibitory activity of this compound FA-5859 was 17% at 250 μg / ml, 25% at 500 μg / ml and 36% at 1000 μg / ml compared with no inhibitor.

The acutely toxic LD _{50 level} of FA-5859 was 400 mg / kg or more as an intravenous intravenous injection.

FA-5859 or its salts are useful, for example, as fatty acid degradation inhibitors. When using FA-5859 or its salts as fatty acid degrading inhibitors, for example, about 0 2 to 200 mg / kg as FA-5859 can be determined using the path of diabetes in mammals (eg, rats, rats, humans, etc.). In the case of administration of FA-5859 or a salt thereof, the administration of FA-5859 or a salt thereof is carried out by mixing with self or host pharmacologically acceptable pharmaceutically acceptable excipients, excipients, and diluents. Formulations such as capsules, liquids and the like can be administered orally, or parenterally, for example, as injections.

When preparing the oral preparation, for example, a tablet, a binder (e.g., hydroxy propyl cellulose, hydroxy propyl methyl cellulose, magrogor, etc.), a disintegrant (e.g., starch, carboxymethyl cellulose calcium, etc.) , Excipients (e.g., promising, starch, etc.), lubricants (e.g., magnesium stearate, talc, etc.) can be suitably blended.

When preparing parenteral preparations such as injections, isotonic agents (e.g., glucose, D-sorbitol, D-mannitol, sodium chloride, etc.), preservatives (e.g., benzyl alcohol, chlorobutanol, methyl paraoxybenzoate, and paraoxybenzoic acid propyl) Etc.), a buffer (e.g., phosphate buffer, sodium acetate buffer, etc.) can be suitably blended.

In addition, FA-5859 can be used as a biochemical reagent in elucidating the metabolism of fatty acids. For example, FA-5859 can be added to the reaction solution to facilitate carnitine deficiency. You can be more clear. In addition, the physiological function of mitochondria and peloxysomes in fatty acid oxidation is hardly clear. Therefore, the combination of fatty acids to mitochondria is inhibited by the addition of FA-5859 to the function of feloxysomes and the oxidation process of peloxysomes and mitochondria. To clarify the relationship. In these cases, although the reaction system commonly used in fatty acid oxidation reaction depends on the concentration of intracellular granular components, in general, a concentration of about 0.5 mg / ml to 50 mg / ml of FA-5859 is advantageously used.

FA-5859 is also a useful substance as a synthetic intermediate of compounds that exhibits favorable fatty acid degradation inhibitory activity.

Deacetyl FA-5859 or its salt can be obtained by hydrolyzing EA-5859 or its salt.

This hydrolysis reaction can be applied to any method of decomposing an amide bond. For example, the hydrolysis reaction can be performed by using an acid group, an ion exchange resin, or the like. Examples of the acid include inorganic acids such as sulfuric acid and hydrochloric acid. Examples of the base include potassium hydroxide, sodium hydroxide, barium hydroxide, and the like. Dow Chemical Co., USA), Amberlite IR-120 (Rom & Haas. Company, USA), Diamond-Sk 1A, SK 1B (Mitsubishi Chemical Industries, Ltd., Japan), etc. are mentioned.

When using the above acid, it is preferable to perform this reaction in aqueous solution, and it is preferable to perform it in the mixed solvent of water, methanol, ethanol, butanol, etc. as an anti-aqueous solvent, for example. The reaction is usually carried out at about 60 to 200 ° C, more preferably at about 90 to 120 ° C for about 30 minutes to 30 hours, more preferably at about 3 to 16 hours.

When using the above base, it is preferable to carry out in aqueous solution, and it is preferable to carry out in mixed solvent with a water-soluble solvent, for example, methanol, ethanol, butanol, etc. The reaction is usually carried out at about 60 to 200 ° C, more preferably at about 90 to 120 ° C, for about 30 minutes to 30 hours, more preferably at about 3 to 16 hours.

In the case of using the above-mentioned ion exchange resin, the ion exchange resin is heated in a state where the ion exchange resin is suspended in an aqueous solution of the raw material compound. The reaction is usually performed at about 60 to 200 캜, more preferably at about 90 to 120 캜 and about 30 to about 30 hours, More preferably, it is performed in about 3 to 16 hours.

In order to obtain teacetyl FA-5859 or its salt from the reaction solution, commonly used means, such as ion exchange resin, adsorption, concentration, crystallization, etc. are used. It can be isolated, but it is easy to separate and collect in the form of salt.

As a specific example of obtaining the target substance from the reaction terminating solution, for example, the target substance is adsorbed using a strong acidic ion exchange resin or the like, and elution is carried out using, for example, hydrochloric acid or the like, to form a positive ninhydrin reaction. A substance can be obtained. For example, from a hydrolysis reaction solution using hydrochloric acid, the reaction solution is more simply concentrated under reduced pressure to distill excess hydrochloric acid, and then a solvent such as methanol, ethanol, diethyl ether or the like is added as a crystal. Hydrochloride can be obtained.

This gives deacetyl FA-5859 in the form of a free or salt.

The salt of deacetyl FA-5859 can also be converted to the free form. In this method, the acid or base forming the salt may be adsorbed onto the ion exchange resin using, for example, an ion exchange resin.

