JPH06287186A - Depsidon system compound - Google Patents

Abstract

PURPOSE: To provide the new compound having ability to accelerate the uptake of a lipoprotein having a low specific gravity from blood into liver, and useful as an agent for lowering lipids.

CONSTITUTION: The objective compound is expressed by the formula; elemental analysis: observed value (%) C-68.69, H-6.31, O-24.91; theoretical value (%) C-68.75, H-6.25, O-25; molecular weight: 384; solubility: insoluble in water, soluble in methanol, chloroform or the like; color reaction: positive in iodo reaction, sulfuric acid reaction or the like, negative in ninhydrin reaction; classification of basicity, acidity and neutrality: weak acidic; appearance: pale yellowish oil. The compound is obtained by culturing a new microorganism, Penicillium- purporogenum-TF-0374(FERM-BP-4540) under aerobic conditions, preferably at pH 6-7 and at 26-28°C for 2 days, then separating from cells and purifying according to a conventional method.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel depsidone compound having an action of promoting low density lipoprotein (LDL) uptake from blood to liver.

[0002]

2. Description of the Related Art Hyperlipidemia is known as a factor associated with arteriosclerotic diseases and has been proved to be a risk factor for ischemic heart disease. Therefore, since blood lipid lowering agents are effective for these pathological conditions, various drugs have been discovered so far. However, since it is not possible to completely control blood lipids clinically, further development of new lipid lowering agents is desired.

As a compound similar to the depsidone compound of the present invention, penicide (Tetrahedron Letters, No.
45, 3941 to 942, 1974), Purpactin A (Journal of Antibiotex, Vol. 44, No. 2, 136 to 159, 199).
1 year) and dehydroisopenicide (Phytochemistry, 30 volumes, No. 6, 2096 to 2098 pages, 1991) are known, and among these compounds, LDL uptake from the blood to the liver is known. There is no known substance having a promoting action.

SUMMARY OF THE INVENTION An object of the present invention is to provide a depsidone compound having an action of promoting LDL uptake from blood into the liver.

[Means for Solving the Problems] As a result of repeated exploratory research to achieve the above-mentioned object, the present inventors found that the specific microorganisms found by the present inventors were HEPATOMA G2 cultured cells (hereinafter, HEP G2 L) for (human hepatoma cells)
The present invention was completed by finding out that a novel substance having a DL uptake promoting action was produced.

That is, the present invention uses the formula

[0005]

The compound represented by the formula (hereinafter referred to as MC-142).

The strain producing the novel substance MC-142 of the present invention is a strain newly isolated from the soil collected by the present inventors, and the name of the microorganism is " Picicillium purpurogenum T".
F-0374 "and microorganism accession number" FERM BP-45 "
40 "has been deposited at the Institute of Biotechnology, Industrial Technology Institute. The mycological properties of this strain are shown below. 1) Morphology This strain grows well on potato / glucose agar medium, oatmeal agar medium, YpSs agar medium and the like, and spore formation is also very good. When colonies formed by culturing the strain on malt extract agar medium at 25 ° C. for 7 days are observed under a microscope, hyphae have partition walls, are highly branched, and show white to bright yellow. Conidia-forming cells are branched from aerial hyphae or basal hyphae from the tips of conidia stalks that have risen to form a polyhedra-symmetric type via Metre, and from the tips, phialo-type cells are formed. The conidia are formed in a chain, and a morphology called Penicilli, which is characteristic of Penicillium, is recognized.
The conidia peduncle has a partition wall, the surface is smooth, and the diameter is 2.0 to 4.
The length is 0 μm and the length is 40 to 270 μm. Mettle is 9.0 to 13.0 μm × 2.0 to 3.2 μm, and phialide is 10.0 to 13.0 (to 20.0) μm ×
When the particle diameter is 1.8 to 2.8 μm, the penicilli are relatively compressed due to dense branching. Conidia are elliptical to subspherical, rarely spherical, with smooth to slightly rough surface
The size is 2.4 to 3.2 (to 4.0) μm × 1.8 to
It is 3.0 μm.

