JPH03279379A - New compound indanonaphthol a and b - Google Patents

Abstract

NEW MATERIAL:Indanonaphthol A and its salt having the following physical and chemical properties. Appearance, yellow powder; molecular formula, C19H12O6; molecular weight, 336 (FAB-MS method); elemental analysis (%), C 66.57, H 4.37; solubility, soluble in alcohols (e.g. methanol, ethanol, propanol and butanol), dimethyl sulfoxide, DMF, chloroform, ethyl acetate, acetone and ethyl ether and insoluble in n-hexane and water; color reactions, positive to sulfuric acid, iodine, potassium permanganate and ferric chloride reactions; etc. USE:Antibacterial agent. PREPARATION:A microbial strain belonging to genus Hormoonema [e.g. Hormoonema sp. SANK 50190 (FERM P-11359)] is aerobically cultured at 22-23 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to new compounds Indanonaftol A and B having antibacterial activity, and a method for producing the same.

(Prior Art) Conventionally, many compounds having a naphthalene skeleton and compounds having an indene skeleton have been known, but a compound in which a naphthalene skeleton and an indene skeleton are bonded through a spiro bond is not known.

(Problems to be Solved by the Invention) The present inventors have discovered that Hormonema (Hormonema) isolated from seawater
The present invention was completed by discovering that new compounds indanonaphthol A and B, which have antibacterial activity, are produced from a culture of SANK 50190 strain belonging to the genus M. onema.

(Means for Solving the Problems) Indanonaphthol A and B of the present invention have the following structural formula and properties.

1. Indanonaphthol A 1) Property of substance: yellow powder 2) Molecular formula: C19H1□06 3) Molecular weight: 336 (measured by FAB-MS method) 4) Elemental analysis: (%) Actual value C66,57H4,37 Calculation Value C67,86H3,60 5) Ultraviolet absorption spectrum: λ□. nm(E',,;)
The ultraviolet absorption spectrum measured in methanol is as shown below.

232, 260 (sh), 333.3986) 1H-
Nuclear magnetic resonance spectrum = (δ: ppm) The nuclear magnetic resonance spectrum (500 MHz) measured in deuterated dichloromethane using tetramethylsilane as an internal standard is as shown below.

5.01 (LH, s), 6.84 (LH, dd), 6.
86 (1H, d), 6.93 (1H, d), 6.94 (
1H, d), 6.95 (LH, d), 7.23 (LH
, d), 7.57 (LH, dd), 7.67 (LH,
dd), 8.64 (1H, s), 11.84 (1H, 5
)7) 13c-nuclear magnetic resonance spectrum = (δ: ppm
) Nuclear magnetic resonance spectrum (125,6
MHz) is as shown below.

66.60(d), 73.41(s), 114.57
(s), 116.62(d), 118.40(d), 1
18.84(d), 120.22(d), 122.82
(s), 126.62(d), 132.93(d), 1
37.73(s), 139.66(d), 140.13
(d), 151.03(s), 157.26(s), 1
63.22(s), 198.04(s), 19
8.36(s), 8) Solubility: Soluble in alcohols such as methanol, ethanol, propanol, butanol, dimethyl sulfoxide, dimethyl formamide, chloroform, ethyl acetate, acetone, ethyl ether, insoluble in n-hexane and water. .

9) Color reaction: Positive for sulfuric acid, iodine, potassium permanganate, and ferric chloride reactions.

10) Thin layer chromatography: Rf value; 0.54 Adsorbent: Silica gel (manufactured by Merck & Co., Ltd., Art.

(C11,
0, MeOH) 4) Molecular formula: C, H1□○6 5) Molecular weight: 336 (measured by EI-MS method)
6) Elemental analysis: (%) (However, Ct s H1
20s·H2O) Actual value C63,14H3,86 Calculated value C64,41H3,98 7) Ultraviolet absorption spectrum: λmaxnm (E:J The ultraviolet absorption spectrum measured in methanol is as shown below.

227, 313.339.351 8) 1H-nuclear magnetic resonance spectrum: (δ: ppm)
Nuclear magnetic resonance spectrum (270
MHz) is as shown below.

