JPH09124683A - Unknown compound a38840 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an unknown compound A38840 which is useful in prevention and treatment for hyperlipemia such as hypercholesterolemia and arteriosclerosis by culturing a bacterium in Streptomyces capable of producing A38840 substance and collecting the product from the culture mixture. SOLUTION: This novel compound A38440 has a chemical structure of the formula and the following descriptions: soluble in acidic lipid, the molecular formula: C47 H76 O17 , molecular weight: 912 (measured by the high-resolution FAB-MS technique), solubility: soluble in methanol, ethanol, dimethyl sulfoxide, slightly soluble in ethyl acetate and water and insoluble in chloroform and hexane and is useful in treatment for hyperlipemia such as hypercholesterolemia caused by trouble in lipid metabolism and prevention of atherosclerosis. This compound is prepared by culturing a microorganism producing A38840 in Streptomyces such as Streptomyces sp. SANK60892 (FERM BP-5525) and collecting the product from the culture mixture.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to A38840 or a pharmacologically acceptable salt thereof, which is effective for treating hyperlipidemia such as hypercholesterolemia due to lipid metabolism disorder and preventing atherosclerosis.

[0002]

It is known that arteriosclerosis is caused by the accumulation of cholesterol in the body. Most arteriosclerosis is atherosclerosis, in which cholesterol accumulates in the arterial wall to form atherosclerotic spots, and the accumulated cholesterol is low-density lipoprotein ( Low De
It is said to be derived from particles called nsity Lipoprotein (LDL). Therefore, the higher the LDL concentration in the blood, the faster the atherosclerosis progresses.

A transgenic mouse into which the human LDL receptor gene has been introduced has been prepared, and by artificially increasing the expression of the LDL receptor gene, LDL in the blood is
It has been reported to have fallen significantly [Science, 25
0, 1273 (1990)]. As a drug for lowering LDL in blood, 3-hydroxy-3-methylglutaryl coenzyme A reductase (3-Hydroxy-3-Methylglut), which is a rate-limiting enzyme in the biosynthetic pathway of endogenous cholesterol, is used.
aryl-Co A reductase: HMG CoA reductase)
Examples thereof include mevalotene (registered trademark; generic name pravastatin sodium) and mevacol (registered trademark; generic name lovastatin) that are currently commercially available. These substances indirectly increase the number of LDL receptors by inhibiting the biosynthesis of cholesterol in the liver, thereby lowering the LDL concentration in the blood.

Examples of the saponin-based compound include Astragalus membranceu
s), Wisteria brachyb
otrys), Crotalaria thebaic
a) Asturagaloside VIII (12)
-Oleanene-3, 22, 24-triol, 3-O- α-L-Rhamnopyranosyl-
1,2-β-D-xylopyranosyl-1,2-β-D-glucuronopyranos
ide) (Kitagawa et al., Chem. Pharm. Bull., Volume 31, Issue 2, 716-
722 (1983)) or isolated from Oxytropis ochrocephala 12-
Oleanene-3,22,24-triol, 3-O-α
-L-rhamnopyranosyl-1,2-α-L-arabinopyranosyl-1,4-β-D-glucuronopyranoside (1
2-Oleanene-3, 22, 24-triol, 3-O-α-L-Rhamnopyranosyl
-1,2-α-L-arabinopyranosyl-1,4-β-D-glucuronopyra
noside) (R. Sun) et al., Planta Med., 58
Volume, pages 211-213 (1992)) is known, but there is no description of efficacy.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have found that LDL
A substance having an action of directly enhancing the expression of the receptor gene and specifically reducing LDL in blood is searched for, and A38840 is produced as a novel compound from the culture solution of the SANK 60892 strain belonging to the genus Streptomyces. And A38840 and its pharmacologically acceptable salts have an excellent LDL receptor gene expression-enhancing effect, and are effective for treating hyperlipidemia such as hypercholesterolemia due to lipid metabolism disorder and for atherosclerosis The present invention has been completed by finding out that.

[0006]

The present invention relates to a compound of the formula (I)

[0007]

Embedded image

A38840 or a salt thereof having

Further, the present invention is directed to A388 having formula (I)
40 or a salt thereof and a use thereof.

A38840 having the formula (I) of the present invention has the following physicochemical properties.

(1) Property of substance: acidic and fat-soluble. (2) Molecular formula: C ₄₇ H ₇₆ O ₁₇ high-resolution FAB-MS [(M + Na) ⁺ ] method,
As a result of measurement using the estimation formula C ₄₇ H ₇₆ O ₁₇ Na (as Na salt), the theoretical value was 935.4980, and the measured value was 935.5015.