Although the culture was extended to 3 weeks, formation of sexual reproductive organs was not observed. 2) Growth state on medium The following Table 1 shows the results of macroscopic observation when the cells were cultured on various mediums at 25 ° C for 14 days. For the color display, the systematic color names of the JIS Standards Color Book (1985) of the Japanese Standards Association were cited.

[0009]

[Table 1]

3) Physiological properties Growth temperature range and optimum temperature The strain of this strain is 14 in Sabouraud liquid medium at pH 6.0.
It grows in the range of ~ 39 ° C, and the optimum temperature is 28-35 ° C. Growth pH range and optimum pH This strain has a pH of 2 to 8 at 26 ° C. in YpSs liquid medium.
The optimum pH is 6-8. 4) Distinction between aerobic and anaerobic; From the morphological characteristics above aerobic and the properties in culture, this strain is P
It has been revealed that it is a bacterium of the genus enicillium . Shunichi Udagawa and Keisuke Tsubaki, "Fungal Encyclopedia" (1978), K. B. Rape
r, C. "A MANUAL OF THE PENICILLIA" by Thom (1949
Year) and J. I. Pitt's "A LABORATORY GUIDE TO
COMMON Penicillium SPECIES ”(1985) was compared with many known strains. As a result, this strain becomes apparent that shows the closest properties to Penicillium purpurogenum Stoll, this strain "Penicillium Purpurog
enum TF-0374 ”. The production of MC-142 is substantially the same as the case of producing a general fermentation product, and Penicillium purpurogenum TF-0374 is used in a medium containing various nutrients.
By culturing under aerobic conditions.

A liquid medium is mainly used as a medium, and a carbon source,
It consists of a nitrogen source and an inorganic salt, and vitamins, precursors and antifoaming agents can be added if necessary, and the pH is adjusted to around 6. As the carbon source, for example, glucose, maltose, dextrin, glycerin, starch and the like are used alone or in combination. Examples of nitrogen sources include yeast extract, peptone, meat extract, soybean powder, corn stee
Liquor, urea, ammonium salt, etc. may be used alone or in combination. Examples of the inorganic salt include monopotassium phosphate,
Magnesium sulfate, sodium chloride, calcium carbonate, etc. may be used alone or in combination. As the defoaming agent, adecanol, a silicon compound or the like can be used.

As the culturing method, aerobic culturing such as shaking culturing and aeration stirring culturing is suitable, and the pH is 4 to 8, 25 to 3
2-3 days at 0 ° C, preferably pH 6-7, 26-28
Incubate at C for 2 days. MC- produced by this culture
In order to isolate 142, a general method for collecting a fermentation product may be used. That is, after culturing, MC-142 is extracted from the cells separated by centrifugation or filtration with an organic solvent such as lower alcohol and acetone, and the extract is concentrated under reduced pressure to remove the organic solvent, and then ethyl acetate and benzene. , Redissolved in a water-insoluble organic solvent such as chloroform, and concentrated under reduced pressure to form a syrup. This syrup was again dissolved in an organic solvent such as ethyl acetate, benzene, chloroform, acetone, and methanol, and subjected to column chromatography using silica gel, high-performance liquid chromatography packed with silica gel ODS for reverse phase partition, and gel filtration column chromatography. By attaching it, MC-142 can be purified and isolated. MC-14, which is the target substance of the present invention obtained by the above purification
The structural formula of ^No. 2 was determined from the analysis results of its elemental analysis value, molecular weight, ultraviolet absorption spectrum, ¹ H-NMR spectrum, ¹³ C-NMR spectrum and the like.