5.21 (1H, s), 6.77 (1H, d), 6.
96 (1H, d), 7.11 (1H, t), 7.14 (
1H, d), 7.25 (1H, d), 7.28 (1H
, d), 7.40 (1H, d), 7.67 (1H, t)
, 9) 13C-nuclear magnetic resonance spectrum: (δ: pp
m) Nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide using tetramethylsilane as an internal standard (4
00MHz) is as shown below.

70.8. 108.2.110.1.112.0.1
13.6°115.9.116.2.118.6.11
8.9.121.0゜124.3.131.6.138
．． 3.140.9.142.5゜151.2.152.
7.156.8.189.610) Infrared absorption spectrum Niji max Cm' The infrared absorption spectrum measured by the potassium bromide (KBr) tablet method is as shown below.

3400, 1710.1611.1460.1281.
1236゜022 11) Solubility: Soluble in alcohols such as methanol, ethanol, propanol, butanol, dimethyl sulfoxide, dimethyl formamide, chloroform, ethyl acetate, acetone, ethyl ether, insoluble in n-hexane and water.

12) Color reaction: Positive for sulfuric acid, iodine, potassium permanganate, and ferric chloride reactions.

13) Thin layer chromatography: Rf value; 0.47 Adsorbent: Silica gel (manufactured by Merck & Co., Ltd., Art.

5715) Developing solvent; dichloromethane: methanol = 90:10 Indanonaphthol A is quickly converted to indanonaphthol B in an organic solvent such as chloroform, ethyl acetate, or methanol.

Indanonaphthol A and B of the present invention have various isomers. In the above formula, all of these isomers and mixtures of these isomers are represented by a single formula. Therefore, the present invention includes all of these isomers and mixtures of these isomers.

Indanonaphthol A and B of the present invention can be converted into salts according to conventional methods. Examples of such salts include salts of alkali metals such as lithium, sodium, and potassium; salts of alkaline earth metals such as calcium and barium; aluminum salts; salts of basic amino acids such as lysine and arginine; etc. I can give it to you. Preferred are pharmacologically acceptable salts.

The SANK 50190 strain of the present invention, which produces indanonaphthol A and B, is obtained by filtering seawater collected from the coast of Yonako, Shizuoka Prefecture using a membrane filter with a pore size of 0.45 μm, and applying the filter to a YM agar medium. The cells were placed on top of the celluloid, cultured at 23°C for 7 days, and then separated.

SANK, a producer of indanonaphthol A and B
The mycological properties of strain 50190 are as follows.

The size of a colony cultured on a malt extract agar medium at 24°C for 7 days was 9 mm, and the size of a colony cultured on the same medium at 18°C for 7 days was 17 mm.

The colonies are smooth, often with a swollen and sticky center, and sparsely form short aerial hyphae.

Colony color is black, dark olive-gray, with gray-white margins.

Young hyphae are transparent, but as they grow, they turn brown and the cell walls become thicker.

The width of the hyphae is 4 to 12 μm, and the width of the cell is longer than the length. In rare cases, there is a vertical bulkhead. Hyphae are branched.

Sexual reproductive organs do not form on malt extract agar, potato/carrot extract agar, or cornmeal agar.

Conidia are present in one or more rarely two locations in an undifferentiated conidiogenic mother cell.
It is formed by an extrusion method (basipetal) from the beginning.

Conidia are oval in shape, measuring 4 to 6 x 6 to 8 μm, and have a smooth surface.

Conidia detach from the conidiogenic mother cell and may swell and form a thick cell wall. Often, the conidia divide and join together in two.

Conidial germination is similar to yeast budding and does not form endospores.

As a result of comparing the above thin shapes with those of known strains, we found that
reobasidium and A11ied Ge
nera, 5 studies in Mycology
.

This bacterium was identified as a strain belonging to the genus Hormonema according to the classification described in Section 141 of [1977]. However, the definition of species in the genus Hormonema is still not clear. Therefore, this strain was classified as Hormonema sp.
SANK 50190 (Feikoken Bibori No. 11359,
FERM P-11359).

The color tone display is based on the "Color Standard" published by the Japan Color Research Institute.
I followed.

