WO2006126654A1 - Novel compound colletoic acid - Google Patents

Abstract

A novel compound having an inhibitory effect on 11β-hydroxysteroid dehydrogenase type 1 (hereinafter referred to as '11β-HSD1'); a pharmaceutical comprising the compound as an active ingredient (preferably a therapeutic/ameliorating agent for diabetes, obesity, hyperlipemia, hypertension or metabolic syndrome that develops these conditions compositery; a method for producing the compound; a novel microorganism capable of producing the compound; and others.

Description

 Specification

 New compound

 Technical field

 [0001] The present invention relates to a novel compound having an inhibitory action on 11 j8-hydroxysteroid dehydrogenase typel (hereinafter referred to as "11 j8- HSD1"), a medicine containing the compound as an active ingredient (preferably diabetes, obesity , Treatment of hyperlipidemia, hypertension, or metabolic syndrome that develops these pathological conditions in combination ”, a method for producing the compound, a novel microorganism that produces the compound, and the like.

 Background art

 [0002] In recent years, with changes in dietary habits, lifestyle and living environment, hyperglycemia, obesity, hyperlipidemia, hypertension, and metabolic syndrome patients with these multiple risk factors have increased worldwide. This is a social problem (Non-patent Document 1). Any treatment! / Food therapy and exercise therapy are used at first and are less effective! / Pharmacotherapy is combined with epilepsy or severe cases.

 [0003] Diabetes is classified into insulin-dependent (type I, IDDM) and insulin-independent (type II, NIDDM), and more than 90% of diabetics are the latter. Insulin injection is used to treat IDDM, and sulfo-urea drugs that promote insulin secretion, thiazolidinedione drugs that improve insulin resistance, and daricosidase inhibitors that inhibit digestion and absorption of sugars. In addition, biguanide drugs that suppress gluconeogenesis in the liver are used (Non-patent Document 2). However, none of these drugs is always effective, and the effectiveness of these drugs decreases over time, increasing the number of patients who have serious complications.

 In the treatment of obesity, central feeding inhibitors are used in severe patients! However, due to the limited use period and rebound, a sufficient effect has not been obtained.

[0005] As a treatment for hypertension, calcium antagonists that dilate blood vessels, diuretics that promote excretion of salt, suppress sympathetic nerves and reduce heart rate) 8 blockers, diminish peripheral blood vessels by suppressing sympathetic nerves An alpha blocker, angi to suppress vasoconstriction by angiotensin And ottensin converting enzyme inhibitors and angiotensin receptor antagonists. However, it is difficult to control blood pressure in the early morning, which is the most frequent stroke period, and the current state of antihypertensive therapy is still insufficient.

 [0006] Hyperlipidemia treatment includes HMG Co A reductase inhibitor that inhibits cholesterol synthesis in the liver, fibrate drugs that inhibit synthesis of neutral fat in the liver, and promotes bile acid excretion Anion exchange resin is used. The ultimate goal of hyperlipidemia treatment is to prevent arteriosclerotic diseases such as coronary artery disease and cerebral infarction, but arteriosclerosis is not only hyperlipidemia but also hypertension, diabetes, obesity Since various risk factors, such as aging, overlap, it is important to keep an eye on these other risk factors, and multifaceted efforts are required (Non-patent Document 3).

[0007] Glucocorticoids (cortisol in humans and corticosterone in rodents) release amino acids from muscle and promote the release of fatty acid and glycerol into blood through the expression of various proteins However, it promotes gluconeogenesis in the liver using these substrates, which in turn promotes an increase in blood sugar. In addition, it matures immature adipocytes in adipose tissue and leads to obesity, and in the kidney, it acts on mineralocorticoid receptors to increase blood pressure. In addition to glucocorticoid production and secretion control in the hypothalamus, pituitary gland, adrenal cortex pathway, 11 j8-hydroxysteroid dehydrogenase in target organs such as liver, adipose and tissue, and lung There is a mechanism of glucocorticoid recycling (conversion from inactive to active) by type 1 (hereinafter referred to as “11 j8—HSD1”) (Non-patent Document 4). 11 — HSD1 inhibitors suppress the action of glucocorticoids in these tissues, thereby suppressing hyperglycemia, obesity, hyperlipidemia and epilepsy or hypertension, and combining these pathologies It is expected to exert multifaceted effects on metabolic syndrome that has developed spontaneously. In adipose tissue-specific 11 β HSD1 high-expressing mice, visceral fat obesity, impaired glucose tolerance, insulin resistance and increased blood pressure have been reported and support these possibilities (Non-Patent Document 5, Or non-patent document 6). 11 Aminothiazol sulphonamide derivatives (Non-patent Document 7) and triazole derivatives (Patent Document 1) are known as 18-HSD1 inhibitors, but are not yet in practical use. Patent Document 1: International Patent Publication Number WO03Z065983 Pamphlet

 Non-patent document 1: “Nature”, 414 卷, p. 782 (2001)

 Non-Patent Document 2: "The American Journal of"

Medicine) ”, 108 23, 23S (2000)

 Non-Patent Document 3: “The Journal of Clinical Endocrinology & Metabolism”, 89, 2601 (2004)

 Non-Patent Document 4: “Endocrinology”, 142 卷, 1371 (2001) Non-Patent Document 5: “Science”, 294 卷, 2166 (2001)

 Non-Patent Document 6: “The Journal of Clinical Investigation”, 112 卷, 83 (2003)

 Non-Patent Document 7: “Journal of medicinal chemistry”, 45 卷, page 3813 (2002)

 Disclosure of the invention

 Problems to be solved by the invention

[0008] The present inventors have conducted extensive research for the purpose of searching for a substance having 11 β-HSD1 inhibitory activity, and that a novel compound isolated and purified from a microorganism culture has the activity. The present invention was completed.