Since deacetyl FA-5859 free form can form a salt, it can be set as the salt pharmacologically acceptable by a routine means. Examples of the acid for forming this salt include hydrochloric acid, sulfuric acid, nitric acid, hydroxyl acid, acetic acid, succinic acid, citric acid, fumaric acid, and the like.

Next, the inhibition of fatty acid degradation of the bioactive substance deacetyl FA-5859 using mitochondrial fraction of rat rat soy homogenate was published in Japanese Biochemical Society, Vol. 9, Biomechanics, P-75 (1975). In accordance with the method described in Tokyo Chemical Driver) and IB Britz et al., Proceedings of the National Academy of Sciences, USA, Volume 54, p-1226, 1965. To measure. In other words, SD rats (7 strains, males) were fasted for 24 hours, and then slaughtered by bleeding, and immediately taken out of soy sauce. Homogenized by Teflon rod homogenizer. Fermented by centrifugation at 600 x g for 20 minutes and 30,000 x g for 30 minutes was suspended in a concentration of 0.2 g / 0.5 ml of soy sauce wet weight solution in the above sucrose solution, and 0.5 ml of the enzyme solution was used for reaction. Used. 30 μmol potassium phosphate feed solution (pH 7.5), 30 μmol KCl, 3 μmol MgCl ₂ , 120 μmol per book, 0.03 μmol L-peracid, 3 μmol ATP, 3 μmol L-carnitine, Coenzyme A (Co enzyme A) 0.6μ Mole, 7.5 μmol NAD, ^1-14 C-palmitic acid (0.2 μCi, pH 7.5 with bovine serum albumin added to Ohl 1: 5) 0.6 μmol, 0.1 ml aqueous solution containing water or inhibitor, and enzyme solution 0.5 25 ml of the reaction mixture in ml was placed in an airtight test tube immersed in the amine hydrooxide 10-X [Faka Co., Ltd. (Frank)], and the reaction was aerobicly shaken at 37 ° C. for 20 minutes. The activity was measured by adding ¹⁴ ml of 70% perchloroic acid to stop the reaction and measuring ¹⁴ CO ₂ produced. In comparison with the case of inhibiting activity of deacetyl FA-5859, that is, without adding an inhibitor, the concentration of inhibiting 50% of the activeol was 4 to 8 g / ml.

The acutely toxic LD _{50 level} of deacetyl FA-5859 was 400 mg / kg or more by intravenous injection of a rat.

Deacetyl FA-5859 or salts thereof of the present invention are useful, for example, as fatty acid degradation inhibitors. The use of deacetyl FA-5859 or its salts as fatty acid degradation inhibitors, for example, for the treatment of diabetes in mammals (e.g., rats, rats, humans), about 0.2 to 200 mg / kg is administered as the daily dose. In addition, in administering deacetyl FA-5859 or its salt, it is mixed by itself or a host of pharmacologically acceptable carriers, excipients, and diluents, for example, by reproducing tablets, granules, capsules, liquids, and the like. It can be administered orally or parenterally, for example, as an injection.

When preparing the oral preparations such as tablets, for example, binders (e.g., hydroxypropyl cellulose, hydroxypropylmethylcellulose, magrogor, etc.), disintegrants (e.g., starch, carboxymethylcellulose calcium) Excipients (eg, lactose, starch, etc.). Lubricants (for example, magnesium stearate, talc, etc.) can be mix | blended suitably.

When preparing parenteral preparations such as injectables, isotonic agents (e.g. glucose, D-sorbitol, D-mannitol, sodium chloride, etc.), preservatives (e.g. benzyl alcohol, chlorobutanol, methyl paraoxybenzoate, paraoxybenzoic acid). Profile, etc.) Buffers (e.g., phosphate buffers, sodium acetate buffers, etc.) can be suitably blended.

The deacetyl FA-5859 of the present invention can also be used as a biochemical reagent for elucidating the metabolism of fatty acids. For example, by adding deacetyl FA-5859 to the reaction solution, the carnitine deficiency state can be easily made, and thus the function of carnitine in fatty acid oxidation can be further clarified. In addition, the physiological function of mitochondria and veloxysomes in fatty acid oxidation is hardly clear. Therefore, by inhibiting the combination of fatty acids to mitochondria with the addition of deacetyl FA-5859, the function of veloxysomes and the effects of veloxysomes and mitochondria The relationship with the oxidation process can be clarified. In these cases, depending on the concentration of intracellular granular components using a reaction system commonly used in fatty acid oxidation reaction, generally, the concentration of about 0.1 μg / ml to 1000 μg / ml of deacetyl FA-5859 is advantageously used.

The deacetyl FA-5859 of the present invention is also a useful substance as a synthetic intermediate of a compound that exhibits more advantageous fatty acid degradation inhibitory action.

In order to obtain compound (1) by deprotecting the compound (2) or a salt thereof, which is the method of the present invention, for example, a means applied to conventional peptide synthesis reactions such as hydrolysis or catalytic reduction and acid treatment can be applied. have. This hydrolysis reaction can be applied to any method of decomposing an amide bond. For example, it can carry out by using an acid, a base, an ion exchange resin, etc. Examples of the acid include inorganic acids such as sulfuric acid and hydrochloric acid, and examples of the dibasic group include potassium hydroxide, sodium hydroxide and barium hydroxide. Examples of the ion exchange resin include Dow-X-50 ( Dow Chemical Co., USA), Amberlite IR-120 (Rom and Haas Company, USA), Diamond-SK 1A, SK 1B (Mitsubishi Chemical Industries, Ltd., Japan), etc. are mentioned.