The SANK 50190 strain has been explained above, but it is well known that the properties of fungi of the genus Hormonema are not constant and can easily change naturally or artificially, so the strains that can be used in the present invention are This includes all strains that produce indanonaphthol A and B belonging to the genus Hormonema.

To obtain the novel compounds of the invention indanonaphthol A and B, the cultivation of these microorganisms is carried out in media such as those used for producing other fermentation products. Such a medium contains carbon sources, nitrogen sources, and inorganic salts that can be assimilated by microorganisms.

Generally, carbon sources include glucose, fructose, maltose, sucrose, mannitol, glycerol, dextrin, oats, rye, corn starch, potatoes, corn flour, soybean flour, cottonseed oil,
Molasses, citric acid, tartaric acid, etc. can be used alone or in combination. Generally, it varies from 1 to 10% by weight of the amount of medium.

As a nitrogen source, substances containing proteins are generally used in the fermentation process. Suitable nitrogen sources include soybean flour, bran,
These include peanut flour, cottonseed oil, cottonseed flour, casein hydrolyzate, firmamine, fishmeal, corn steep liquor, peptone, meat extract, yeast, yeast extract, malt extract, sodium nitrate, 9-ammonium nitrate, ammonium sulfate, and the like.

The nitrogen source is used singly or in combination in a range of 0.2-6% by weight of the amount of the medium.

Nutrient inorganic salts incorporated into the culture medium include sodium, ammonium, calcium, phosphate, sulfate,
These are ordinary salts from which ions such as chloride and carbonate can be obtained. It also contains trace amounts of metals such as potassium, calcium, cobalt, manganese, iron, and magnesium.

For liquid culture, silicone oil, vegetable oil, surfactant, etc. are used as antifoaming agents.

Hormonema sp.
The pH of the medium in which the SANK50190 strain is cultured to produce indanonaphthol A and B can be varied from 5.0 to 7.0.

The growth temperature of the fungus ranges from 5°C to 26°C, but growth is good in the range of 15°C to 23°C, and 18°C to 23°C is suitable for the production of indanonaphthol A and B.

Indanonaphthol A and B can be obtained by culturing aerobically, and commonly used aerobic culture methods such as solid state culture, shaking culture, aerated agitation culture, etc. can be used.

For small-scale cultivation, it is best to culture with shaking at 22°C to 23°C for several days.

Cultivation begins with a 1-2 stage seed development process in Erlenmeyer flasks. The medium for the seed development stage can contain a combination of carbon and nitrogen sources. Seed flasks are shaken in a constant temperature incubator at 23° C. for 5 days or until full growth. The grown seeds are used to inoculate a second seed medium, or production medium. If an intermediate development step is used, it is grown in essentially the same way and used in part to inoculate the production medium. The inoculated flask is shaken at a constant temperature for several days, and at the end of the incubation 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, a nutrient medium can be created in a tank.

The nutrient medium is sterilized by heating to 125° C., and after cooling, the sterile medium is inoculated with previously grown seeds. Cultivation is carried out at 22°C with aeration and stirring. This method is suitable for obtaining large amounts of compounds.

Changes over time in the amounts of indanonaphthol A and B produced over the course of culture can be measured using high performance liquid chromatography. Normally, the production of indanonaphthol A and B reaches its maximum value after 72 to 150 hours of culture.

After culturing, the liquid part in the culture solution and indanonaphthol A and B present in the bacterial bodies are separated from the bacterial bodies and other solid parts by a filtration operation or centrifugation using diatomaceous earth as a filtering agent. It is obtained by extracting and purifying indanonaphthol A and B present in the filtrate or supernatant and in the bacterial cells using their physicochemical properties.

For example, the indanonaphthols A and B present in the filtrate or supernatant may be used alone or in combination with organic solvents that are immiscible with water under acidic PH conditions, such as ethyl acetate, chloroform, ethylene chloride, methylene chloride, etc. Extraction and purification can be achieved by combination. Alternatively, as an adsorbent, for example, activated carbon or adsorption resin Amberlite
HP-20, HP-50 (manufactured by Mitsubishi Kasei Corporation), etc. are used. A liquid containing indanonaphthol A and B is passed through a layer of adsorbent as described above to adsorb and remove impurities, or after adsorbing indanonaphthol A and B, methanol water, acetone water, n- It can be obtained by elution using butanol water or the like. Also,
Indanonaphthol A and B present in the bacterial body are 5
It is obtained by extracting with 0-90% aqueous acetone or aqueous methanol and removing the organic solvent, and then performing the same extraction and purification operation as the filtrate.