 Means for solving the problem

[0009] The present invention provides:

 (1) The following formula (I):

[0010] [Chemical 2]

で表わされる化合物またはその塩、

Or a salt thereof,

(2) a compound having the following physical properties or a salt thereof,

 1) Material properties: Acid fat-soluble colorless powder

 2) Solubility: soluble in methanol, dimethyl sulfoxide, ethyl acetate, hardly soluble in hexane

3) Molecular formula: C H O

 15 24 3

 4) Molecular weight: 252 (measured by FAB mass spectrometry)

 5) High-resolution [M + Na] +, measured by the FAB mass spectrum method, is as follows.

Actual value: 275.16325

Calculated value: 275.16231

6) Optical rotation: [α] ²⁵ + 17.8 ° (cO.73, methanol)

 D

 7) UV absorption spectrum: terminal absorption

 8) Infrared absorption spectrum:

 The infrared absorption spectrum measured by the thin film method shows the following maximum absorption.

3282, 2957, 2877, 2637, 1687, 1548, 1450, 1410, 1392, 1367, 1308, 1276, 1256, 1215, 1193, 1169, 1100, 1078, 1053, 1034, 985, 962, 92 6cm

 9) Nuclear magnetic resonance spectrum:

The ¹ H-nuclear magnetic resonance spectrum measured in the deuterated dimethyl sulfoxide using the residual proton signal (2.50 ppm) as an internal standard is shown below.

0.78 (3H, d, J = 6.5Hz), 0.85 (3H, d, J = 6.9Hz), 1.51 (IH, m), 1.58 (2H, m), 1.63 (3H, s), 1.68 (1H, m ), 1.74 (IH, m), 1.78 (IH, m), 1.80 (1H, m), 1.84 (1H, m), 1.92 (2H, m), 2.48 (lH, t, J = 8.4Hz), 4.00 (IH, m), 5.35 (IH, m) ppm

10) ¹³ C—Nuclear magnetic resonance spectrum:

The ¹³ C-nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide using the deuterated dimethyl sulfoxide signal (39.5 ppm) as an internal standard is shown below. 19.2 (q), 19.6 (q), 23.7 (t), 25.2 (q), 26.1 (t), 27.1 (d), 27.3 (t), 45.8 (t), 47. l (s), 55. 17 (d), 55. 19 (d), 66.8 (d), 122. 6 (d), 135.9 (s), 17 6.1 (s ) ppm,

(3) It is characterized by culturing a bacterium producing the compound described in (1) or (2) belonging to the genus Colletotrichum and collecting the compound described in (1) or (2) from the culture. A method for producing the compound according to (1) or (2),

(4) The compound producing the compound according to (1) or (2) belonging to the genus Colletotricum (Colletotrichum) is Colletotrichum gloeosporioid s SANK 21404 (FERM BP-10309), ( 3) the production method according to

(5) a microorganism belonging to the genus Colletotrichum and producing the compound according to (1) or (2),

(6) The microorganism according to (5), which is Colletotrichum gloeosporioides SANK 21404 (FERM BP— 10309),

(7) A pharmaceutical comprising the compound according to (1) or (2) or a pharmaceutically acceptable salt thereof as an active ingredient,

(8) The medicament according to (7), which is a therapeutic drug for diabetes, a therapeutic drug for obesity, a therapeutic drug for hyperlipidemia, a therapeutic drug for hypertension or a drug for improving metabolic syndrome,

(9) 11 β-hydroxys teroia dehydrogenase typel | ¾_ "^ agent comprising the compound or salt thereof according to (1) or (2),

About.

(10) The compound described in (1) or (2) or a pharmacologically acceptable drug for the patient A method for the treatment or amelioration of at least one disease selected from the group strength of diabetes, obesity, hyperlipidemia, hypertension and metabolic syndrome by administering a salt.

 [0012]

 (11) The compound according to claim 1 or 2, for the manufacture of a therapeutic or ameliorating drug for at least one disease selected from the group consisting of diabetes, obesity, hyperlipidemia, hypertension and metabolic syndrome Use of its pharmacologically acceptable salt.

 [0013]

 In the present invention, “choletoic acid” (English name: “colletoic acid”) refers to a compound represented by the above formula (I) or a compound having the physicochemical properties described in the above (2).

 [0014] The compound of the present invention can be converted to a salt by a method well known to those skilled in the art using a base. The present invention also includes salts of these compounds. When the salt of the compound of the present invention is used as a pharmaceutical, it is not particularly limited as long as it is medically used and pharmacologically acceptable. Moreover, when using for uses other than a medicine, if it can be used for this use, it will not specifically limit.