The physicochemical properties of MC-142 are as follows. (A) Elemental analysis value: measured value (%) C 68.69, H 6.31, O 2
4.91 theoretical value (%) C 68.75, H 6.25, O 2
5.00 (calculated as C ₂₂ H ₂₄ O ₆ ) (b) EI mass spectrum: EIMS m / z 384 (M ⁺ ) (c) molecular weight: 384 (d) specific rotation: [α] _D ²⁰ = -25 ° (c = 0.04, chloroform) (e) UV absorption spectrum: λ _max 257 nm (ε = 16570) 267 nm sh (ε = 13600) 287 nm sh (ε = 6910) (measured in methanol solution) (f) Infrared Absorption spectrum: The result measured in a potassium bromide tablet is shown in FIG. (G) ¹ H-NMR spectrum: 400M in CDCl ₃ .
The result measured at Hz is shown in FIG. (H) ¹³ C-NMR spectrum: 100 in CDCl ₃ .
The result measured at MHz is shown in FIG. (I) Solubility in solvent: insoluble in water soluble in methanol, ethanol, ethyl acetate, chloroform, benzene (j) color reaction; positive: iodine, sulfuric acid, ferric chloride negative: ninhydrin (k ) Basic, acidic and neutral: weakly acidic (l) Appearance: pale yellow oil

The compound (MC-142) of the present invention is HE
Since it has LDL uptake promoting activity on PG2 cells (human hepatoma cells), it is useful as a lipid lowering agent.

EXAMPLES The present invention will be described in more detail with reference to Examples and Test Examples. Example (1) Glucose 2 g, polypeptone 0.5 g, yeast extract 0.3 g, and monopotassium phosphate 0.1 g per 100 ml.
Penicillium purpurogenum TF-0374 strain was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of a sterile liquid medium of pH 6 consisting of 1 g and 0.05 g of magnesium sulfate.
The culture was carried out at 6 ° C. for 72 hours with shaking. Next, 50 L culture tank and 200 L culture tank were filled with 30 L and 120 L of culture medium having the same composition as the seed culture, and after sterilization, 0.3 L and 1.2 L of the above-mentioned seed culture solution were inoculated, respectively, and 26 ° C., 48 hours Culture was performed with aeration and stirring. (2) After the culture was completed, the cells were separated into a supernatant and cells by a centrifuge. 180 L of the obtained supernatant was adsorbed on 10 L of HP-20, washed with 20 L of purified water, and then eluted with 10 L of acetone. The bacterial cells were extracted with 24 L of acetone, and this extract was diluted with H
It was concentrated under reduced pressure together with the eluate of P-20. After distilling off acetone, the mixture was extracted 3 times with 10 L of ethyl acetate. The ethyl acetate extraction sections were combined, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to dryness to obtain 147 g of a brown syrup. (3) This brown syrup was dissolved in 300 ml of chloroform and adsorbed on a 3800 ml column of silica gel [Kieselgel-60 (trade name, manufactured by Merck)] prepared with chloroform. After washing with 8500 ml of chloroform,
Active fractions eluted with a mixed solvent of chloroform-methanol (99: 1 to 98: 2) were combined and concentrated to dryness to obtain 23.72 g of brown syrup. (4) The brown syrup of the preceding paragraph was dissolved in chloroform and chloroform: n-hexane: methanol (5: 5: 1).
Gel filtration was performed with the same solvent using a 1300 ml column packed with Sephadex LH20 (trade name, manufactured by Pharmacia) prepared with the mixed solvent of. The active fractions were collected to obtain 1.597 g of a brown substance. (5) Dissolve the brown substance in the preceding paragraph in methanol,
Gel filtration was carried out with methanol using a 340 ml column packed with Sephadex LH20 (trade name, manufactured by Pharmacia) prepared by the above procedure. Collect the active fractions and brown substance 5
61 mg was obtained. (6) The brown substance of the preceding paragraph was dissolved in acetonitrile, and this solution was subjected to preparative medium pressure liquid chromatography using 55% acetonitrile as a mobile phase [used device: KP-6H manufactured by Kusano Scientific Instruments; column: YMC-ODS- AQ120A
(50φ × 350 mm)], and UV absorption is 280 nm
While monitoring at 1, flow rate 14.8 ml / min
The peak eluting at 10 minutes was collected. The separated fractions were combined, concentrated under reduced pressure to remove acetonitrile, and then extracted twice with half the amount of ethyl acetate. The ethyl acetate extraction sections were combined, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to dryness to obtain 9.4 mg of a brown substance. (7) The brown substance of the preceding paragraph was dissolved in methanol, and the solution was subjected to preparative high performance liquid chromatography using 60% methanol as a mobile phase [apparatus used: JASCO Corporation 880-P.
U; Column: YMC-ODS-AQ (10φ × 250 m
m)] while monitoring the UV absorption at 280 nm, the peak eluting at 12.4 minutes was collected at a flow rate of 4.6 ml / min. The separated fractions were combined and concentrated under reduced pressure to remove methanol, and then half the volume of ethyl acetate
Extracted twice. The ethyl acetate extraction fractions were combined, dehydrated with anhydrous sodium sulfate, and then concentrated under reduced pressure to dryness to obtain 3.0 mg of MC-142 as a pale yellow oily substance.

