WO1994018190A1

WO1994018190A1 - Depsidone compound

Info

Publication number: WO1994018190A1
Application number: PCT/JP1994/000179
Authority: WO
Inventors: Zengxiang Chen; Bi Mei HE; Tomoko Akama; Takuya Hamaguchi; Ikuyo Taneoka; Akira Kawashima; Kazunori Hanada
Original assignee: Taisho Pharmaceutical Co., Ltd.; Sichuan Industrial Institute Of Antibiotic Of State Pharmaceutical Administration
Priority date: 1993-02-08
Filing date: 1994-02-07
Publication date: 1994-08-18
Also published as: AU5979694A

Abstract

To provide a compound that promotes the incorporation of LDL from the blood into the liver. A depsidone compound represented by formula (1).

Description

Description Debsidone compound Technical field

The present invention relates to a novel depsidone-based compound having an action of promoting the uptake of low-density lipoprotein (LDL) from blood into the liver. Background art

Hyperlipidemia is a known factor linked to atherosclerotic disease and has been proven to be a risk factor for E-cardiac disease. Therefore, blood lipid-lowering agents are effective for these conditions, and various drug powers have been discovered so far. Because of the inability to completely control blood lipids in clinical practice, there is a need for the development of new lipid-lowering agents.

By the way, as analogous compounds to the depsidone compound of the present invention, benicilide (Tetrahedron Letters, No. 45, pp. 3941 to 3942, 1974), purpactin A (Journal of Antibiotex, Vol. 44, N 0.2, 136) 159, 1991) and Dehydroisobenicide (Fuite Chemistry, Vol. 30, No. 6, pp. 2096-2098, 1991) are known. There is no known substance that has the effect of promoting LDL uptake from the liver into the liver.

An object of the present invention is to provide a debsidone compound having an action of promoting LDL uptake from blood into the liver. Disclosure of the invention

As a result of repeated research to achieve the above object, the present inventors found that a specific microorganism discovered by the present inventors was HEPATOMA G2 cultured cells (hereinafter abbreviated as HEPG2) (human liver cancer). The present inventors have found that the present invention produces a novel substance having an LDL uptake promoting effect on cells) and completed the present invention. That is, the present invention provides a 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 has the name of microorganism rPenicillium purpurogenum TF-03.

No. 74 ”and microbial accession number“ FERM BP-4540 ”have been deposited with the National Institute of Advanced Industrial Science and Technology.

The bacteriological properties of this strain are shown below.

1) Form

This strain grows well on potato-glucose agar medium, oatmeal agar medium, YpSS agar medium, etc., and has extremely good spore formation. Observation under a microscope of colonies formed by this strain on a malt dextran agar medium at 25 ° C for 7 days reveals that the hyphae have septum, are highly branched, and have a white to bright yellow color. Present. The conidium-forming cells diverge from the tip of the conidiophores that have diverged from the aerial hyphae or the basal hyphae into a bicyclic unisymmetrical shape via the metre, respectively. It forms a chain of conidia, and has a morphological force called Penicilli, which is characteristic of Benicillium. Conidiophores have septum, surface is smooth, diameter is 2.0 ~ 4.0m, length is 40 ~ 270m. Metri is 9.0-13.0 m X 2.0-3.2 ^ m, and Fearide 10.0-13.0 (~ 20.) Μπ \ X 1.8-2.8 zm. Become. Conidia are elliptical to subspherical, rarely spherical, and the surface is smooth to slightly rough, with a size of 2.4 to 3.2 (4.0) 11 1.8 to 3.0 izm.

Although the culture was extended to 3 weeks, no sexual reproductive organs were formed. 2) Growth on the medium

Table 1 below shows the results of macroscopic observation when cultured at 25 ° C for 14 days on various media. In addition, the color display quoted the system color name of the Japan Standards Association and the JIS color name book (1985).