 [0015] Examples of such salts include alkali metal salts such as sodium salts, potassium salts and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts and iron salts. , Zinc salt, copper salt, nickel salt, cobalt salt, etc .; inorganic salt such as ammonium salt; toctylamine salt, dibenzylamine salt, morpholine salt, darcosamine salt, vinylglycine alkyl ester salt, ethylenediamine salt, N —Methyl darcamine salt, guazine salt, jetylamine salt, triethylamine salt, dicyclohexylamine salt, N, N, -dibenzylethylenediamine salt, black pro-in salt, pro-in salt, diethanol Amine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (hydroxy Organic amine salts such as chill) Aminometan salts; glycine salts, lysine salts, arginine salts, Ol - Chin salts, as possible out to include such amino acid salts such as Asuparagin salt. Preferable are pharmacologically acceptable salts, and more preferable are sodium salt, strength rhodium salt, and ammonium salt.

[0016] In addition, the compound of the present invention and a salt thereof may be left in the air or mixed with water or an organic solvent to combine with water or a solvent to form a hydrate or a solvate. Ah However, these hydrates and solvates are also included in the present invention.

 [0017] Furthermore, the choretic acid of the present invention has various steric structures. In the present invention, equal and unequal mixtures of these stereoisomers are all represented by a single structural formula. However, the compound of the present invention also includes these isomers and mixtures of these isomers. Everything is included.

 [0018] Choletoic acid can be isolated and purified according to a conventional method for culturing microorganisms that produce the substance. The microorganism producing colletic acid is not particularly limited, but is preferably a fungus, more preferably a fungus belonging to the genus Colletotricum, and most preferably Colletotrichum gloeosporioides (Colletotrichum gloeosporioides). ) S ANK 21404 (hereinafter referred to as “SANK 21404 shares”! /).

[0019] SANK 21404 strain is isolated from a plant in Okagaki-machi, Onga-gun, Fukuoka.

[0020] The SANK 21404 strain has the following mycological properties. In describing mycological features, the color indications are: “Kornerup and Vanceyer, Metune Handbook of Color, 3rd Edition, Ellie Metune, London, UK, 1—252 (1978); (Kornerup A, Wanscher JH : Methuen handbook of color. 3rd ed., Eyre Methuen, London, UK., 1—252 (1978))

 [0021] When observing the mycological properties of SANK 21404 strain under culture, the following media are used.

 [0022] Potato Dextrose Agar Medium (hereinafter referred to as “PDA” medium): Dissolve 39 g of Nissi Potato Dextrose Agar Medium (manufactured by Nissui Pharmaceutical Co., Ltd.) in 100 ml of distilled water. Prepare plates after sterilization at ° C for 15 minutes.

 [0023] Modified Weitzman and Silva

 -Hunter) medium) (hereinafter referred to as “WSH medium”): eclipse oatmeal 10 g, magnesium sulfate heptahydrate lg, potassium dihydrogen phosphate lg, sodium nitrate lg and agar 20 g are dissolved in 1000 ml of distilled water. Prepare plates after sterilization at ° C for 15 minutes.

[0024] Potato Carrot Agar medium (hereinafter referred to as “PCA” medium): Peel 30g of carrot, 2.5g of carrot, cut into lcm, add 800ml of distilled water, boil for 1 hour, add distilled water to 1000ml, add 20g of agar, 121 ° C Prepare plate after sterilization for 15 minutes.

 The mycological properties of SANK 21404 strain on plant diseased leaves are described below.

 [0025] The lesion is round to irregular, light brown, and forms a conidia layer on the front or back surface. The conidia layer is superficial to integumental, discoid and forms bristles, and conidia appear as white to pale pink conidia. The bristles are sparsely formed from the conidial pattern, and the dark brown power is black, 27.9-51.3 X 3—4.7 (average = 39.6 X 3.6) m. The conidia pattern is parallel to the surface of the conidia layer, is colorless and does not branch, and forms conidia at the tip. Conidia are oblong to cylindrical, blunt at both ends, colorless single cells, 11-15 X 3.3-4.7 (average = 13.0 X 4) / z m.

 [0026] The SANK 21404 strain exhibits the following mycological properties on each medium.

 [0027] Colonies in PDA medium have a diameter of 82-88 mm when cultured for 1 week at 25 ° C. Koguchi-1 is fluffy and has a greenish gray (29B2) to white color with abundant aerial hyphae. No exudate, sclerotia, or conidia are observed. The back side is dark green (28F4) to greenish gray (28C2) at the center and white at the edge. No soluble dye is observed.

 [0028] Colonies in WSH medium have a diameter of 75-85 mm after 1 week of culture at 25 ° C. Colonies range from wooly to velvety, from gray (lEl) to white. No exudate, nuclei, or conidia are observed. The back side is brownish gray (8F2) at the center and reddish gray (8C2) and white at the edge. No soluble pigment is observed.