When using the above acid, it is preferable to perform this reaction in aqueous solution, and it is preferable to perform it in the mixed solvent of water, methanol, ethanol, butanol, etc. as a water-soluble solvent, for example. The reaction is usually carried out at about 60 to 200 ° C, more preferably at about 90 to 120 ° C for about 30 minutes to 30 hours, more preferably at about 3 to 16 hours.

When using the above base, it is preferable to carry out in aqueous solution, and also it is preferable to carry out in water-containing solvent, for example, mixed solvent of water, methanol, ethanol, butanol, and the like. This reaction is usually carried out at about 60 to 200 ° C, more preferably at about 90 to 120 ° C for about 30 minutes to 30 hours, more preferably at about 3 to 16 hours.

When using said ion exchange resin, it heats in the state which suspended this ion exchange resin in the aqueous solution of a raw material compound. The reaction is usually carried out at about 60 to 200 ° C, more preferably at about 90 to 120 ° C for about 30 minutes to 30 hours, more preferably about 3 to 16 hours.

This catalytic reduction reaction is carried out by introducing hydrogen gas in water or an alcohol such as methanol or ethanol or in the presence of a catalyst such as palladium or palladium carbon in a mixed solvent of water and alcohol and pressurizing it as necessary. The temperature of this catalytic reduction reaction is about 0-50 degreeC, Preferably it is about 20-30 degreeC, Reaction time is about 0.5 to 5 hours, More preferably, it is performed in 1 to 3 hours. Examples of the acid used for this acid treatment include hydrogen sulfide-acetic acid, hydrochloric acid-acetic acid, hydrochloric acid-dioxane, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, and the like. The temperature of the acid treatment is about -10 ° C to 50 ° C, preferably about 0 ° C, buty 25 ° C and the reaction time is suitably changed by the acid used as about 30 minutes to 24 hours.

In order to acetylate a compound (2) or a compound obtained by protecting group desorption reaction, for example, the compound and acetic anhydride, acetyl chloride or acetic acid may be methyl chlorocarbonate, ethyl chlorocarbonate, chloro carbonate, chloro carbonate, chloro It is achieved by reacting anhydride of acetic acid obtained by reacting with alkoxycarbonyl chlorides such as isobutyl carbonate in water or a mixed solvent of water with organic solvents such as acetone, dioxane, acetonitrile, dimethylformamide, tetrahydrofuran and the like. . In the reaction, organic bases such as pyridine, triethylamine and trimethylamine or alkali metals such as sodium, potassium and calcium, and hydroxides, oxides and bicarbonates of alkaline earth metals may coexist as deoxidizers. As reaction temperature, it is 50 degreeC from about -10 degreeC, Preferably it is 25 degreeC from about 0 degreeC.

In order to trimethylate the compound (3) or a salt thereof, which is the method of the present invention, for example, the compound (3) or the salt thereof and dimethyl sulfate, methyl bromide, methyl chloride or methyl oxide are dissolved in water or water, acetonitrile, dioxane, The reaction is carried out by reacting tetrahydrofuran and diethyl in an organic solvent such as amide. In the reaction, alkali gold 의 such as sodium, potassium, calcium, and the like, hydroxides and oxides of alkaline earth metal may coexist as necessary. The reaction temperature is about −10 ° C. to 50 ° C., preferably about 0 ° C. to 20 ° C.

Each target product produced by each of the above reactions can be collected and purified by a commonly used separation, purification means, for example, chromatography, recrystallization, or the like.

The trimethylation reaction of the compound (3) or its salt, and then the hydrolysis reaction carried out as necessary can be applied to any method for decomposing an amide bond. For example, it can carry out by using an acid, a base, an ion exchange resin, etc. Examples of the acid include inorganic acids such as sulfuric acid and hydrochloric acid. Examples of the base include potassium hydroxide, sodium hydroxide, barium hydroxide, and the like. Dow Chemical company, USA), Amberlite IR-120 (Rom and Sus Company, USA) Diamond ion-SK 1 A, SK 1 B (made by Mitsubishi Chemical Industries, Japan), etc. are mentioned. .

When using the above acid, it is preferable to perform this reaction in aqueous solution, and it is preferable to perform it in the mixed solvent of water, methanol, ethanol, butanol, etc. as a water-soluble solvent, for example. The reaction is usually carried out at about 60 to 200 ° C, more preferably at about 90 to 120 ° C for about 30 minutes to 30 hours, more preferably at about 3 to 16 hours.

When using the said base, it is preferable to carry out in aqueous solution, Furthermore, it is preferable to carry out in a mixed solvent of water, methanol, ethanol, butanol, etc. for water-containing functions. This reaction is carried out at about 60 to 200 ° C., more preferably at about 90 to 120 ° C. for about 30 minutes to 30 hours, and more preferably at about 3 to 16 hours.

In the case of using the ion exchange resin described above, it is resisted by heating in a state where the ion exchange resin is suspended in an aqueous solution of a raw material compound. This reaction is usually at about 60 to 200 ° C, more preferably at about 90 to 120 ° C, for about 30 minutes to 30 hours, more preferably at about 3 to 16 hours.