(3) Molecular weight: 912 (high resolution FAB
-Measured by MS method) (4) Ultraviolet absorption spectrum (λ _max nm (ε)): The ultraviolet absorption spectrum measured in methanol is only end absorption.

(5) Infrared absorption spectrum: ν _max cm ^-1 potassium bromide (KBr) The infrared absorption spectrum measured by the tablet method is as shown below. 3425, 2947, 1730, 1676, 1637, 1458, 1414, 1384, 1364, 1257, 1205, 1176, 1138, 1079, 1052, 979.

(6) ¹ H-nuclear magnetic resonance spectrum:
The nuclear magnetic resonance spectrum (360 MHz) measured using tetramethylsilane as an internal standard in (δ, ppm) deuterated methanol is as shown below. 0.82 (s), 0.89 (s), 0.91 (s), 0.94 (m), 0.96 (m), 1.00 (s), 1.04 (m), 1.05 (m), 1.12 (s), 1.25 (s), 1.25 (s), 1.31 (m), 1.33 (m), 1.37 (m), 1.40 (m), 1.46 (m), 1.58 (m), 1.59 (m), 1.63 (m), 1.65 (m), 1.76 (m), 1.77 (m), 1.81 (m), 1.87 (m), 1.97 (s), 2.05 (m), 2.10 (m), 3.20 (d), 3.38 (m), 3.38 (m), 3.4 (m), 3.4 (m), 3.53 (d), 3.55 (m), 3.55 (m), 3.57 (m), 3.64 (m), 3.65 (m), 3.72 (m), 3.74 (m), 3.84 (d), 3.92 (dd), 4.10 (m), 4.14 (d), 4.45 (d), 4.78 (d), 5.12 (brs), 5.25 (t).

(7) ¹³ C-nuclear magnetic resonance spectrum:
(Δ, ppm) The nuclear magnetic resonance spectrum (90 MHz) measured by using tetramethylsilane as an internal standard in deuterated methanol is as shown below. 16.47, 17.59, 18.29, 19.44, 20.43, 23.38, 2
4.87, 25.41, 26.90, 27.09, 29.10, 29.96, 31.40
(s), 32.56, 34.42, 37.52 (s), 38.61 (s), 39.88,
40.79 (s), 42.27, 43.40 (s), 44.76 (s), 46.80, 47.
47, 49.0, 57.42, 64.39, 67.13, 69.61, 71.12,
72.24, 72.30, 74.16, 74.29, 75.85, 77.00 77.01, 77.69, 78.05, 78.43, 92.27, 102.32, 1
02.54, 105.62, 123.77, 145.15 (s), 176.56 (s).

(8) High-performance liquid chromatography: Separation column: Senshupack H-2151 (column φ6 × 150 mm, manufactured by Senshu Scientific Co., Ltd.) Mobile layer: Acetonitrile-0.2M phosphate buffer (pH 6.8) = 34: 66 Flow rate: 1.0 ml / min Detection wavelength: UV 210 nm Retention time (Rf value): 7.9 minutes.

(9) Solubility: methanol, ethanol,
Soluble in dimethyl sulfoxide, poorly soluble in ethyl acetate and water,
Insoluble in chloroform and hexane.

A38840 of the present invention has various stereoisomers. In A38840 of the present invention, equal and unequal mixtures of these isomers are all represented by a single formula. Therefore, in the present invention, all of these isomers and a mixture of these isomers are also included.

Further, in the present invention, when A38840 forms a solvate (for example, a hydrate), these are also included.

For example, the A38840 of the present invention may be left in the atmosphere or recrystallized to absorb water, adsorb water, or form a hydrate. The present invention includes such a solvate.

Further, the present invention includes all compounds which are metabolized in vivo and converted into A38840, so-called prodrugs.

The A38840 of the present invention can be salted according to a conventional method. As the salt of A38840, A3
There is no particular limitation as long as it is medically used and pharmacologically acceptable as compared with 8840, that is, it does not reduce the activity and does not increase the toxicity. It should be noted that there is no limitation when used for purposes other than medicine, for example, when used as an intermediate.

Examples of such salts include alkali metal salts such as lithium, sodium and potassium; alkaline earth metal salts such as calcium, barium and magnesium; aluminum salts; salts of basic amino acids such as lysine and arginine. Amine salts such as ammonia, methylamine and dimethylamine: and the like. Preferably, it is a pharmacologically acceptable salt.

