JPH0532656A - Decalin-based compound - Google Patents

Abstract

PURPOSE:To obtain a new compound useful as an anti-dementia medicine, having promoting effects on production of nerve growth factor. CONSTITUTION:A compound shown by formula I (R is group shown by formula II or formula III). The compound is obtained by culturing a strain separated from fallen leaves of pineapple manufactured in Okinawa Prefecture, Aspergillus versicolor F-5,015 (FERM P-12,361) in a liquid medium under an aerobic condition. The physical and chemical properties of the compound shown by formula I. Elemental analysis value: C 68.07 H 7.51 (calculated as C31H40O8 by measured value %); molecular weight: 540. Melting point: 135-138 deg.C. Specific rotatory power: [alpha]D 25=75.0 (C=1.0, methanol). Solubility: readily soluble in methanol, ethanol and dimethyl sulfoxide, slightly soluble in acetone, chloroform, ethyl acetate and insoluble in water and n-hexane. Color reaction: positive in iodine, sulfuric acid and 2,4-dinitrophenylhydrazine and negative in ninhydrin. Acidity. Yellow powder.

Description

Detailed Description of the Invention

[0001]

The present invention relates to nerve growth factor (hereinafter referred to as N
It is called GF. The present invention relates to a decalin compound having an effect of promoting production.

[0002]

2. Description of the Related Art There are no known compounds that are structurally similar to the compounds of the present invention and have similar effects.

[0003]

DISCLOSURE OF THE INVENTION The object of the present invention is NG
It is intended to provide a novel compound having an F production promoting effect.

[0004]

[Means for Solving the Problems] The present inventors have isolated a large number of strains from the soil in order to achieve the above-mentioned object, and variously studied the metabolites of the strains. As a result, a certain strain produced NGF. The inventors have found that a novel physiologically active substance having a promoting effect is produced and completed the present invention. That is, the present invention is

[0005]

[Chemical 4]

(Where R is an expression

[0007]

[Chemical 5]

A group or formula represented by

[0009]

[Chemical 6]

A group represented by A compound represented by the formula [hereinafter, R is a compound represented by the formula 5
And a compound in which R is a group represented by Chemical formula 6 is referred to as NG-112. ]. The microorganisms producing NG-111 and NG-112 are strains newly isolated from pineapple litter leaves collected by the present inventors in the suburbs of Kin-cho, Okinawa Prefecture,
The name of the microorganism is " Aspergillus versicolor F-5015" and the microorganism deposit number is "Microtech Lab. No. 12361 (FERM P-1
2361) ”has been deposited with the Institute of Microbial Technology, Institute of Industrial Technology.

The bacteriological properties of this strain are shown below. [Morphology] This strain grows well on potato / glucose agar medium and oatmeal agar medium, and spore formation is good on potato / glucose agar medium, oatmeal agar medium, malt extract agar medium, YpSs agar medium. Then it is moderate. This strain is on Czapek / Yeast extract agar medium,
When the colony formed by culturing at 25 ° C. for 7 days is observed with an optical microscope, the mycelium has a septum and is highly branched,
The conidia stalks elongated from the basal hyphae swell in a spherical shape, and radial conidia heads are observed, which are characteristic of Aspergillus spp. At the same time, a large number of penicillate-like morphology in which 1 to 7 phialides are circulated from the aerial hyphae directly or through the short conidia stalk is also recognized. When the conidia head is formed, the conidia stalk is colorless and has a thick wall, the surface is smooth, and the size is 153 to 388 μm × 3.0 to 6.5 μm.
Is. The apex is spherical to slightly subspherical with a size of φ6.
It is 5 μm to 15.0 μm. The size of the metre is 4.0
˜7.0 μm × 3.0 to 3.5 μm. The size of the phialide is 4.5-8.0 μm × 2.0-3.0 μm
Is. Conidia are spherical and have no septum, white immediately after formation,
Eventually it became grayish green, the surface was thorny, and the size was φ2.8.
Is about 4.0 μm. On the other hand, the conidia peduncle when forming a penicilli-like morphology is 0 to 15.0 μm × 1.0 to 2.0.
In many cases, the tip portion is slightly swollen like a crest at μm. The morphology of phialides and conidia is consistent during conidial head formation. Although the culture was extended to 3 weeks, the formation of ascaris was not observed. [Various Properties on Medium] Table 1 shows the results of visual observation when cultured at 26 ° C. for 14 days on various mediums. The colors are displayed by the Japanese Standards Association, "JIS Color Name Book" (19
1985) systematic color names are quoted.