In order to obtain deacetyl FA-5859 or its salt from the reaction solution, conventionally used means such as ion exchange resin, adsorption, concentration, crystallization, and the like are used. When the target object is removed from the reaction solution, either free or salt can be isolated, but it is easier to separate the salt in the form of a salt.

As a specific example of obtaining the target substance from the reaction termination solution, for example, strong targets are adsorbed by using an acidic ion exchange resin or the like, and eluted using, for example, hydrochloric acid or the like, to collect a positive sample by the ninhydrin reaction. For example, the target substance can be obtained. For example, from the hydrolysis reaction solution with hydrochloric acid, the reaction solution is more simply concentrated under reduced pressure, and the excess hydrochloric acid is distilled off, and then methane is added as a crystal to add a solvent such as ethanol or diethyl ether. Hydrochloride of this substance can be obtained.

This gives deacetyl FA-5859 or FA-5859 in the form of a free or salt.

The salts of deacetyl FA-5859 or FA-5859 can also be converted to the free form. This method is good to adsorb | suck the acid or base which forms this salt, for example using ion exchange resin etc. to an ion exchange resin. Since deacetyl FA-5859 free form or FA-5859 free form can form salt, it can be set as the pharmacologically acceptable salt by a conventional means. As an acid which forms this salt, hydrochloric acid, sulfuric acid, nitric acid, aquatic acid, acetic acid, succinic acid, citric acid, fumaric acid, etc. are mentioned, for example.

Compound (2) used as a starting compound in the method of the present invention can be produced by, for example, the following reaction step.

The compound (4) can be produced, for example, by the method described in Synthesis p-266, 1981, or by such a method.

Compound (3) can be manufactured by the reaction process shown next, for example.

Wherein R ₂ has the same meaning as electricity. R <_3> represents the protecting group represented by R <_2> and the protecting group which differs in the property at the time of detachment. As the protecting group represented by R ₃ , for example, benzyloxycarbonyl, t-butoxy carbonyl, P-carnitrobenzyloxy, carbonyl, p-methoxybenzyloxy carbonyl, trityl, tosyl, t-amyl Oxycarbonyl, oxycarbonyl, diphenylphosphinyl, o-nitrophenyl sulfphenyl, phthaloyl and the like.

In order to produce compound (5) from compound (4), for example, acid chlorides such as ethyl chlorocarbonate, methyl colocarbonate, propyl chlorocarbonate, butyl chlorocarbonate and isobutyl chloro carbonate are added to compound (4). The reaction is carried out in organic solvents such as ethyl, methyl acetate, dioxane, tetrahydrofuran and acetonitrile. In this reaction, organic bases such as N-methylmorpholine, N-ethyl morpholine, triethylamine, trimethylamine and pyridine You can coexist. The reaction temperature is about −20 ° C. to 30 ° C., preferably about −10 ° C. to 0 ° C.

Compound (6) is prepared by reacting diazomethane with compound (5). For example, the compound (5) is used at about -20 ° C to 30 ° C in organic solvents such as methyl acetate, methyl acetate, tetrahydrofuran, dioxane, acetonitrile, and dietyl ether, preferably at about -10 ° C. Reaction with diazomethane at 25 ° C. for about 1 hour to 24 hours. Diazomethane may be directly blown into the reaction liquid, or a solution saturated with an organic solvent such as diethyl ether or ethyl acetate may be added to the reaction liquid.

To prepare the compound (7) from the compound (6), for example, a silver salt such as benzoic acid or acetic acid is added to a methanol solution of the compound (6), and trimethylamine, triethylamine, N-methylmorpholine, and N-ethyl mortar. The solution dissolved in organic bases such as porin and pyridine is carried out by acting at about -5 ° C to 5 ° C, preferably at about 0 ° C to 27 ° C in water, for about 30 minutes to 10 hours in the dark.

Compound (8) can be manufactured by quenching compound (7). The kenification is, for example, water or hydroxides such as sodium, potassium and barium in an organic solvent such as methanol, ethanol, dioxane, tetrahydrofuran, acetonitrile or a mixed solvent of water and these organic solvents. The reaction is carried out by reacting at about −10 ° C. to 50 ° C., preferably at about 0 ° C. to 27 ° C. for about 1 hour to 5 hours.

In order to prepare compound (9) from compound (8), a method of selectively varying R ₃ is applied. This method is a method commonly used for peptide synthesis, and can be carried out in the same manner as the reaction for removing the protective group from the aforementioned compound (2).

Compound (2) can be manufactured by carrying out trimethylation reaction of compound (9). This trimethylation reaction can be performed similarly to the reaction at the time of carrying out trimethylation reaction of the compound (3) mentioned above.

In order to replace the compound (8) with the compound (10), the above-mentioned compound (2) can be carried out in the same manner as the reaction for removing the protecting group.

Compound (11) can be manufactured by acetylating reaction of compound (10). This acetylation reaction can be carried out in the same manner as this acetylation reaction in the reaction in which the above-mentioned compound (2) is subjected to the protective group desorption reaction and acetylation reaction if necessary.

Compound (3) can be manufactured by carrying out group deprotection reaction of compound (11). As this protecting group removal reaction, it can carry out similarly to the reaction at the time of manufacturing compound (9) from the compound (8) mentioned above.

Each target product produced by each of the above reactions can be collected and purified by a commonly used separation and purification means, for example, chromatography, recrystallization or the like.