[0029] Colonies in PCA medium have a diameter of 65-67 mm when cultured for 1 week at 25 ° C. The mouth is fluffy and has a gray (grey (lEl)) to white color. No exudate, mycorrhiza, or conidial mass is observed. The back surface is gray yellow (4C3) to gray (grey (4 B1)) at the center and white at the edge. No soluble pigment is observed. The mycelium on the PCA medium is septum, colorless, 1.2-2. Applicators can be single or rare, oval to rod-shaped, full edge or rarely shallow, dark brown to dark brown, 5.3 — 14. 8 X 4.1 — 8 ( Average = 9.5X5.6) / zm. Conidia are single cells, oblong to cylindrical, blunt at both ends, colorless, PDA medium: 9— 18.6X2.9— 6.2 (average = 13.0X4.4) / zm; WSH medium: 10.6— 17.1X3.4 — 5.8 (average = 13.7X4.1) / zm; PCA medium: 11. 2-21.1X3.1-5.4 (average = 13.8X4.0) m, not forming conidia layer, formed directly from the hyphae The

 [0030] The bacteriological features described above are based on Sutton's literature (Sutton: The Genus Gromelera and Do Ittsu Anamorph Koretotricam, ： Baile 1 'Jaeger, Koreto Tricam: Biologics, Pathology and Control: CAB International, Walling Ford, UK, 1992; (Sutton BC: The genus Glomerella and its an amorph Colletotrichum., In: Bailey JA, Jeger MJ, (eds) Colleto trichum: Biology, Pathology and Control: CAB International, Wallingford, UK., 1 — 26. (1992)) was well matched with the description of Co lletotrichum gloeosporioides. Therefore, the strain was identified as Colletotrichum gloeosporioides Collestotrichum gloeosporioides named SANK 21404. SANK 21404 strain 2005 On March 31st, the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (address: Tsukuba Tohoku, Ibaraki, Japan 1 — 1— 1 Chuo No. 6) was deposited internationally with the accession number FERM BP— 10309 It was done.

 [0031] As is well known, fungi are naturally mutated in nature or by artificial manipulation (for example, ultraviolet irradiation, radiation irradiation, chemical treatment, etc.). It is presumed that In the present invention, the SANK 21404 strain includes all the mutant strains. Preferably, the mutant strain included in the SANK 21404 strain of the present invention is a strain characterized by producing cholate acid.

 In addition, these mutant strains include those obtained by genetic methods such as recombination, transduction, transformation and the like. That is, SANK 21404 strain producing cholate, mutants thereof and strains not clearly distinguished from them are all included in SANK 21404 strain.

[0033] As described above, choletic acid can be produced by culturing a bacterium that produces the substance. The culture of SANK 21404 strain can be carried out using a medium usually used for producing secondary metabolites of microorganisms. Such a medium contains a carbon source, a nitrogen source and an inorganic salt that can be assimilated by the microorganism.

[0034] Examples of the carbon source include glucose, fructose, maltose, sucrose, man-toll, glycerin, dextrin, oats, rye, starch, potato, corn flour, cottonseed oil, molasses, citrate, tartaric acid and the like. These can be used alone or in combination. The amount of carbon source added is usually in the range of 1 to 10% by weight of the medium.

 [0035] As the nitrogen source, a substance or an inorganic salt containing a protein and a hydrolyzate thereof is usually used. Examples of such nitrogen sources include soy flour, bran, peanut flour, cottonseed flour, casein hydrolyzate, pharmamine, fish meal, corn steep liquor, peptone, meat extract, yeast, yeast extract, malto extract, gelatin. Sodium nitrate, ammonium nitrate, ammonium sulfate and the like, and these can be used alone or in combination. The amount of nitrogen source added is usually in the range of 0.2 to 10% by weight of the medium.

 [0036] As the nutrient inorganic salt, for example, salts capable of obtaining ions such as sodium ion, ammonium ion, calcium ion, phosphate ion, sulfate ion, salt ion, and carbonate ion are used. . In addition, salts containing trace amounts of metals such as potassium, calcium, conoleto, manganese and magnesium can also be used.

 [0037] When liquid culture is performed, an antifoaming agent can be used as necessary, and as such an antifoaming agent, for example, silicon oil, vegetable oil, a surfactant or the like is used.

[0038] Choleto acid can be obtained by aerobic culture of SANK 21404 strain. As such a culture method, for example, a solid culture method, a shaking culture method, an aeration stirring culture method, or the like is used. For small-scale cultures, shaking culture is preferably performed at 9 to 35 ° C (preferably 15 ° C to 33 ° C, optimally 23 ° C). Incubation begins in a conical flask with one or more stages of seed development. The seed culture is performed by adding a sterilized seed culture medium to the flask, inoculating the SANK 21404 strain, and then shaking in a constant temperature incubator for several days or until it has grown sufficiently. Part or all of the obtained seed culture solution is used to inoculate the seed culture medium or production culture medium in the next stage. The culture should be sterilized The seed culture solution can be inoculated into a flask containing the production culture medium and shaken at a constant temperature for several days or until it has grown sufficiently.

 [0039] Mass culture is preferably performed in a suitable jar or tank equipped with a stirrer and a ventilator. Media can be made in jars or tanks, sterilized by heating to 121 ° C, and cooled before use. For cultivation, inoculate the seed culture medium in the production culture medium in the jar or tank and agitate at 9 to 35 ° C (preferably 15 ° C to 33 ° C, optimally 23 ° C). Can be done. This method is suitable for obtaining large amounts of compounds.

 [0040] After completion of the culture, the target culture can be obtained by extracting and purifying the obtained culture, the supernatant obtained from the culture by centrifugation or filtration, or the whole culture. A compound can be obtained.

 [0041] The amount of choletic acid produced with the progress of culture is determined by collecting a part of the culture solution, extracting the compound, performing high performance liquid chromatography, and measuring the compound. It's possible to quit.