Indanonaphthol A and B thus obtained
Furthermore, adsorption force ram chromatography using carriers such as silica gel and magnesium-silica gel-based Florisil, partition column chromatography using Sephadex LH-20 (manufactured by Pharmacia), and normal phase and reverse phase force chromatography. It can be purified by high performance liquid chromatography using ram.

Indanonaphthol A and B can be separated and purified by repeatedly using the above separation and purification means alone or in appropriate combinations.

Indanonaphthol A and B of the present invention are new compounds that have not been described in any literature, and exhibit antibacterial activity against Gram-positive bacteria in animals (e.g., humans, dogs, cats, rabbits, etc.), and therefore are effective against various bacterial infections. It is useful as an antibacterial agent for controlling diseases.

When indanonaphthol A and B of the present invention are used as a medicine, they can be prepared by themselves or mixed with appropriate pharmaceutically acceptable carriers, excipients, and diluents according to a conventional method, and prepared into powders, granules, tablets, capsules, etc. It can be safely administered orally or parenterally in the form of an injection. The dosage varies depending on the target disease, route of administration, frequency of administration, etc., but for example, for adults, 100 mg to 2000 mg per day.
It is preferable to administer g once or in divided doses depending on the symptoms.

(Example) Next, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these.

Example 1. Indanonaphthol B A) Cultured Hormonema sp. SANK 50190 strain was aseptically inoculated into 30 500 ml Erlenmeyer flasks containing 100 ml of a sterilized medium with the composition described below.
The cells were cultured at 22° C. for 5 days in a rotary shaking incubator at 200 rpm (7 cm radius of rotation).

Medium composition Glucose 10 g Polypeptone 5 g Yeast extract 3 g Malt extract 3 g Deionized water 1000 ml pH 5,6 B) Add Celite 545 (
(manufactured by Jones Manville Project Corporation, USA) was added and filtered to obtain a cake of bacterial cells. 80% acetone water (IL) was added to the obtained bacterial cell cake, and the mixture was extracted with stirring for 0.5 hours. The same operation was performed twice, and 2 L of the resulting extract was used to distill off acetone under reduced pressure using a rotary evaporator. Water was added to the residue to make 500 ml, the pH was adjusted to 3.0 with hydrochloric acid, and the mixture was extracted twice with 500 ml of ethyl acetate. The obtained ethyl acetate layer was washed with IL saturated brine, further dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure using a rotary evaporator to obtain 500 mg of an oil.

The obtained oil was dissolved in the same solvent and adsorbed on a column filled with 20 g of silica gel filled with methylene chloride, and then developed and eluted with the same solvent. The eluate was fractionated into 15ml portions, and the fraction Nα containing indanonaphthol B
Collect 16 to 19 and concentrate to dryness under reduced pressure to obtain 100 g of coarse yellow powder.
mg was obtained. 100 mg of yellow coarse powder was further subjected to high performance liquid chromatography (Senshu Pack ODS H-4).
251 (manufactured by Senshu Kagaku Co., Ltd.; eluent: 40% acetonitrile) to obtain 80 mg of crystalline indanonaphthol B.

Example 2. Example 1. Indanonaphthol A and BA) Cultured Hormonema sp. SANK 50190 strain. It was carried out in the same way.