[0012]

[Table 1]

[Physiological Properties] 1) Growth pH Range and Optimum pH This strain grows in YpSs liquid medium in a pH range of 3 to 10, and the optimum pH is 5 to 6. 2) Growth temperature range and optimum temperature This strain grows in a Sabro-liquid medium in the range of 13 to 33 ° C, and the optimum temperature is 24 to 27 ° C. 3) Distinction between aerobic and anaerobic; aerobic From the above morphological characteristics and culturing characteristics, it is clear that this strain is a subspecies of Aspergillus spp. Keisuke Tsubaki's "Fungus Encyclopedia" (1978
Year), K. B. Raper, D.M. I. Fennell "THE GENUS
Aspergillus "(1965) and M.G. A. Klich,
J. I. Pitt's `` ALABORATORY GUIDE TO COMMON Asperg
illus SPECIES AND THEIR TELEO MORPHS "(1988
Year)) and compared with many known strains.
As a result, this strain was Aspergillus versicolor (Vuill.)
It became clear that it shows the properties closest to Tiraboschi,
This strain was named " Aspergillus versicolor F-5015".

The production of NG-111 and NG-112 is
According to the case of producing a general fermented product, Aspergillus versicolor F-5015 is cultured under aerobic conditions in a medium containing various nutrients. The liquid medium is mainly a liquid medium, consisting of carbon source, nitrogen source, and inorganic salt,
If necessary, vitamins, precursors and defoamers can be added, and the pH is adjusted to around 6. As a carbon source,
For example, glucose, maltose, dextrin, glycerin, starch and the like are used alone or in combination. As the nitrogen source, for example, yeast extract, peptone, meat extract, soybean powder, corn steep liquor, urea, ammonium salt, etc. may be used alone or in combination. As the inorganic salt, for example, 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, aeration stirring culturing and the like is suitable, and the pH is 3 to 10, 25 to 25.
28 ° C for 2-3 days, preferably pH 4-6, 26-2
Incubate at 7 ° C for 3 days. NG produced by this culture
In order to isolate -111 and NG-112, it may be carried out according to a general method for collecting a fermentation product. That is, after the completion of the culture, the culture filtrate and the bacterial cells are separated by centrifugation or filtration, and the filtrate is extracted with a non-water-soluble organic solvent such as ethyl acetate, and concentrated under reduced pressure to form a syrup. The 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, gel filtration column chromatography, and thin layer chromatography to obtain NG-111 and NG-112 can be purified and isolated.

NG-111 and NG-112, which are the target substances of the present invention obtained by the above-mentioned method, have their elemental analysis values, molecular weights, ultraviolet absorption spectra and ¹ H-NMR.
The structural formula was determined as shown in Chemical formula 4 from the analysis results of the spectrum, ¹³ C-NMR spectrum and the like. NG-111 and N
The physicochemical properties of G-112 are as follows.