Medium Growth (] π: -diameter) Coloni--color cell Secretory pigment

Nature of the surface

,

Eleven

'Lay good ^ (L59-white (N9.5) to deep bluish purple good purple

¾ =>

66.7 nm) Very light yellow (5Ρ3 / 8) From preferred red sugar sugar agar photo (10YR9 / 3) Light around the center (10RP medium is dark grayish yellow near fluffy medium) Yellow (5Υ9 / 6) 3/10)

(2.5GY4 / 3) Auto ΐ dark grey-yellow (2.5 bright grey-yellow good purple violet agar GY4 / 3). Perimeter (5Υ8 / 3) Preferred red medium is bright yellow (5Y (10RP

8.5 / 10) 3/10) Wheat ^ ヱ cow Medium (46.6- light yellow (5V9 / 6 bright reddish yellow medium dark purple agar 48.3nim)). Partially light (10YR8 / 11). Moderate red ground Fluffy red purple red

(10RP6 / 10) 'Purple red like 3/10)

(10 P4.5 / 12) γ p S Dark grayish blue (2.5 pale yellow red good purple agar medium G3.5 / 3). Perimeter (5Y 8/7) Preferred red part is bright yellow (5Φ Near the heart (10RP Υ8.5 / 10) Very dark yellow 3/10)

(10YR2 / 2) Sabrow Medium (.8-light reddish yellow BJ3 light reddish yellow non-secreted agar medium 52.3mm) (10YR8.5 / 6) (10YR8 / 11) bad hairy 3) physiological properties

① Growth temperature range and optimum temperature

This strain grows in a sub-mouth liquid medium with a pH of 6.0 at a temperature in the range of 14 to 39 ° C, and the optimum temperature is 28 to 35 ° C.

② Growth pH range and optimal pH

The strain grows in YpsS liquid medium at 26 ° C in the range of ρΗ2 to 8, and the optimum pH is 6 to 8.

4) distinction between aerobic and anaerobic; aerobic

From the above morphological characteristics and culture characteristics, it was clarified that this strain is a strain of the genus Penicillium. Thom's "A MANUAL OF THE PENICILLIAJ (1949)" and JI Pitt's "A LABORATORY GUIDE TO COMMON Penicillium SPECIESJ (1985)" were compared with many known strains. Penicillium

It is clear that the strain has the properties closest to purpurogenum Stoll.

₀ named "Penigillium purpurogenum TF-0374"

The production of MC-142 is carried out by cultivating Penicillium purpurogenum TF-0374 in a medium containing various nutrients under aerobic conditions in accordance with the production of general fermentation products.

The medium is mainly a liquid medium and consists of a carbon source, a nitrogen source, and inorganic salts. If necessary, it can be added with bisamines, precursors, and defoamers. The pH is adjusted to around 6. As the carbon source, for example, glucose, maltose, dextrin, glycerin, starch or the like is used alone or as a mixture. As the nitrogen source, for example, yeast exo-peptone, meat extract, soy flour, corn liquor, urea, ammonium salt or the like is used alone or in combination. As the inorganic salt, for example, potassium phosphate, magnesium sulfate, sodium chloride, calcium carbonate and the like are used alone or in combination. As an antifoaming agent, Adekinol, a silicon compound, or the like can be used.

The culture method is suitable for aerobic cultivation such as shaking culture and aeration and stirring culture, and is performed at pH 4-8, 25-30 ° C for 2-3 days, preferably at pH 6-7, 26-28 ° C. Incubate for 2 days. The MC-142 produced by this culture can be isolated according to a general method for collecting fermentation products.

That is, after completion of the culture, MC-142 is extracted from the cells separated by centrifugation or filtration with an organic solvent such as a lower alcohol or acetone, and the extract is concentrated with Mil to remove the organic solvent. The solution is dissolved in a water-insoluble organic solvent such as benzene, benzene, orifice, and concentrated under reduced pressure to form a syrup. This syrup is again dissolved in an organic solvent such as ethyl acetate, benzene, black form, acetone, methanol, etc., and column chromatography using silica gel, high-performance liquid chromatography filled with silica gel 0DS for reverse phase distribution. MC-142 can be purified and isolated by subjecting it to a gel filtration power and a ram chromatograph.