 [0042] After completion of the culture, choletic acid present in the liquid part of the culture liquid, in the microbial cells, or both, uses the whole culture ending liquid or the microbial cells and other solid parts as diatomaceous earth as a filter aid. It is possible to extract and purify from the filtrate or supernatant obtained by filtration or centrifugation, and bacterial cells, using high-performance liquid chromatography as an index, and using their physical and physical properties.

[0043] Choletic acid present in the filtrate or supernatant is not miscible with water under acidic conditions, and is an organic solvent such as ethyl acetate, chloroform, ethylene chloride, methylene chloride, butanol alone or , And can be extracted and purified by a combination thereof. Alternatively, as an adsorbent, for example, Amberlite XAD-2, XAD-4 (manufactured by Rohm 'and' Haas Co., Ltd.), which is activated carbon or an adsorbent resin, Diaion HP-10, HP-20 CHP20P, HP-50, Sepabeads SP-207 (manufactured by Mitsubishi Chemical Corporation) and the like can also be used for extraction and purification. When extracting and purifying using an adsorbent, the liquid containing the target compound is removed by passing through the adsorbent layer and adsorbing the impurity to the adsorbent, or the target compound is adsorbed and washed away. Thereafter, the target compound can be extracted and purified by eluting the target compound with methanol water, acetone water, butanol water or the like. [0044] Choletic acid present in the microbial cells is extracted with 50 to 90% aqueous acetone or aqueous methanol, and after removing the organic solvent by concentration, the extraction and purification operation is performed in the same manner as described above. Obtainable. For example, the target compound can be extracted by adding an appropriate amount (preferably a final concentration of 50%) of acetone or methanol after completion of the culture. After completion of the extraction, the target compound can be extracted and purified by performing a filtration operation using diatomaceous earth as a filter aid and subjecting the obtained extract to the same extraction and purification operation as the filtrate.

 [0045] The solution containing choletic acid extracted and purified by the above method is a carrier such as silica gel, Cefadex LH-20 (manufactured by Amersham Biosciences), florisil, cosmosyl (manufactured by Nakarai Tesque). Column chromatography using sorbent; adsorption column chromatography using a carrier such as Diaion HP—20, CHP20P (Mitsubishi Chemical Corporation); Cefadex G-10 (Amersham Biosciences) Gel filtration chromatography using Toyopearl HW40F (Tosoh Corp.), etc .; Dowex 1 (Dow Chemical Corp.), Diaion PA316 (Mitsubishi Chemical Corp.), DEAE Cef Adex A— 25 (Amershamno Science Co., Ltd.), etc .; and high-performance liquid chromatographs using normal and reverse phase columns As possible out be further purified by I over like.

 [0046] The above-mentioned separation and purification means can be separated or purified by using the above-mentioned separation and purification means alone or in combination as appropriate, and repeatedly in some cases.

 [0047] While typical methods for producing choletic acid have been described above, the production methods are not limited to these, and other production methods already known to those skilled in the art can also be used.

 [0048] The choretic acid or a salt thereof of the present invention has 11 β-HSD1 inhibitory activity.

[0049] Glucocorticoids (cortisol in humans and corticosterone in rodents) are known to have various physiological activities that regulate blood sugar levels, blood pressure, and the like. For example, it promotes the release of amino acids from muscles and the release of fatty acids and glycerol from adipose tissue into the blood, and promotes gluconeogenesis in the liver using these substrates, resulting in an increase in blood sugar levels. Physiological activity that promotes elevation is known. Glucocorticoids are also known to have the activity of maturating immature adipocytes in adipose tissue to lead to obesity and the activity of acting on mineralocorticoid receptors in the kidney to increase blood pressure. Being Yes. 11 j8 — HSD1 is an enzyme that is mainly expressed in tissues such as liver, adipose tissue, and lung, and has an activity to convert inactive glucocorticoids into active forms. 11 — HSD1 inhibitors are By suppressing the action of glucocorticoids in tissues, hyperglycemia, obesity, hyperlipidemia, and sputum or hypertension are suppressed. It also exerts multifaceted effects on metabolic syndrome, which is a condition with these symptoms. “Metabolic syndrome” is a symptom in which the metabolic functions of carbohydrates and lipids in the living body are abnormal. Although the diagnostic criteria differ among multiple international organizations, they have multiple symptoms of impaired glucose tolerance (or insulin resistance), obesity, hypertension, hypertriglyceridemia, and low HDL cholesterolemia. Match. Details of the symptoms are described in "The Lancet, vol.365, pp.1415-1428".

 [0050] The present invention also relates to a medicament containing as an active ingredient choletic acid or a pharmaceutically acceptable salt thereof. Since cholate has the activity described above, the drug is useful as an 11 j8-HSD1 inhibitor or a therapeutic or prophylactic agent for diabetes, obesity, hyperlipidemia, hypertension, and Z or metabolic syndrome. It is. The present invention also provides a method for inhibiting 11 β-HSD1 in vivo by administering the medicament, or the treatment or prevention of diabetes, obesity, hyperlipidemia, hypertension, and epilepsy or metabolic syndrome. Regarding the method. Furthermore, the present invention relates to 11 β-HSD1 inhibitor or choletic acid or a pharmaceutically acceptable salt thereof for producing a therapeutic or prophylactic agent for diabetes, obesity, hyperlipidemia, hypertension, and epilepsy or metabolic syndrome. Use of the salt.