NG-111 (a) Elemental analysis value: measured value (%) C 68.07, H 7.51 theoretical value (%) C 68.87, H 7.46 (C₃₁H₄₀O₈(B) FAB mass spectrum: FAB (+) m / z 563 (M + Na)⁺  FAB (-) m / z 539 (M-H)^-  (C) Molecular weight: 540 (d) Melting point: 135-138 ° C (e) Specific optical rotation: [α]_D ^{twenty five}= 75.0 ° (c = 1.0, methanol) (f) Ultraviolet absorption spectrum: λmax 196 nm (ε = 11000) 240 nm (ε = 20600) 328 nm (ε = 15600) (measured in methanol solution) (g) Infrared absorption spectrum: measured in potassium bromide tablets
The results obtained are shown in FIG. (H)¹H-NMR spectrum: heavy dimethyl sulfoxide
The result measured at 400 MHz in SID is shown in FIG. (I)¹³C-NMR spectrum: heavy dimethyl sulfoxide
The result measured at 100 MHz in SID is shown in FIG. (J) Solubility in solvent: Easily soluble; methanol, ethanol, dimethylsulfoxy
Insoluble; acetone, chloroform, ethyl acetate insoluble; water, n-hexane (k) color reaction; positive: iodine, sulfuric acid, 2,4-dinitrophenylhydra
Zin Negative: Ninhydrin (l) Distinction between basic, acidic and neutral: Acidic (m) Color of substance: Yellow powder

NG-112 (a) Elemental analysis value: measured value (%) C 68.43, H 7.92 theoretical value (%) C 68.61, H 7.80 (C₃₁H₄₂O₈(B) FAB mass spectrum: FAB (+) m / z 565 (M + Na)⁺  FAB (-) m / z 541 (MH)^-  (C) Molecular weight: 542 (d) Melting point: 136 to 140 ° C. (e) Specific optical rotation: [α]_D ^{twenty five}= 60.2 ° (c = 0.47, methanol) (f) Ultraviolet absorption spectrum: λmax 205 nm (ε = 7590) 231 nm (ε = 10800) 339 nm (ε = 79700) (measured in methanol solution) (g) Infrared absorption spectrum: measured in potassium bromide tablets
The results obtained are shown in FIG. (H)¹H-NMR spectrum: heavy dimethyl sulfoxide
The result of measurement at 400 MHz in SID is shown in FIG. (I)¹³C-NMR spectrum: heavy dimethyl sulfoxide
The result of measurement at 100 MHz in SID is shown in FIG. (J) Solubility in solvent: Easily soluble; methanol, ethanol, dimethylsulfoxy
Insoluble; acetone, chloroform, ethyl acetate insoluble; water, n-hexane (k) color reaction; positive: iodine, sulfuric acid, 2,4-dinitrophenylhydra
Zin Negative: Ninhydrin (l) Distinction between basic, acidic and neutral: Acidic (m) Color of substance: Yellow powder

[0019]

The Alzheimer-type dementia, which is increasing in number in recent years, is said to occur because the cholinergic nerve cells in the basal forebrain are selectively lost. Since NGF has an action of suppressing the loss of nerve cells, the compound of the present invention having a production promoting effect is useful as an anti-dementia drug.

[0020]

EXAMPLES Hereinafter, the present invention will be described in more detail by showing Examples and Test Examples. Example 2 g of glucose per 100 ml, 0.5 of polypeptone
g, yeast extract 0.3 g, monopotassium phosphate 0.1 g,
Aspergillus versicolor F-5015 strain was inoculated into a sterile liquid medium of pH 6 containing 0.05 g of magnesium sulfate, and 2
Culture was carried out at 6 ° C. for 72 hours with shaking. Next, using a 5 L culture mini jar, 100 ml of the seed culture solution was inoculated into 3 L of sterile medium having the same composition as the seed culture, and agitated and aerated for 72 hours at 26 ° C. After completion of the culture, the supernatant and the cells were separated with a centrifuge. The resulting supernatant was extracted twice with an equal amount of ethyl acetate, the ethyl acetate extraction sections were combined, dehydrated with anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to give a yellow syrup 45.
g was obtained.