The SANK 60892 strain producing the novel compound A38840 of the present invention is isolated from soil. Morphological characteristics of the SANK 60892 strain,
The physiological properties are as follows.

1. Morphological features SANK 60892 strain is an International Streptomyces Project (ISP: International
Streptomyces Project) The following morphological characteristics were exhibited by culturing at 28 ° C. for 14 days on a defined medium. When observed by an optical microscope, the basal hyphae of SANK 60892 are well elongated and branched and show light brown, grayish yellow brown or olive gray color, but no rupture or zigzag elongation like Nocardia strain is observed. The aerial hyphae simply branch. The spore linkage has a spiral morphology, approximately 10
Form a chain of 50 to 50 or more spores.
Observation by a scanning electron microscope shows that the spores have an elliptical shape and the surface structure thereof is spiny. The size of spores is 0.8 to 1.0 × 0.8 to 1.2 μm. Spores are formed only on aerial hyphae. No special organs such as sporangia, aerial mycelial axle branching, aerial hyphae rupture, and sclerotia were not observed.

[0026] 2. Culture properties on various culture media Table 1 shows the culture properties of the SANK 60892 strain on an agar medium after culturing at 28 ° C for 14 days.

[0027]

[Table 1] ────────────────────────────────── Type of medium Item ^{* 1} Properties of SANK 60892 strain ─ ───────────────────────────────── Yeast extract ・ G ： Very good, flat, grayish yellow tea (2.5 Y 5/3) ^{* 2} Malt extract agar AM: Richly formed, velvety, light yellowish tea (2.5Y7 / 2) (ISP2) R: Yellowish tea (10YR 6/6) SP: Yellowish tea (10YR 6/6) ── ──────────────────────────────── Oatmeal agar G: Very good, flat, olive ash (5Y 7/3) AM: Richly formed, velvety, tea taste ash (2.5Y6 / 2) R: Yellow tea (2.5Y 6/4) SP: Not produced ────────────────── ──────────────── Starch / inorganic salt agar G: Good, flat, thin (2.5Y 8/2) AM: Richly formed, velvety, light brown (2.5Y8 / 2) R: Light brown (2.5Y 9/4) SP: Not produced ───────────── ────────────────────── Glycerin ・ G: Very good, flat, grayish yellow tea (2.5Y 6 /
3) Asparagine agar AM: Richly formed, velvety, tea taste ash (10YR6 /
2) (ISP5) R: Yellow tea (2.5Y 7/4) SP: Not produced ─────────────────────────────── ──── Peptone yeast G: Good, flat, tea taste ash (2.5Y 6/2) Extract / iron agar AM: Not formed (ISP6) R: Tea taste ash (10YR 4/2) SP: Dark yellow tea (7.5) YR 3/2) ────────────────────────────────── Tyrosine / agar G: Very good, flat, Grayish yellow tea (2.5Y 5/3) (ISP7) AM: Abundantly formed, fluffy, light yellowish green tea (2.5Y7 / 2) R: Dark brown ash (7.5YR 3/1) SP: Brown ash (10YR 4 / 2) ────────────────────────────────── Sucrose G: Not very good, flat, brownish white (2.5Y 9/1) Nitrate agar AM: Good, velvety, light yellowish tea (2.5Y7 / 2) R: Brownish white (2.5YR 9 / 1) SP: Not produced ────────────────────────────────── Glucose G: Good, flat, Tea taste white (10YR 9/2) Asparagine agar AM: Good, velvety, bright tea taste ash (10YR 8/2) R: Tea taste white (2.5Y 9/1) SP: Not produced ───────── ────────────────────────── Nutrient agar G: Good, flat, light yellowish tea (2.5Y 7/3) (DIFDO) AM: Slightly Formation, white R: Light yellowish tea (2.5Y 8/4) SP: Light yellowish tea (2.5Y 8/6) ────────────────────────── ───────── Potato extract G: Poor, flat, brownish white (2.5Y 9/1) Ginseng extract agar AM: Very good, fluffy, bright brownish ash (10YR 8/2) R : Chami White (2.5Y 9/1) SP: Not produced ────────────────── ─────────────── Water agar G: Poor, flat, brownish white (2.5Y 9/1) AM: Good, fluffy, bright brownish ash (10YR 8/2) R : Chami White (2.5Y 9/1) SP: Not produced ─────────────────────────────────── * 1: G: growth, AM: aerial mycelium, R: back, SP: soluble pigment * 2: display of color that conforms to the Munsell system 3. Physiological properties SANK observed for 2 to 21 days after culturing at 28 ° C
The physiological properties of strain 60892 are shown in Table 2.