 [0051] The choletic acid of the present invention or a pharmacologically acceptable salt thereof can be administered in various forms. Examples of the administration form include tablets, capsules, granules, emulsions, pills, powders, syrups (solutions) or the like, or injections (intravenous, intramuscular, subcutaneous or intraperitoneal administration), Examples include parenteral administration such as instillation and suppository (rectal administration). These various preparations are usually used in the pharmaceutical formulation technical field such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc. It can be formulated with adjuvants that can be used.

[0052] When used as a tablet, for example, lactose, sucrose, sodium chloride salt, dalcose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, key acid, etc. are used as carriers. Agents: Water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polypyrrolidone, etc .; dry starch, sodium alginate, agar powder, lamina lan Powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, etc .; disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil; Absorption accelerators such as quaternary ammonium salts and sodium lauryl sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite, and colloidal key acid; purified talc and stearate , Boric acid powder, polyester It can be used lubricants such as glycols. Further, if necessary, it can be made into a tablet with a normal coating, such as a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, or a multilayer tablet.

[0053] When used as pills, as carriers, for example, excipients such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, kaolin, talc; binders such as gum arabic powder, tragacanth powder, gelatin, ethanol, etc. ; Disintegrants such as laminaran agar can be used.

 [0054] When used as a suppository, carriers that are conventionally known in this field can be widely used as carriers, such as polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like. Can do.

 [0055] When used as an injection, it can be used as a solution, emulsion or suspension.

 These solutions, emulsions or suspensions are preferably sterilized and isotonic with blood. The solution used for the production of these solutions, emulsions or suspensions is not particularly limited as long as it can be used as a medical diluent, such as water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, Examples thereof include polyoxylated isostearyl alcohol and polyoxyethylene sorbitan fatty acid esters. In this case, the preparation may contain a sufficient amount of sodium chloride, glucose or glycerin to prepare an isotonic solution, and it also contains ordinary solubilizing agents, buffers, soothing agents, etc. May be

[0056] Further, in the above-mentioned preparations, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, etc., as necessary. Can also be included, and other pharmaceuticals can also be included.

 [0057] The amount of the active ingredient compound contained in the above-mentioned preparation is not particularly limited, and can be appropriately selected over a wide range.

 [0058] The amount used varies depending on symptoms, age, body weight, administration method, dosage form, etc., but is usually 2000 mg or 40 mg / kg (preferably lOOmg or 2 mg / kg) per day for adults. The lower limit is 0. lmg or 0.002mg / kg (preferably lmg or 0.02mg / kg, more preferably lOmg or 0.2mg / kg) once or several times a day depending on the symptoms. Can be administered.

 [0059]

 The invention's effect

 [0060] The present invention provides a novel compound having 11 β-HSD1 inhibitory activity.

 The compound or a pharmacologically acceptable salt thereof is considered to have an effect of treating or ameliorating diabetes, obesity, hyperlipidemia, and hemorrhoids or hypertension. Furthermore, the compound is considered to have a therapeutic or ameliorating effect on metabolic syndrome in which these symptoms are combined.

 [0061]

 BEST MODE FOR CARRYING OUT THE INVENTION

[0062] Next, the present invention will be described in more detail with reference to Examples, Test Examples and Formulation Examples, but the present invention is not limited thereto.

 Example

 (Example 1) Culture of SANK 21404 strain

 (1) Type 1 culture

Aseptically inoculate SANK 21404 strain grown on PDA slant medium into a 100 ml Erlenmeyer flask using platinum ears, sprinkling the mycelium with agar, pulverizing and adding 30 ml of the seed medium shown in Table 1. Fungus. The first type culture was performed by culturing at 23 ° C, 210 rpm for 5 days in a rotary shaker. Aseptically remove 1 ml of the obtained first-type culture solution, aseptically pour into a 2 ml cryotube containing lml of 20% glycerin solution, suspend, and store frozen at 100 ° C as a final concentration of 10% glycerin solution. (Hereinafter referred to as frozen first-class culture) The nutrient solution is called “frozen seed”).

 [Table 1] Composition of seed medium Soluble starch 20 g

 Glycerin 30 g

 Glucose 30 g

 Soy flour 10 g

 Gelatin 2.5 g

 Yeast extract 2.5 g

 NH NO 2.5

 4 3 g

 Agar 3 g

 Antifoam (CB442) 5 mg

 Tap water 1000 ml pH unadjusted

 Sterilization: Sterilized at 121 ° C for 20 minutes.

 [0064]

 (2) Second kind culture

 After thawing the frozen seed at room temperature, the entire amount was aseptically inoculated into a 100 ml Erlenmeyer flask containing 30 ml of the seed medium shown in Table 1 using a pipette. This culture broth was cultured at 23 ° C, 210 rpm for 5 days in a rotary shaker (second-type culture) to obtain a second-type culture solution.

 [0065]

 (3) Main fermentation culture

 Aseptically inoculated 2 ml each of the obtained second culture medium into two 500 ml Erlenmeyer flasks containing 80 ml of the main fermentation medium shown in Table 2. The culture broth was cultured at 23 ° C, 210 rpm for 7 days in a rotary shaker (main fermentation culture) to obtain a culture end solution.