The target substance of the present invention, MC-142, obtained by the above purification was analyzed for its elemental analysis value, molecular weight, ultraviolet absorption spectrum, 1H-NMR spectrum, 13C-NMR spectrum, etc. The structural formula was determined from the results.

The physicochemical properties of MC-142 are as follows.

(a) Elemental analysis value:

Actual value (%) C 68.69, H 6.31, 0 24.91

Theoretical value (%) C 68.75, H 6.25, 0 25.00

(Calculated as C22H2406)

(b) EI mass spectrum:

E IMS m / z 384 (M +)

(c) molecular weight: 384

(d) Specific rotation:

[a] D20 = -25 ° (c = 0.04, black-hole form)

(e) UV absorption spectrum:

max 257 nm (e = 16570)

267 nm s h (e = 13600)

287 nm s h (e = 6910)

(Measured in methanol solution)

(f) Infrared absorption spectrum: Fig. 1 shows the results measured in potassium bromide tablets.

(g) lH—NMR spectrum:

FIG. 2 shows the result of measurement at 400 MHz in CDC13.

(h) 13C—NMR spectrum:

FIG. 3 shows the results of measurement at 100 MHz in DC13.

(i) Solubility in solvent:

Insoluble in water Soluble in methanol, ethanol, ethyl acetate, black form, benzene

(j) a color reaction;

Positives: iodine, sulfuric acid, ferric chloride

Negative: ninhydrin

(k) Basic, acidic, neutral: weakly acidic

(1) Appearance: pale yellow oil Best mode for carrying out the invention

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples.

Example

(1) In a 500 ml 1 Erlenmeyer flask containing 10 Om1 of a sterile liquid medium of pH 6 consisting of 2 g of glucose, 0.5 g of polypeptone, 0.3 g of yeast extract, 0.1 g of monopotassium phosphate and 0.05 g of magnesium sulfate per 10 Om1 Penicillium

Purpurogenum TF-0374 strain was inoculated and cultured with shaking at 26 ° C for 72 hours. Then 50

In a L-volume culture tank and a 200-L culture tank, a medium having the same composition as the seed culture was placed in 30 L and 120 L, respectively, and after sterilization, 0.3 L and 1.2 L of the seed culture were inoculated at 26 ° C. The cells were cultured with aeration and stirring for 48 hours.

(2) After completion of the culture, the supernatant was separated from the 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 dissolved with 10 L of acetone. The cells were extracted with 24 L of acetone, and this extract was combined with the eluate of HP-20 and concentrated if-if. After distilling off the acetone, dehydrated with 1 OL 3 times extracted _c anhydrous sulfate Na Bok potassium combined the acid Echiru extracted partitioned with acetate Echiru of, reduced to dryness under reduced pressure 147 g of a brown syrup were obtained.

(3) Dissolve the brown sample in 300 ml of chloroform and adsorb it on a 3800 ml 1 column of Siri force gel [Kieselgel-1 60 (trade name, manufactured by Merck)] prepared in black hole form. Was. After washing with 8500 ml of black-mouthed form, the active fractions eluted with a mixed solvent of black-mouthed formum methanol (99: 1 to 98: 2) were combined and concentrated to dryness to obtain 23.72 g of brown syrup.

(4) Dissolve the brown syrup of the preceding paragraph in black-mouthed form and fill with Sephadex LH20 (trade name, manufactured by Pharmacia) prepared with a mixed solvent of black-mouthed form: n-hexane: methanol (5: 5: 1) Using a 1300 ml column, gel filtration was performed with the same solvent. The active fraction was collected to obtain 1.597 g of a brown substance.

(5) The brown substance described in the preceding paragraph is dissolved in methanol, and gel filtration is carried out with methanol using a 340 ml column filled with Sephadex LH20 (trade name, manufactured by Pharmacia) prepared with methanol. I went. The active fraction was collected to obtain 561 mg of a brown substance.