[0028]

[Table 2] Physiological properties SANK 60892 strain ──────────────────────────── Hydrolysis of starch Positive liquefaction of gelatin Negative nitrate Reduced positive milk Coagulation of negative milk Peptone of milk negative melanin-like pigment productivity positive substrate degradability: casein negative tyrosine negative xanthine negative growth temperature range (medium 1) 15-41 ° C proper growth temperature (medium) 1) 18-33 ° C Growth in the presence of sodium chloride (medium 1) 5% ───────────────────────────── Medium 1: Yeast Extract / malt extract agar (ISP2).

Further, the assimilation of carbon source of the SANK 60892 strain was observed after culturing at 28 ° C. for 14 days using Prideham-Gotrieve agar medium (ISP9).
As shown in FIG.

[0030]

[Table 3] Utilization of carbon source of SANK 60892 strain +: Utilized,-: Not utilized.

[0031] 4. Chemotaxonomic properties The cell wall of the SANK 60892 strain is the method of Hasegawa et al. [Hasegawa et al., The Journal of General and Applied Microbiology.
ral and Applied Microbiology), 29, 319-322.
(1983)], LL-diaminopimelic acid was detected. In addition, the major sugar component in the whole cells of the SANK 60892 strain was determined by the method of MP Rechevalier [MPLechevalier, Journal of Laboratory and Clinical Medicine (Journal of Lab
oratory and Clinical Medicine), 71, 934-944.
(1968)], no characteristic component was detected.

From the above mycological properties, SANK 6089
Among the actinomycetes, the two strains are Streptomyces.
It was clarified that it belongs to the genus. Therefore, the SANK 60892 strain is Streptomyces sp. SANK.
It was identified as strain 60892. SANK 60892 shares in Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry
Deposited as ERM BP-5225.

Although the SANK 60892 strain has been described above, it is well known that the various properties of fungi of the genus Streptomyces are not constant, and that they easily change naturally and artificially, and are used in the present invention. Possible strains include all strains producing A38840 belonging to the genus Streptomyces.

[0034]

DETAILED DESCRIPTION OF THE INVENTION To obtain A38840 of the present invention, culturing of these microorganisms is carried out in a medium such as those used to produce other fermentation products. Such a medium contains a carbon source and inorganic salts that can be assimilated by microorganisms.

Generally, as carbon sources, glucose, fructose, maltose, sucrose, mannitol,
Glycerol, dextrin, oats, rye, corn starch, potato, corn flour, cottonseed oil, molasses, citric acid, tartaric acid and the like can be used alone or in combination. Generally, a medium amount of 1-1
The amount is variable at 0% by weight.

As the nitrogen source, a protein-containing substance is generally used in the fermentation process. Suitable nitrogen sources include soybean flour, bran, peanut flour, cottonseed flour, casein hydrolyzate,
Examples include pharmamin, fish meal, corn steep liquor, peptone, meat extract, yeast, yeast extract, malt extract, sodium nitrate, ammonium nitrate, ammonium sulfate and the like. The nitrogen sources are used alone or in combination in the range of 0.2-6% by weight of the medium amount.

The nutrient inorganic salts incorporated into the medium are ordinary salts capable of obtaining ions such as sodium, potassium, calcium, magnesium, ammonium, phosphate, sulfate, chloride and carbonate. It also contains trace amounts of metals such as cobalt, manganese, and iron.

In liquid culture, silicone oil, vegetable oil, surfactant or the like is used as an antifoaming agent.

Streptomyces sp.
tomyces sp. ) The pH of the medium for culturing the SANK60892 strain and producing A38840 is 5.0-7.
It can be changed to 0.

The growth temperature of the bacterium is 15 ° C. to 41 ° C., but the growth range of 18 ° C. to 33 ° C. is good, and 22 ° C. to 30 ° C. is suitable for the production of A38840. A38840 can be obtained by aerobically culturing, but a commonly used aerobic culturing method, for example, a solid culturing method, a shaking culturing method, an aeration and stirring culturing method or the like is used.

In small-scale culture, it is preferable to carry out shaking culture at 28 ° C. for several days. Culture was performed in an Erlenmeyer flask equipped with a baffle (water flow control wall) for 1-2.
Begin with the staged seed development process. The medium at the seed development stage can use a carbon source and a nitrogen source together. Shake the seed flask in a constant temperature incubator at 28 ° C for 7 days, or
Or shake until it grows sufficiently. The grown seed is used to inoculate a second seed or production medium. If an intermediate development step is used, it is grown in essentially the same way and partially used to inoculate the production medium. Shake the inoculated flask at constant temperature for several days,
When the incubation is over, the contents of the flask are centrifuged or filtered.