Main fermentation medium [Table 2] Composition of main fermentation medium Glycerin 50 g

Raw potatoes (baron) * 50 g

Manoleto extract 5 g

Yeast extract 5 g

Antifoam (CB442) 5 mg

Tap water 1000 ml pH unadjusted

 Sterilization: Sterilized at 121 ° C for 20 minutes.

* The raw potato (baron) was peeled and cut into lcm dice and crushed with tap water.

 (Example 2) Isolation and purification of choletic acid

 In the following purification, the active fraction was monitored by using HPLC under the following conditions.

 [Active fraction monitoring conditions]

 Column: Capsule pack (CAPCELL PAK) C UG120:

 18

 4.6 X 150mm (Shiseido Co., Ltd.)

 Solvent: 0.05% 45% acetonitrile water containing 0.05% formic acid

 Flow rate: 1. OmlZ min

 Detection: UV absorption 210nm

Retention time: 7.9 minutes Acetone (200 ml) was added to the culture completion solution (160 ml) obtained in Example 1, and the mixture was allowed to stand for 30 minutes without stirring and then centrifuged (3000 rpm, 10 minutes). The obtained supernatant was adjusted to pH 3 with 6N H C1, and extracted with ethyl acetate (200 ml). The resulting aqueous layer was extracted again by adding ethyl acetate (200 ml), and the organic layer obtained by the two extraction operations was merged. It was. The organic layer (600 ml) was washed with saturated brine (400 ml), dehydrated with anhydrous sodium sulfate (80 g), and dehydrated for 1 hour. After removing anhydrous sodium sulfate by filtration, the filtrate was concentrated to dryness under reduced pressure to obtain an oily substance (188 mg). This oily substance was dissolved in 40% aqueous methanol (10 ml) adjusted to pH 3 containing 0.3% triethylamine monophosphate, and then adjusted to pH 3 containing 0.3% triethylamine-phosphate. This was applied to a Cosmosil 140C18 (manufactured by Nacalai Testa Co., Ltd.) column (50 ml) equilibrated with 30% acetonitrile water. The ram was washed with the same solvent (250 ml) and then developed with the same solvent (200 ml), and the eluate was fractionated every 20 ml (Fr. 1 to 11). Then, similarly, containing 0.3% triethylamine-phosphate, 35% acetonitrile water adjusted to PH3 (200 ml, Fr. 12 to 21), containing 0.3% triethylamine-phosphate, adjusted to pH 3 40% acetonitrile water (200 ml, Fr. 22 to 31) was developed in this order, and the eluate was fractionated every 20 ml, and the fractions (600 ml) of Fr. Obtained. The obtained fraction was concentrated to distill off acetonitrile, followed by extraction with ethyl acetate (300 ml). The aqueous layer was extracted again with ethyl acetate (200 ml), and the organic layers obtained by the two extraction operations were combined. The organic layer (500 ml) was washed with saturated brine (400 ml), dried over anhydrous sodium sulfate (60 g) and dehydrated for 1 hour. After removing anhydrous sodium sulfate by filtration, the filtrate was concentrated to dryness under reduced pressure to obtain a crude product (14 mg). This crude product was dissolved in methanol (500 μ1), and then the solution was divided into 5 portions of 100 1 and equilibrated with 48% acetonitrile water containing 0.5% formic acid (CAPCELL PAR C UG120 , 20 Χ 250 m

 18

m (Made by Shiseido Co., Ltd.) The column was developed at a flow rate of 10. OmlZ, the purple external absorption at 210 nm was detected, and the active fraction eluted at a retention time of 19.8 minutes was fractionated. The obtained fractions having the same retention time were combined, concentrated under reduced pressure, and freeze-dried to obtain colorless powder (7.4 mg) as choletic acid.

 (Test Example 1) 11 β HSD1 enzyme inhibition experiment

(1) Enzyme source

A plasmid in which cDNA encoding the full length of human 11 β- HSD1 was incorporated into the mammalian cell expression vector pCIneo (Promega) was introduced into HEK293 cells using Lipofectamine plus reagent (Invitrogen) according to the package insert. To collect Frozen at 80 ° C. As a control, cells into which the pCIneo vector was introduced were similarly prepared. Cells are thawed, suspended in buffer containing 20 mM final concentration of Hepes, ImM ethylenediaminetetraacetic acid, 2 mM magnesium chloride, and protease inhibitor cocktail (Roche), and placed on ice with N2 cavity. 4. Pressure was applied for 30 minutes to disrupt the cells. The cell lysate was centrifuged at 1,000 X g for 10 minutes at 4 ° C, and the supernatant was recovered. The resulting supernatant was further centrifuged at 10, 5000 Xg for 30 minutes at 4 ° C, and the resulting precipitate was suspended in Atsey buffer (50 mM Tris buffer, 10% glycerol) as a microsomal fraction. The enzyme source.