As is apparent from the above, Compound (8) can be used as a synthetic intermediate in preparing useful compounds.

In the reaction step, salts of these compounds may be used. As this salt, salts, such as sodium, potassium, calcium, barium, triethylamine, pyridine, hydrogen chloride, hydrogen embrittlement, and hydrogen iodide, are mentioned, for example.

Next, the present invention will be described in more detail with reference to examples and examples. And in the following examples the% of the composition of the medium represents the weight / volume%.

Example 1

Daily platinum of Emerisella Cuadrillinea IFO 5859, which had grown sufficiently in advance on a slope medium made of Potato sucrose agar and formed spores, was prepared with glucose 2.0%, maldos 3.0%, and raw soy flour 1.5 500 ml of culture medium consisting of%, corn steep liquor 1.0%, polypeptone 0.5%, yeast extract 0.3% and saline 0.3%, pH 6.0 was inoculated into a 21 sterile saccagucci flask, which was sterilized and 2 at 28 ° C. on a reciprocating shaker. Incubated daily. L.5 liters of this culture was 3.0% oleic acid, 0.5% raw soy flour, 0.5% malt extract, 0.5% polypeptone, 0.2% yeast extract, KH ₂ PO ₄ 0.1%, FeSO ₄ · 7H ₂ O 0.05%, MnSO ₄ 100 l of the main culture medium consisting of 0.05% of nH ₂ O, 0.05% of MgSO ₄ 7H ₂ O, and pH 4.5 was injected into a sterilized 200-l fermenter. The main fermentation was carried out at 28 ° C., aeration (100 l / min), and agitation (200 r.pm) for 114 hours at an internal pressure of 1.0 kg / cm 2. Two batches of the main fermentation broth obtained in this manner are combined and sterilely filtered to obtain a culture filtrate containing FA-5859.

Example 2

125 l of the culture filtrate obtained in Example 1 was passed through a column of Amberlite IR-120 (H ⁺ type) (20 l), washed with 40 l of water, and eluted with 60 l of N-ammonia water. The eluate was concentrated under reduced pressure to 30 l to distill the anemones, pass through the activated carbon column for chromatography (301), wash the column with rule 60 l, and elute with 90 l of 50% methanol water. The eluate was partitioned by 10 liters, and the effective fractional fractions Nos. 5 to 6 were collected and concentrated under reduced pressure to give 25.5 g of crude syrupy material. This crude was dissolved in 100 ml of pH 4.0 acetic acid buffer (0.05 M) and passed through a column of Dowex 50 × 2 (500 ml) buffered with acetic acid buffer (0.1 M) at pH 4.0. Elution was carried out sequentially using the same buffer pH 4.0 l, pH 4.3 1.5 l, pH 4.6 1.5 l, pH 5.0 1.5 l. The eluate was separated by 100 ml, and fractions No. 32 to 63 were collected, and the mixture was washed with 600 ml of Amberlite IR-120 (H ⁺ type) (300 ml) and eluted with 1.5 L of 0.5 N-ammonia water. The eluate was concentrated under reduced pressure to 500 ml, and the concentrate was washed with 200 ml of water through Dow X 1 × 2 (OH ⁺ type) (200 ml). The passing liquid and washing liquid were combined, concentrated under reduced pressure, and freeze-dried. When the syrup-like substance obtained here was left to stand at room temperature, 10.7 g of FA-5859 glassware of colorless hygroscopic crystal phase was obtained. The infrared absorption spectrum of this material is shown in FIG.

Example 3

After dissolving 210 mg of FA-5859 vitreous obtained in Example 2 in 10 ml of water and cooling with ice, 1 ml of 1N hydrochloric acid was added, concentrated under reduced pressure, 10 ml of ethanol was added, and the crystals obtained by standing at room temperature were recrystallized from water and ethanol to give FA- of a colorless needle. 225 mg of 5859 hydrochloride were obtained. Melting point, 215 ° C. (decomposition). The infrared absorption spectrum of this material is shown in FIG.

Example 4

According to the method described in Example 1, it was confirmed that FA-5859 was produced as a result of culturing using the microorganism shown below.

Emerisella Cuadrilineata IFO 30911, Emerisella Cuadrilineata IFO 30912, Emerisella Cuadrilineata IFO 30850, Emerisella Cuadrilineata IFO 30851, Emerisella needleless Bar Acresta IFO 30063, Emerisella Nidlans Bar Acrestata IFO 30844, Emerischela Chrysler Minuta IFO 30839, Emerisella Nidrants Bar Nidrance IFO 30872, Emerisella Needle Lance Bar Lata IFO 30847, Emerisella Rugularosa IFO 8626, Emerisella Rugularosa IFO 8629, Emerisella Rugularosa 1FO 30913, Emerisella Rugularosa IFO 30852, Emerisela Lugulrosa IFO 30853, Emerisella Nidrance IFO 5719, Emerisela Nidrance.IFO 7077, Emerisela Nidrance IFO 30062, Emerisela Sablata IFO 30906.