In the case of large-scale culture, it is preferable to culture in a suitable tank equipped with a stirrer and an aeration device. According to this method, the nutrient medium can be prepared in the tank. The nutrient medium is sterilized by heating to 125 ° C., and after cooling, the sterile medium is inoculated with seeds that have been previously grown. Culture is 28
It is carried out by aeration stirring at ℃. This method is suitable for obtaining large amounts of compounds.

After completion of the culturing, A38840 existing in the liquid portion of the culture broth was separated by separating the bacterial cells and other solid portions by a filtration operation using diatomaceous earth as a filter aid or by centrifugation to obtain the filtrate or supernatant. A38840 present in
It can be obtained by extracting and purifying by utilizing its physicochemical properties. For example, A38 present in the filtrate or supernatant
840 is an organic solvent that is immiscible with water under acidic pH conditions,
For example, extraction and purification can be performed with ethyl acetate, chloroform, ethylene chloride, methylene chloride-butanol, etc., alone or in combination. Alternatively, as an adsorbent, for example, activated carbon or an adsorbent resin such as Amberlite XAD-2, XAD-4 (manufactured by Rohm and Haas Co.) or the like, Diaion HP-10, HP-2.
0, CHP-20, HP-50 (manufactured by Mitsubishi Kasei Co., Ltd.)
Etc. are used. Obtained by passing a liquid containing A38840 through an adsorbent layer as described above to remove impurities by adsorption, or adsorbing A38840 and then eluting it with methanol water, acetone water, butanol water, etc. .

The A38840 thus obtained is
Further, silica gel, adsorption column chromatography using a carrier such as magnesium-silica gel florisil, partition column chromatography using Sephadex LH-20 (Pharmacia), Cosmo Seal 140C ₁₈ -OPN (trade name, Nakarai Tesque Co., Ltd.
Column chromatography using a reversed-phase carrier such as those manufactured by K.K.) and high-performance liquid chromatography using normal-phase and reversed-phase columns.

A38840 can be separated and purified by repeatedly using the above separation and purification means alone or in appropriate combination.

When A38840 of the present invention is used as a medicine, it is mixed with itself or an appropriate pharmaceutically acceptable carrier, excipient, diluent according to a conventional method, and powder, granules, tablets, capsules, injections are prepared. Can be safely administered orally or parenterally. The dose varies depending on the target disease, administration route, number of administrations, etc., but for adults, the lower limit of 0.1 mg per day (preferably 1 m
It is preferable to administer g, more preferably 10 mg) to an upper limit of 2000 mg (preferably 200 mg, more preferably 20 mg) once or in several divided doses depending on the symptoms.

[0047]

EXAMPLES The present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited thereto.

Example 1 (1) Cultured Streptomyces sp. SANK 60892 strain sterilized T-12 medium having the composition shown in Table 4 below 8
A 500 ml Erlenmeyer flask containing 0 ml was brought into contact with a platinum loop and precultured at 28 ° C. for 3 days on a rotary shaking culture machine at a rotation speed of 200 rpm (7 cm, radius of gyration). 80 ml of T-12 medium of the same composition that was sterilized from this preculture liquid
2 flasks were inoculated into 70 500 ml Erlenmeyer flasks each containing 2 ml, and main culture was carried out at 28 ° C. for 7 days with a rotary shaking culture machine.

[0049]

[Table 4] T-12 medium ─────────────────────── glucose 3.0% raw yeast 1.0% soybean flour 3.0% calcium carbonate 0 4% magnesium sulfate heptahydrate 0.2% antifoaming agent 0.1% ────────────────────── pH 7.2.