Test Example (LDL using HEP G2 cells
Uptake promoting action) (Sample) 1.0 mg of MC-142 obtained in the example was dissolved in ethanol and prepared so as to have a desired concentration. (Test cells) HEP G2 human hepatoma cells (culture medium used) Dulbecco's modified Eagle medium (containing 10% fetal bovine serum) (Test method) HEP G2 cells were prepared at a concentration of 4 × 10 ⁵ / ml. , 0.5 ml each in a 24-well plate (made by Corning) with a diameter of 20 mm, and 37 ° C
The cells were cultured in a 5% carbon dioxide incubator for 48 hours. Then add 10 μl of the sample diluted in advance to the target concentration, and add 10
% Lipoprotein defensive serum (LPDS,
Dulbecco's Modified Eagle Medium (D.
The medium was replaced with 0.3 ml of MEM), and the cells were further cultured for 24 hours. Add 1 μg of DiI-labeled LDL (Funakoshi Chemical) and incubate for 4 hours, then remove the medium and
The cells were lysed with 0.4 ml of M sodium lauryl sulphate solution. 0.3 ml of the obtained cell lysate was diluted 2-fold with purified water, and a fluorometer (Shimadzu RF
-5000) and the fluorescence intensity was measured, and the remaining 0.1 ml was used for protein quantification by the Lowry method. HEP without adding sample
G2 cells prepared at a concentration of 4 × 10 ⁵ / ml were used alone as a control, and the incorporated DiI-L was used as a control.
The DL amount was calculated from the fluorescence intensity by a calibration curve, and the specific activity per unit protein amount relative to the control was expressed as the uptake promoting activity. (Results) The results are shown in Table 2.

[0015]

[Table 2]

[Brief description of drawings]

FIG. 1 shows an infrared absorption spectrum of MC-142 measured with a KBr tablet.

FIG. 2 MC-measured in CDCl ₃ at 400 MHz.
¹ shows a ¹ H-NMR spectrum of 142.

FIG. 3 MC-measured in CDCl ₃ at 100 MHz.
¹⁴ shows a ¹³ C-NMR spectrum of 142.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12R 1:80) 7804-4B (72) Inventor Tomoko Akama 3-24-1 Takada, Toshima-ku, Tokyo No. Taisho Pharmaceutical Co., Ltd. (72) Inventor Takuya Hamaguchi 3-24-1, Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd. (72) Inventor Ikuyo Taneoka 3-24-1, Takada, Toshima-ku, Tokyo Taisho Pharmaceuticals Co., Ltd. (72) Inventor Akira Kawashima 3-24-1 Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd. (72) Inventor Kazuki Hanada 3-24-1 Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Within the corporation

Claims (1)

[Claims] 1. A formula The compound represented by.