Example 5

Aspergillus sp. No. 3704 strain (IFO 31171, FERM BP-185, KAIST 830426-07810) was inoculated into 500 ml of the same culture medium as in Example 1 in a sterile, 2-liter sagagucci flask, which was then circulated on a reciprocating shake incubator. Incubated at 28 ° C. for 2 days. 1.51 of this culture was 3.0% soybean oil, 0.5% soy flour, 0.5% malt extract, 0.5% polypeptone, 0.2% yeast extract, KH ₂ PO ₄ 0.1%, FeSO ₄ 7H ₂ O 0.05%, MnSO _{4 100} l of the main culture medium consisting of 0.05% ₂ O and 0.05% MgSO ₄ H ₂ O, pH 4.5 were injected and transplanted into a sterile 200 fermentor. This main fermentation was incubated for 114 hours under conditions of 28 ° C., aeration (100 l / min) and agitation (200er.pm), and internal pressure 1.0 kg / cm 2. When the obtained culture liquid was made into sterile cells by filtration, the liquid 80 of the culture containing FA-5859 was obtained.

Example 6

80 l of the filtrate obtained in Example 5 was treated and purified in the same manner as in Example 2, and 3.15 g of a syrup-like FA-5859 vitreous was dissolved in 150 ml of water, ice-cooled, 15 ml of N-hydrochloric acid was added, concentrated under reduced pressure, and 150 ml of ethanol was added and left to stand. Recrystallization of the crystals from water and ethanol gave 3.3 g of colorless needle supernatant FA-5859 hydrochloride. Melting point 214 ° C. (decomposition).

Elemental Analysis Value (%):

C; 45.39, H; 7.73, N; 11.50, Cl; 44.77.

Example 7

1.60 g of FA-5859 glass was dissolved in 40 ml of maintenance shop hydrochloric acid, and left at 95 ° C. for 16 hours. The reaction solution was concentrated under reduced pressure, and a small amount of water was added to the residue, followed by concentration under reduced pressure again. A mixed solvent of methanol and diethyl ether was added to the residue, and the precipitated crystals were filtered out. Recrystallization from ethanol yielded 1.20 g of dihydrochloride of acetyl FA-5859.

+6.3°(=1.0, N-AcOH).Melting point 219-220 ° C,

+ 6.3 ° (= 1.0, N-AcOH).

Elemental Analysis: C ₇ H ₁₈ O ₂ N ₂ Cl ₂

Theoretical value (%): C; 36.05, H; 7.77, N; 12.01, Cl, 30.40

Found (%): C; 36.09, H; 7.72, N; 11.81, Cl, 29.80 Absorption Spectrum: Specific absorption is not shown in the ultraviolet and visible region from 210 nm to 700 nm.

Reference Example 1

(1) 6.1 g of (L) -α-benzyloxycarbonyl ino-β-t-butoxycarbonylaminopropionic acid was dissolved in 100 ml of ethyl acetate, cooled to -10 ° C, and 1.8 g of N-methylmorpholine 1.9 g of ethyl chlorocarbonate was added, and it stirred at -10 degreeC for 1 hour. The insoluble portion was filtered off and a large excess of diazomethane-diethyl ether solution was added to the filtrate and stirred at 0 ° C. for 1 hour and then at room temperature for 12 hours. The solvent was distilled off under reduced pressure, and the residue was dissolved in 50 ml of methanol, and 2 ml of triethylamine solution of 200 ml of silver salt of benzoic acid was added thereto, followed by stirring at room temperature, followed by 4 hours in the dark. The insolubles were filtered off, the filtrate was concentrated under reduced pressure, and the residue was dissolved in 100 ml of ethyl acetate. The ethyl acetate solution was washed with 10% citric acid water, 5% sodium bicarbonate and water in that order and dried over anhydrous sodium sulfate. When ethyl acetate was distilled off, crystals of (L) -β-benzyloxycarbonylamino-γ-t-butoxycarbonylamino lactate methyl ester precipitated. Recrystallized from ethyl acetate-petroleum ether. Quantity 4 8 g (73%).

Melting Point 100-101 ℃

+6.0℃ (c=1, 디메틸포름아미드 중)

+ 6.0 ° C. (c = 1 in dimethylformamide)

Elemental analysis: using C ₁₈ H ₂₆ O ₆ N ₂

Calculated (%): C; 59.00, H; 7.15, N; 7.65

Found (%): C; 59.30, H; 7.07, N; 7.74

(2) 3.60 g of (L) -β-benzyloxycarbonylamino-γ-t-butoxycarbonylaminolactic acid methyl ester obtained above was dissolved in 20 ml of methanol, and 12 ml of 1N-sodium hydroxide was added at 0 ° C. After stirring at room temperature for 3 hours, the mixture was concentrated with citric acid and 100 ml of ethyl acetate was added. The ethyl acetate solution was washed with water, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. Precipitated crystals are filtered off and recrystallized from ethyl acetate to obtain crystals of (L) -β-benzyloxycarbonylino-γ-t-butoxycarbonylaminolactic acid. Quantity 2.96 g (85%)

Melting point 136-137 ℃

+6.0℃ (c=1, 디메틸포름아미드 중)

+ 6.0 ° C. (c = 1 in dimethylformamide)