(6) Dissolve the brown substance described in the preceding paragraph in acetonitrile, and use this solution for preparative medium pressure liquid chromatography using 55% acetonitrile as a mobile phase. —ODS—AQ12OA (5O0X35Om m)], the beak eluted at 110 minutes under the conditions of a flow rate of 14.8 ml and 1 Zmin was collected while monitoring the UV absorption at 280 nm. The fractions obtained by the fractionation were combined, concentrated under reduced pressure to remove acetonitrile, and then extracted twice with a half amount of ethyl acetate. The ethyl acetate extracted sections were combined, dehydrated with anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain 9.4 mg of a brown substance.

(7) The brown substance described in the preceding paragraph was dissolved in methanol, and this solution was subjected to preparative high-performance liquid chromatography using 60% methanol as a mobile phase [Equipment: 880-PU, manufactured by JASCO Corporation; Column: YMC —ODS—AQ (100 × 25 Omm)], the beak eluted in 12.4 minutes was collected at a flow rate of 4.6 m 1 / min while monitoring the UV absorption at 280 nm. The fractions obtained by the fractionation were combined, concentrated under reduced pressure to remove methanol, and then extracted twice with a half amount of ethyl acetate. The ethyl acetate extracted sections were combined, dehydrated with anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain 3.0 mg of MC-142 as a pale yellow oily substance. Test example (LDL uptake promoting effect using HEPG2 cells)

(Sample)

L.Omg of MC-142 obtained in the example was dissolved in ethanol and used to obtain the target concentration.

(Test cell)

HEP G2 human hepatoma cells

(Culture used)

Dulbecco's Modified Eagle Medium (containing 10% fetal serum)

(Test method)

Dispense 0.5 ml of a culture solution prepared from HEP G2 cells to a concentration of 4X105Zm1 into a 24-well plate (manufactured by Corning) having a diameter of 2 Omm, and incubate at 37 ° C in a 5% carbon dioxide gas incubator. Cultured for 48 hours. Then, replace the medium with 0.3 ml of Dulbecco's modified single-glue medium containing 10% ribonucleic acid tindeficient serum (LPDS, International Bio Inc.) to which 1 Ozl of the sample previously diluted to the target concentration was added. And cultured for an additional 24 hours. 1 ₂ 01 LDL labeled with I (Funakoshi Pharmaceutical) medium was removed after incubation for 4 hours was added, the cells were lysed with 2 mM Soddy um Rau Lil monkey Fe Ichito溶solution 0.4 m 1. 0.3 ml of the obtained cell lysate was diluted twice with purified water, and the fluorophore was measured with a fluorometer (Shimadzu RF-5000). It was subjected to quantification. Using only the culture solution prepared by adding HEP G2 cells to which no sample was added to a concentration of 4 × 105 / m1 as a control, the amount of incorporated Di I-LDL was calculated from the fluorescence intensity using a calibration curve. The specific activity per unit protein amount was expressed as the uptake promoting activity.

(Result)

The results are shown in Table 2. Table 2

Industrial applicability

As is clear from the test examples, the compound of the present invention (MC-142) has an excellent LDL uptake promoting activity in the liver, and thus can provide a drug useful as a lipid-lowering agent.

The method of administration of MC-142 is oral administration. The dosage form can be selected from tablets, granules, powders, capsules, syrups, and suspending agents. In the preparation of various dosage forms, conventional excipients (eg, microcrystalline cellulose, starch, lactose, mannitol, etc.), binders (eg, hydroxypropyl cellulose, polyvinyl virolidone, etc.) It can be produced by a usual production method using a powder (eg, magnesium stearate, talc, etc.), a disintegrant (eg, calcium carboxymethylcellulose) and the like.

The dose of MC-142 is 100 to 50 Omg for treating adults, and is administered once or several times a day. This dosage can be adjusted appropriately according to the patient's age, weight and condition. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the infrared absorption spectrum of MC-142 measured with a KBr tablet. FIG. 2 shows the 1H-NMR spectrum of MC-142 measured in CDC 13 at 400 MHz.

FIG. 3 shows the 13C-NMR spectrum of MC-142 in CDC13 measured at 10 MHZ.

Claims

The scope of the claims

(1 set

Depsidone compound represented by

Equation (2)

Lipid-lowering agent comprising a debsidone compound represented by the formula