20 ml of this yellow syrup is chloroform
And was adsorbed to a 250 ml column of silica gel [Kieselgel (trade name; manufactured by Merck)] prepared with chloroform. After washing with 500 ml of chloroform, the active fraction was eluted with 500 ml each of a mixed solvent of chloroform-methanol (90:10), chloroform-methanol (95:15) and chloroform-methanol (80:20). The active fractions were collected and concentrated under reduced pressure to dryness, and NG-1
10 g of crude powder of 11 and 112 was obtained. Crude powders of NG-111 and NG-112 obtained by silica gel column purification were dissolved in 8 ml of methanol. This solution was applied to Sephadex LH-20 (trade name; manufactured by Pharmacia) prepared with methanol, subjected to gel filtration, and the active fractions were collected and concentrated to obtain 4.5 g of a coarse powder.

4.5 g of the crude powder obtained by this gel filtration column purification was added to a silica gel plate [Kieselgel 60F].
20 cm × 20 cm × 0.5 mm (trade name; manufactured by Merck & Co., Inc.)] and developed in a solvent system of chloroform-methanol-water-triethylamine (70: 30: 5: 0.1). The active fractions having Rf values of 0.66 and 0.61 were scraped off, and 100 ml of water and 100 ml of ethyl acetate were added to each of the fractions for extraction. The ethyl acetate layer was dehydrated with anhydrous sodium sulfate and then concentrated to dryness, and NG-11
1 (Rf value 0.66) 400 mg, NG-112 (R
71 mg of f value 0.61) was obtained.

Test Example [NGF production promoting effect test] (Sample) NG-111 and NG-11 obtained in the examples
2 were each dissolved in dimethylsulfoxide, and this was mixed in the medium described below and diluted 200-fold, and NG-111 or NG-112 was added at 0.003 μg / ml to 0.1 μg.
The medium containing the sample was prepared so as to have a concentration of / ml. (Test cells) LM cells (mouse fibroblasts) (Used medium) 199 medium (manufactured by Gibco) containing 0.5% bactopeptone, 50 units / ml penicillin, and 50 μg / ml streptomycin (test method) LM cells were pre-cultured in the above medium, 24
Pore plate (Corning, area 2c per culture hole)
m ² ), seed about 7 × 10 ⁴ cells / culture hole, and incubate at 37 ° C.
The cells were cultured in 5% CO ₂ for 3 days to be confluent. The medium was then replaced with medium containing various concentrations of analyte.
After culturing for 24 hours, the NGF concentration in the medium was measured by enzyme immunoassay [S. Furukawa et al. Neurochem, Volume 40, Volume 73
4 to 744 (1984)].
The activity was shown with the control being 100%.

[0024]

[Equation 1]

(Results) The results are shown in Table 2.

[0026]

[Table 2]

[Brief description of drawings]

FIG. 1 shows an infrared absorption spectrum of NG-111 measured in a potassium bromide tablet.

FIG. 2 shows the ¹ H-NMR spectrum of NG-111 measured at 400 MHz in heavy dimethyl sulfoxide.

FIG. 3 shows the ¹³ C-NMR spectrum of NG-111 measured at 100 MHz in heavy dimethyl sulfoxide.

FIG. 4 shows an infrared absorption spectrum of NG-112 measured in a potassium bromide tablet.

FIG. 5 shows the ¹ H-NMR spectrum of NG-112 measured at 400 MHz in heavy dimethyl sulfoxide.

FIG. 6 shows the ¹³ C-NMR spectrum of NG-112 measured at 100 MHz in heavy dimethyl sulfoxide.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12R 1:66) 7804-4B (72) Inventor Mayumi Ito 3-24-1 Takada, Toshima-ku, Tokyo Taisho product (72) Inventor Kazuki Hanada 3-24-1 Takada, Toshima-ku, Tokyo Taisho Seiyaku Co., Ltd.

Claims (1)

What is claimed is: (Claim 1) (In chemical formula 1, R is A group represented by or Represents a group represented by. ) The compound represented by.