C ₁₇ H ₂₄ O ₆ N ₂

Calculated (%): C; 57.94, H; 6.87, N; 7.95

Found (%): C; 57.97, H; 6.76, N; 8.13

Reference Example 2

0.75 g of (L) -β-benzyloxycarbonylamino-γ-t-butoxycarbonylaminolactic acid obtained in Reference Example 1 was dissolved in 10 ml of trifluoroacetic acid and allowed to stand at room temperature for 10 minutes. The reaction solution was dried under reduced pressure, and the residue was dried again under reduced pressure. It was dissolved in 7 ml of 10% sodium hydroxide, cooled to 0 ° C., and 0.65 ml of dimethyl sulfate was added and stirred at 0 ° C. for 1 hour and then at room temperature for 1 hour. The reaction solution was transferred to Amberlite IR-120 (H + type) column chromatography (60 ml), washed with water and eluted with N-ammonia water. Then, the elution fractions were collected from 150 ml to 220 ml, concentrated under reduced pressure, and the residue was dried under a reduced pressure to obtain 0.44 g. (L) -β-benzyloxycarbonylamino-γ-trimethylaminolactic acid of was obtained as a flaky substance. Thin layer chromatography (carrier silica gel 60F ₂₅₄ , Melk, West Germany)

1) Rf = 0.13 (n-propanol: = 4: 1)

Rf = 0.26 (n-propanol: water: 15)

N-ammonia = 70: 28: 2).

Reference Example 3

(1) 1.4 g of (L) -β-benzyloxycarbonylamino-γ-t-butoxycarbonylaminolactic acid obtained in Reference Example 1 was dissolved in 30 ml of 80% methanol water and subjected to catalytic reduction in the presence of palladium nodule. It was done. The catalyst was filtered off and the methanol was distilled off and 840 mg of sodium hydrogen carbonate and 10 ml of water were added to the residue. 10 ml of acetonitrile and 0.5 ml of acetic anhydride were added to the solution at 0 ° C, and stirred at 0 ° C for 1 hour and at room temperature for 12 hours. Acetonitrile was distilled off, 50 ml of diethyl ether was added to the residue, and the aqueous layer was neutralized with 0.1 N hydrochloric acid. Extracted three times with 50 ml of ethyl acetate, the ethyl acetate layer was dried over anhydrous sodium sulfate. Ethyl acetate was distilled off, crystallized with petroleum benzine and recrystallized from ethyl acetate to obtain crystals of (L) -β-acetylamino-y-t-butoxycarbonylaminolactic acid. Quantity 88 g (86%).

Melting point 140-141 ℃

(c = 1, in dimedylformamide).

Elemental analysis: using C ₁₁ H ₂₀ O ₅ N ₂

Calculated (%): C; 50.75, H; 7.75, N; 10.76

Found (%): C; 50.53, H; 7.35, N; 10.68

(2) 700 mg of (L) -β-acetylamino-γ-t-butoxycarbonylaminolactic acid obtained above was dissolved in 10 ml of trifluoroacetic acid and left at room temperature for 30 minutes. Trifluoroacetic acid was distilled off and the residue was dried under reduced pressure and dissolved in 100 ml of water⁺brother ) Into a column (300 ml). After washing with water, eluted with 0.5N ammonia, and the eluate was concentrated under reduced pressure. The precipitated crystals are filtered off and recrystallized from methanol to obtain crystals of (L) -β-acetylamino-γ-aminolactic acid. Quantity 348 mg.

Melting Point 177-178 ℃ (Decomposition)

]²⁴ _D-15.4

(c=0.7, 0.1N-염산).[

²⁴ _D -15.4

(c = 0.7, 0.1 N hydrochloric acid).

Elemental Analysis: Using C ₆ H ₁₂ O ₃ N ₂

Calculated (%): C; 44.99, H; 7.55, N; 17.49

Found (%): C; 44.83, H; 7.62, N; 17.22

Example 8

(1) 0.39 g of (L) -β-benzyloxycarbonylamino-γ-trimethylaminolactic acid obtained in Reference Example 2 was dissolved in 5 ml of 5.7 N hydrochloric acid, and the melt was warmed at 90 DEG C for 30 minutes. The reaction solution was concentrated under reduced pressure, and a small amount of water was added to the residue, followed by concentration under reduced pressure again. A mixed solvent of methanol and diethyl ether was added to the residue, and the precipitated crystals were filtered to obtain 228 mg of (L) -γ-trimethylamino-β-aminolactic acid (deacetyl FA-5859).

Melting Point 219-220 ℃ (Decomposition)

]²⁴ _D+6.7

(c=1, N-초산중).[

] ²⁴ _D +6.7

(c = 1, in N-acetic acid).

Elemental analysis: using C ₇ H ₁₈ O ₂ N ₂ Cl ₂

Calculated (%): C; 36.05, H; 7.77, N; 12.01, Cl: 30.40

Found (%): C; 35.96, H; 7.58, N; 11,88, Cl: 30.13

염산염(FA-5859

염산염)의 결정을 얻다. 수량 510mg.(2) 699 mg of the dihydrochloride of deacetyl FA-5859 obtained in Example 7 was dissolved in 20 ml of water, and a solution obtained by dissolving 1.1 g of sodium bicarbonate and 0.45 ml of acetic anhydride in 10 ml of acetonitrile was added while stirring at 0 ° C. Stirred at 0 ° C. for 1 hour and at room temperature overnight. Acetonitrile was distilled off and flowed into a column (80 ml) of Dow X 50 × 2 (H ⁺ type). The column was washed with 300 ml of water and eluted with 0.5N ammonia. 240 ml of the elution fractions were collected from 220 ml, concentrated under reduced pressure, and the residue was dissolved in 50 ml of water and freeze-dried. The obtained malty substance was dissolved in 2.8 ml of 1N hydrochloric acid, and dried under reduced pressure. The residue was crystallized using methanol-diethyl ether to give (L) -y-trimethylamino-β-acetylaminolactic acid.