(2) Isolation and Purification To 5.6 liters of the obtained culture broth, 500 g of Celite 545 (manufactured by Jones Manville Project Corporation, USA) was added as a filter aid, followed by filtration to obtain 4 liters of the filtrate. Obtained. An equivalent amount (4 liters) of ethyl acetate was added to this filtrate, pH was adjusted to 3.0 with hydrochloric acid, and extraction was performed with ethyl acetate. The aqueous layer of the extract was extracted again with 4 liters of ethyl acetate, the obtained ethyl acetate was combined, and dried over anhydrous sodium sulfate. The ethyl acetate layer was concentrated under reduced pressure. Obtained extract 1.
9 g was dissolved in a small amount of methanol, and Sep-pak-C
-18 (Vac, 35 cc, manufactured by Waters Co.) 10
20 g, 30%, 40%, 50%, 7
Eluted with 0% acetonitrile. The elution fraction of 50% acetonitrile was concentrated and then freeze-dried to give 44m.
g of powder was obtained. Next, this powder was fractionated using high performance liquid chromatography. That is, the above crude powder was dissolved in a small amount of 1 ml of methanol, and
Each was injected into a Senshu Pack ODS type H-4251 (manufactured by Senshu Kagaku Co., Ltd., φ10 × 250 mm). 34% acetonitrile-0.04 as developing solvent
Flow rate 3 ml / using M phosphate buffer (pH 6.8)
Deployed in minutes. A38840 eluted at 10 to 12 minutes. Acetonitrile was distilled off from this section under reduced pressure, and the obtained aqueous solution was desalted using Sep-pak-C-18 described above. That is, the aqueous solution is treated with Sep-pak-C-1.
It was adsorbed on 8, then washed with water, and then eluted with 40% acetonitrile. After concentrating the eluate under reduced pressure and freeze-drying, A
38840 was obtained as a powder in an amount of 15.2 mg.

[0051]

[Test Example] Test Example 1 Chloramphenicol Acetyl Transfer
Assay using gelase (reporter assay)
Measurement of LDL receptor gene enhancing activity by quantification of LDL receptor gene expression level is basically performed by Su.
Dhoff et al. [Cell, 48, 1061 (1987)]
Was used. That is, a DNA region called a sterol regulatory element (SRE), which is present in the promoter region of the LDL receptor gene, is chemically synthesized using a DNA synthesizer (manufactured by Applied Biosystems) to obtain a bacterial clone. Plasmid pUCCAT having a lamphenicol resistance gene
I inserted it in. Next, the prepared recombinant plasmid was transferred to Chinese Hamster Ovary cells (Chinese Hamster Ov
ary Cell: CHO) using a gene transfer device (GTE-1 (trade name), manufactured by Shimadzu Corporation). The level of the LDL receptor gene in this cell is chloramphenicol acetyltransferase (Chloramphenicol Ac
It can be quantified by measuring the activity of etyltransferase (CAT). This method is generally called a reporter assay method and was developed by Gorman et al. [Molecular and Cellular Biology, Vol. 2, p. 1044 (19
(1982)]. The method for preparing the extract of cells having the LDL receptor gene and the CAT gene on the chromosome and the method for measuring chloramphenicol acetyltransferase were in accordance with the method of Gorman et al. In addition, this cell culture was performed as follows. That is, an appropriate number of cells were seeded in a 4-well plate, and after 24 hours, a culture solution (90% α-MEM was used.
(Minimum Essential Medium) + 10% fetal bovine serum (Fe
tal Calf Serum (FCS) was removed, and the cells were further cultured for 24 hours in a culture medium (95% α-MEM + 5% FCS) supplemented with the test substance. Cell culture was performed in a 5% carbon dioxide incubator kept at 37 ° C. The test substance was added to the culture medium, a cell extract was prepared 24 hours later, and the activity of chloramphenicol acetyltransferase was measured. As a result, the chloramphenicol acetyltransferase activity was 1.7 times that of the control without addition. The activity of was increased. Therefore, it was shown that the expression of the LDL receptor gene was increased.

Test Example 2 Luciferase Assay Method
Measurement of LDL receptor gene enhancing activity by luciferase assay method is one of the reporter assay methods described in Test Example 1 above, in which a luciferase gene derived from firefly is used instead of the chloramphenicol acetyltransferase gene. Yes [JR
de Wet et al., Molecular and Cellular Biology, Volume 7,
725 pages (1987)]. L chemically synthesized in Test Example 1 above
A plasmid in which the firefly luciferase gene was bound to the promoter region of the DL receptor gene was prepared and introduced into Chinese hamster ovary cells (CHO) by the same method. The level of LDL receptor gene in this cell can be quantified by measuring the activity of luciferase.