B) Celite 54 was added to 2.5 L of the isolated culture solution as a filtering medium.
5 (manufactured by Jones Manville Project Corporation, USA) was added and filtered to separate the filtrate and bacterial cells. 80% acetone water I for bacterial cell division
L was added thereto, and the mixture was stirred at room temperature for 1 hour and extracted. The obtained extract was filtered and separated into bacterial cells and extraction filtrate. The acetone was distilled off under reduced pressure using a rotary evaporator, and the aqueous solution 5
00 ml was obtained. After adjusting the resulting aqueous solution to pH 3.0, it was extracted twice with 500 ml of ethyl acetate to obtain an IL ethyl acetate layer. The ethyl acetate layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure using a rotary evaporator to obtain 120 mg of an oily substance. On the other hand, 2.5L of filtrate was adjusted to pH 3.0 with hydrochloric acid,
The mixture was extracted twice with ethyl acetate I to obtain a 2 L ethyl acetate layer. The ethyl acetate layer was washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated to dryness using a rotary evaporator under reduced pressure to obtain 160 mg of an oil. A total of 280
mg of oil was obtained. The obtained oil was mixed with Sephadex LH-2050g and methylene chloride:ethyl acetate (
It was dissolved in a column filled with a 1:1) solvent mixture and adsorbed. Then, it was developed and eluted with the same solvent. The eluate was fractionated into 10 ml portions, and fractions Nα26 to 30 containing indanonaphthol A were collected and concentrated to dryness under reduced pressure to obtain 3 mg of powdered indanonaphthol A. Indanonaphthol B is fraction Nα37~
46 was eluted, and the fractions were collected and concentrated to dryness under reduced pressure to obtain 40 mg of indanonaphthol B as a yellow powder.

(Effects of the Invention) From the above, the novel compounds indanonaphthol A and B of the present invention exhibit antibacterial activity and are useful as antibacterial agents against various bacteria.

Claims (1)

[Claims] 1. Indanonaphthol A having the following physical and chemical properties
and its salt. 1) Properties of substance: yellow powder 2) Molecular formula: C_1_9H_1_2O_6 3) Molecular weight: 336 (measured by FAB-MS method) 4)
Elemental analysis: (%) Actual value C66.57H4.37 Calculated value C67.86H3.60 5) Ultraviolet absorption spectrum: λ_m_a_xnm (E^
1^%_1_c_m) The ultraviolet absorption spectrum measured in methanol is as shown below. 232,260 (sh), 333,398 6)^1H-Nuclear magnetic resonance spectrum: (δ: ppm) The nuclear magnetic resonance spectrum (500 MHz) measured in deuterated dichloromethane using tetramethylsilane as an internal standard is: It is as shown below. 5.01 (1H, s), 6.84 (1H, dd), 6.
86 (1H, d), 6.93 (1H, d), 6.94 (
1H, d), 6.95 (1H, d), 7.23 (1H,
d), 7.57 (1H, dd), 7.67 (1H, dd
), 8.64 (1H, s), 11.84 (1H, s) 7
)^1^3C-nuclear magnetic resonance spectrum: (δ:ppm)
The nuclear magnetic resonance spectrum (125.6 MHz) measured in deuterated dichloromethane using tetramethylsilane as an internal standard is as shown below. 66.60(d), 73.41(s), 114.57(
s), 116.62(d), 118.40(d), 11
8.84(d), 120.22(d), 122.82(
s), 126.62(d), 132.93(d), 13
7.73(s), 139.66(d), 140.13(
d), 151.03(s), 157.26(s), 16
3.22 (s), 198.04 (s), 198.36 (
s), 8) Solubility: Soluble in alcohols such as methanol, ethanol, propanol, butanol, dimethyl sulfoxide, dimethyl formamide, chloroform, ethyl acetate, acetone, ethyl ether, insoluble in n-hexane and water. 9) Color reaction: Positive for sulfuric acid, iodine, potassium permanganate, and ferric chloride reactions. 10) Thin layer chromatography: Rf value: 0.54 Adsorbent: Silica gel (manufactured by Merck & Co., Art. 5715) Developing solvent: dichloromethane:methanol = 90:10 2. Indanonaphthol B having the following formula and its salt . ▲There are mathematical formulas, chemical formulas, tables, etc.▼ 3. Indanonaphthol A and B producing bacteria belonging to the genus Hormonema are cultured, and claims 1 and 2 are obtained from the culture.
The method for producing indanonaphthol A and B according to claims 1 and 2, characterized in that indanonaphthol A and B described in . 4. In claim 3, the indanonaphthol A and B-producing bacteria belonging to the genus Hormonema are Hormonema sp.
FERMP-11359).