[0068] (2) Enzyme inhibition experiment The reaction was carried out on a 96-well PCR plate (Perkin Elmer) with a reaction volume of 20 μL! / ヽ. Both samples were prepared with Atsy buffer (50 mM Tris buffer, pH 7. 4, 10% glycerine). NADPH at a final concentration of 5 mM, glucose 6 phosphate at 50 mM, glucose 6-phosphate dehydrogenase (Sigma) at 10 units ZmL, magnesium salt at 20 mM, and microsomes at 5 μg protein per well The fraction was added to the plate, a solution containing the test compound dissolved in dimethyl sulfoxide was added to a final concentration of 0.1%, and preincubated for 15 minutes at room temperature. After completion of preincubation, cortisone with a final concentration of 500 nM was added to start the reaction, and the reaction was performed at room temperature for 60 minutes. The plate was heated at 99 ° C for 10 minutes to stop the reaction, and the amount of cortisol produced was measured with Discovery (Packard) using a Cortisol prototype kit (CIS Bio International) according to the attached document. Coretic acid showed 99% inhibition at 10 μΜ against the production of I l j8 — HSD1-expressing cortisol, which was not seen in the microsomal fraction derived from similarly treated control cells.

[0069] (Formulation example) Capsule

 Choleto acid 100 mg

 Lactose 100 mg

 Corn starch 148.8 mg

Magnesium stearate 1.2 mg Total amount 350 mg

 After mixing the powder of the above formulation and passing it through a 60 mesh sieve, this powder is put into a gelatin force capsule to form a capsule.

 [0070]

 Industrial applicability

[0071] The compound of the present invention or a salt thereof is useful as an effective component of a therapeutic or ameliorating agent for diabetes, obesity, hyperlipidemia, hypertension, and Z or metabolic syndrome that develops them in combination. It is.

Claims

The scope of the claims  Formula (I)  [Chemical 1]

 Or a salt thereof. [2] A compound or salt thereof having the following physical properties.  1) Material properties: Acid fat-soluble colorless powder  2) Solubility: soluble in methanol, dimethyl sulfoxide, ethyl acetate, hardly soluble in hexane 3) Molecular formula: C H O  15 24 3  4) Molecular weight: 252 (measured by FAB mass spectrometry)  5) High-resolution [M + Na] +, measured by the FAB mass spectrum method, is as follows.  Actual value: 275. 16325  Calculated value: 275. 16231 6) Optical rotation: [α] ²⁵ + 17.8 ° (c O. 73, methanol)  D  7) UV absorption spectrum: terminal absorption  8) Infrared absorption spectrum:  The infrared absorption spectrum measured by the thin film method shows the following maximum absorption. 3282, 2957, 2877, 2637, 1687, 1548, 1450, 1410, 1392, 1367, 1308, 1276, 1256, 1215, 1193, 1169, 1100, 1078, 1053, 1034, 985, 962, 92 6cm  9) Yeah! Nuclear magnetic resonance spectrum: The residual proton signal (2.50 ppm) in deuterated dimethyl sulfoxide was used as an internal standard. The ¹ H-nuclear magnetic resonance spectrum measured in this way is as shown below.  0. 78 (3H, d, J = 6.5 Hz), 0.85 (3H, d, J = 6.9 Hz), 1. 51 (IH, m), 1. 58 ( 2H, m), 1.63 (3H, s), 1.68 (1H, m), 1.74 (IH, m), 1.78 (IH, m), 1.8 0 (1H, m), 1.84 (1H, m), 1.92 (2H, m), 2.48 (lH, t, J = 8.4Hz), 4.00 ( IH, m), 5.35 (IH, m) ppm 10) ¹³ C—Nuclear magnetic resonance spectrum: The ¹³ C-nuclear magnetic resonance spectrum measured in heavy dimethyl sulfoxide using the heavy dimethyl sulfoxide signal (39.5 ppm) as an internal standard is shown below.  19.2 (q), 19.6 (q), 23.7 (t), 25.2 (q), 26.1 (t), 27.1 (d), 27.3 (t), 45.8 (t), 47. l (s), 55. 17 (d), 55. 19 (d), 66.8 (d), 122. 6 (d), 135.9 (s), 17 6.1 (s ) ppm  [3] The bacterium according to claim 1 or 2, which belongs to the genus Colletotrichum, is cultured, and the compound according to claim 1 or 2 is collected from the culture. Item 3. A method for producing the compound according to Item 1 or 2. [4] The bacterium according to claim 1 or 2, which belongs to the genus Colletotrichum, is Colletotrichum gloeosporioid s SANK 21404 (FERM BP-10309). The manufacturing method as described in. [5] A microorganism belonging to the genus Colletotrichum and producing the compound according to claim 1 or 2. [6] Colletotrichum gloeosporioides SA The microorganism according to claim 5, which is NK 21404 (FERM BP-10309). [7] A medicament comprising the compound according to claim 1 or 2 or a pharmacologically acceptable salt thereof as an active ingredient.  [8] The medicament according to claim 7, which is a therapeutic drug for diabetes, a therapeutic drug for obesity, a therapeutic drug for hyperlipidemia, a therapeutic drug for hypertension or a drug for improving metabolic syndrome. [9] An 11β-hydroxysteroid dehydrogenase type 1 inhibitor comprising the compound or salt thereof according to claim 1 or 2. [10] The compound or pharmacologically acceptable salt thereof according to claim 1 or 2 for a patient A method for treating or ameliorating at least one disease selected from the group consisting of diabetes, obesity, hyperlipidemia, hypertension and metabolic syndrome.  The compound according to claim 1 or 2 for the manufacture of a therapeutic or ameliorating drug for at least one selected disease, such as diabetes, obesity, hyperlipidemia, hypertension and metabolic syndrome. Use of a salt that is physically acceptable.