As a reagent for assaying luciferase enzyme, Picagene luciferase assay system (manufactured by Toyo Ink Co., Ltd.) was used. As the chemiluminescence measuring device (luminometer), "Luminoscan" (trade name, manufactured by Lab Systems) was used. The cell culture and disruption methods were performed as follows. That is, LD
Chinese hamster ovary cells (CH containing L receptor gene promoter and luciferase gene)
O) was inoculated into a 24-well plate in an appropriate amount, and after about 24 hours, the culture medium (90% α-MEM + 10% FCS) was removed and an appropriate amount of assay sample was added (9).
5% α-MEM + 5% FCS). In addition, 24
After culturing for a period of time, the culture solution was removed and the buffer solution (137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM
The cells were washed twice with disodium hydrogen phosphate and 1.4 mM potassium dihydrogen phosphate, and 50 μl of cell lysate (25
mM Tris-phosphate (pH 7.8),
2 mM dithiothreitol (DT)
T), 2 mM 1,2-diaminocyclohexane-N,
N, N ', N'-tetraacetic acid, 10% glycerol,
After adding 1% Triton X-100),
The supernatant obtained by centrifugation with a low-speed centrifuge was used as a crude cell extract in the luciferase assay. The protein concentration of the cell extract was measured using the Protein Assay Kit (manufactured by Bio-Rad). 20 μl of the crude cell extract was put into a 96-well plate for luciferase assay, and the plate was set to luminoscun scan. Then, a luminescent substrate solution (20 mM Tricine, 33.3 mM was used.
DTT, 1.07 mM magnesium carbonate / magnesium hydroxide pentahydrate ((MgCO ₃ ) ₄ Mg (OH) ₂ / 5H ₂ O),
270 μM coenzyme A, 2.67 mM magnesium sulfate, 470 μM luciferin, 0.1
mM ethylenediamine tetr
aacetic acid (EDTA), 530 μM adenosine triphosphate (ATP) 100 μ
The solution was dispensed in liters and the reaction was started at room temperature. After starting the reaction
Read the integrated value of light emission for 5 seconds (unit: rlu),
The value (rlu / mg) divided by the protein mass (unit: mg) was calculated. As a result, the test substance was found to have an activity of increasing the luciferase activity by 2.5 times as compared with the control without addition.

Test Example 3 Radioiodinated LDL
Enhancement of LDL receptor by BINDING ASSAY method
Activity measurement To confirm whether the LDL receptor gene expression-enhancing effect actually leads to an increase in LDL receptor protein,
LDL receptor radiolabeled with LDL ([[
¹²⁵ I] LDL) was bound to directly confirm the elevation of LDL receptor. From 140 ml of human fresh blood, the method of Goldstein et al. [Method in Enzymolo
gy, 98, 241 (1983)], cholesterol-free human serum (Lipoprotein Deficient Serum:
LPDS) and human LDL were prepared. The iodine labeling of LDL and the binding experiment to cells also followed the method of Goldstein et al. The human fibroblast NHDF strain is cultured in an appropriate amount of culture medium (90% E-MEM (Eagle Minimum Essent).
ial medium) + essential amino acids (GIBCO) + L-glutamine (SIGMA) + sodium hydrogen carbonate (SIGMA) +
Penicillin / streptomycin (SIGMA) + 10%
FCS). A 24-well plate was inoculated with an appropriate number of cells, the serum concentration was lowered to 5%, and the sample was added. After 24 hours, remove the medium and remove 200 μl
[9% culture medium (90% E-MEM + 10% LPDS)]
¹²⁵ I] -LDL (10 μg / ml) was added. 37
After standing at ℃ for 2 hours, 1 ml of buffer solution B (150 mM sodium chloride, 50 mM Tris-hydrochloric acid (Tris), 2 mg)
/ Ml Bovine Serum Albumin: B
SA, (Fraction V manufactured by Seikagaku Corporation)] three times, and further washed once with buffer solution A (buffer solution B containing no BSA). The cells were lysed by adding 0.5 ml of 0.1 M saline, the suspension was transferred to a tube, and radioactive iodine was measured by a gamma counter. The count obtained when unlabeled LDL (500 μg / ml) was added to the above culture medium was finally subtracted as non-specific binding. A part of the cell lysate was used for protein quantification. Incorporated [ ¹²⁵ I] from radioactivity measured by gamma counter
-Calculate the amount of LDL, divide by the amount of protein in the cell, LDL
The activity was expressed as (ng) / Protein (mg).
As a result, the test substance increased the binding of [ ¹²⁵ I] -LDL to the human fibroblast NHDF strain and the intracellular uptake by 5-fold as compared with the control without addition.

[0055]

INDUSTRIAL APPLICABILITY The A38840 of the present invention or a salt thereof is useful as a therapeutic and / or prophylactic agent for hyperlipidemia such as hypercholesterolemia and arteriosclerosis.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1: 465) (72) Inventor Nao Okazaki 33 Miyukigaoka, Tsukuba City, Ibaraki Sankyo Co., Ltd. ( 72) Inventor Ryuzo Enoda 33 Miyukigaoka, Tsukuba City, Ibaraki Prefecture Sankyo Co., Ltd.