Hydrochloride (FA-5859

Hydrochloride) to obtain crystals. Quantity 510mg.

Melting Point 222-223 ° C (Decomposition)

]²⁴ _D-200

(c=0.75, 물).[

²⁴ _D -200

(c = 0.75, water).

Elemental Analysis: Using C ₉ H ₁₉ O ₃ N ₂ Cl ₂

Calculated (%): C; 45.28, H; 8.02, N; 11.73, Cl; 14.85

Found (%): C; 45.13, H; 8.24, N; 11.79, Cl; 14.73

Example 9

염산염(FA-5859

염산염)의 결정을 얻다. 수량 220mg(1) 288 mg of (L) -β-acetylamino-γ-aminolactic acid obtained in Reference Example 3 was dissolved in 8 ml of 10% sodium hydroxide. The solution was cooled to 0 deg. C, 0.8 ml of dimethyl sulfate was added, stirred at 0 deg. C for 30 minutes, and then at room temperature for 30 minutes, and then referred to Dow X 50 × 2 (H ⁺ type) column chromatography (80 ml). The column was eluted with 0.5N-ammonia water after washing with water, and the elution fractions from 250ml to 240ml were collected and concentrated under reduced pressure. The residue was dissolved in 1.5 ml of N-hydrochloric acid and dried again under reduced pressure. The dried material was determined from methanol-diethyl ether to give (L) -γ-trimethylamino-β-acetylaminolactic acid.

Hydrochloride (FA-5859

Hydrochloride) to obtain crystals. Quantity 220mg

Melting point 217-218 ° C. (decomposition).

]²⁴ _D-20.2

(c=0.96, 수중)[

²⁴ _D -20.2

(c = 0.96, underwater)

Elemental Analysis: Using C ₉ H ₁₉ O ₃ N ₂ Cl

Calculated (%): C; 45.28, H; 8.02, N; 11.73, Cl: 14.85

Found (%): C; 44.97, H; 8.02, N; 11.49, Cl: 14.84

염산염(FA-5859

염산염) 100mg을 5.7N 염산 5ml에 용해하여 100℃에서 6시간 처리하여 재차 감압 농축하였다. 잔류물에 메탄올과 디에틸에테르와의 혼합 용매를 가하고 석출하는 결정을 여취하여 95mg의 (L)-γ-트리메틸아미노-β-아미노락산

2염산염(데아세틸 FA-5859

2염산염)의 결정을 얻다.(2) (L) -γ-trimethylamino-β-acetylaminolactic acid obtained above

Hydrochloride (FA-5859

Hydrochloride) 100 mg was dissolved in 5 ml of 5.7 N hydrochloric acid, treated at 100 ° C. for 6 hours, and concentrated under reduced pressure again. A mixed solvent of methanol and diethyl ether was added to the residue, and the precipitated crystals were filtered out to obtain 95 mg of (L) -γ-trimethylamino-β-aminolactic acid.

Dihydrochloride (Deacetyl FA-5859

Dihydrochloride) to obtain crystals

Melting Point 218-219 ℃ (Decomposition)

]²⁴ _D-6.3

(c=1, N1-초산중)[

²⁴ _D -6.3

(c = 1, N1-acetic acid)

Elemental analysis: using C ₇ H ₁₈ O ₂ N ₂ Cl ₂

Calculated (%): C; 36.05, H; 7.77, N; 12.01, Cl; 30.40

Found (%): C; 36.12, H; 7.88, N; 11.85, Cl; 30.11

Example 10

Tablets are prepared by conventional means using the following ingredients.

FA-5859 300mg

스타치 50mgCone

Starch 50mg

Lactose 28mg

Hydroxypropyl Cellulose 20mg

스테아레이트 2mgmagnesium

2 mg stearate

400 mg

(Per tablet)

Adults take 4 to 8 tablets per day after meals (three times a day).

Example 11

Tablets are prepared by conventional means using the following ingredients.

Deacetyl FA-5859 300mg

스타치 50mgCone

Starch 50mg

Lactose 28mg

Hydroxypropyl Cellulose 20mg

스테아레이트 2mgmagnesium

2 mg stearate

400 mg

(Per tablet)

Adults take 2 to 4 tablets per day after meals (three times a day).

Claims (4)

Expressions belonging to the genus Emerisella, genus or Aspergillus

A method for producing a bioactive substance FA-5859, comprising culturing the bioactive substance FA-5859 producing bacterium represented by a medium, generating and accumulating the bioactive substance FA-5859 in a culture. expression

Hydrolysis reaction of the compound or salt thereof represented by

The manufacturing method of the compound shown by these, or its salt. expression

A compound represented by (wherein R ₂ represents a protecting group other than an acetyl group) or a salt thereof is reacted differently with a protecting group or subsequently acetylated.

(Wherein R ₁ represents hydrogen or an acetyl group) A method for producing a compound or salt thereof. expression

A compound represented by the above formula or a salt thereof is subjected to trimethylation reaction or subsequent hydrolysis reaction.

(Wherein R ₁ represents hydrogen or an acetyl group) A method for producing a compound or salt thereof.

Images