Claims (7)

[Claims] 1. A compound of the formula (I) Having A. 38840 or a salt thereof. 2. A38840 having the following physicochemical properties.
Or its salt. (1) Properties of substance: acidic and fat-soluble. (2) Molecular formula: C ₄₇ H ₇₆ O ₁₇ high-resolution FAB-MS [(M + Na) ⁺ ] method,
As a result of measurement using the estimation formula C ₄₇ H ₇₆ O ₁₇ Na (as Na salt), the theoretical value was 935.4980, and the measured value was 935.5015. (3) Molecular weight: 912 (measured by high resolution FAB-MS method) (4) Ultraviolet absorption spectrum (λ _max nm (ε)): The ultraviolet absorption spectrum measured in methanol is only terminal absorption. (5) Infrared absorption spectrum: ν _max cm ^-1 potassium bromide (KBr) The infrared absorption spectrum measured by the tablet method is as shown below. 3425, 2947, 1730, 1676, 1637, 1458, 1414, 1384, 1364, 1257, 1205, 1176, 1138, 1079, 1052, 979. (6) ¹ H-nuclear magnetic resonance spectrum: (δ, ppm) The nuclear magnetic resonance spectrum (360 MHz) measured by using tetramethylsilane as an internal standard in deuterated methanol is
It is as follows. 0.82 (s), 0.89 (s), 0.91 (s), 0.94 (m), 0.96 (m), 1.00 (s), 1.04 (m), 1.05 (m), 1.12 (s), 1.25 (s), 1.25 (s), 1.31 (m), 1.33 (m), 1.37 (m), 1.40 (m), 1.46 (m), 1.58 (m), 1.59 (m), 1.63 (m), 1.65 (m), 1.76 (m), 1.77 (m), 1.81 (m), 1.87 (m), 1.97 (s), 2.05 (m), 2.10 (m), 3.20 (d), 3.38 (m), 3.38 (m), 3.4 (m), 3.4 (m), 3.53 (d), 3.55 (m), 3.55 (m), 3.57 (m), 3.64 (m), 3.65 (m), 3.72 (m), 3.74 (m), 3.84 (d), 3.92 (dd), 4.10 (m), 4.14 (d), 4.45 (d), 4.78 (d), 5.12 (brs), 5.25 (t). (7) ¹³ C-nuclear magnetic resonance spectrum: (δ, ppm) A nuclear magnetic resonance spectrum (90 MHz) measured by using tetramethylsilane as an internal standard in deuterated methanol is as shown below. 16.47, 17.59, 18.29, 19.44,
20.43, 23.38, 24.87, 25.41, 26.90, 27.09, 29.1
0, 29.96, 31.40 (s), 32.56, 34.42, 37.52 (s), 3
8.61 (s), 39.88, 40.79 (s), 42.27, 43.40 (s), 44.7
6 (s), 46.80, 47.47, 49.0, 57.42, 64.39, 6
7.13,69.61, 71.12, 72.24, 72.30, 74.16, 74.2
9, 75.85, 77.00 77.01, 77.69, 78.05, 78.43, 92.27, 102.32, 1
02.54, 105.62, 123.77, 145.15 (s), 176.56 (s). (8) High-performance liquid chromatography: Separation column: Senshu Pack H-2151 (column φ6 × 150 mm, manufactured by Senshu Kagaku Co., Ltd.) Mobile layer: Acetonitrile-0.2M phosphate buffer (pH 6.8) = 34: 66 Flow rate: 1.0 ml / min Detection wavelength: UV 210 nm Retention time (Rf value): 7.9 minutes (9) Solubility: Soluble in methanol, ethanol and dimethylsulfoxide, poorly soluble in ethyl acetate and water , Insoluble in chloroform and hexane. 3. Streptomyces
es) A3840-producing bacterium belonging to the genus A3840 is collected from the culture.
8840 manufacturing method. 4. A38840 belonging to the genus Streptomyces
The producing bacterium is Streptomyces sp.
myces sp. ) SANK 60892 strain (FER
MBP-5225), The manufacturing method of Claim 3. 5. A medicine comprising A38840 or a salt thereof as an active ingredient. 6. A therapeutic and / or prophylactic agent for hyperlipidemia, which comprises A38840 or a salt thereof as an active ingredient. 7. A therapeutic and / or prophylactic agent for arteriosclerosis containing A38840 or a salt thereof as an active ingredient.