JPH0449289A - New compound apiodionen - Google Patents

Abstract

NEW MATERIAL:Apiodionen of the formula. USE:Useful as an anti-neoplastic agent showing an inhibitory effect on topoisomerases I and II in animals (e.g. human, dog, cat or rabbit) or as an antiinflammatory agent, and showing a chemiluminescence inhibition effect. PREPARATION:An apiodionen-producing bacterium belonging to the genus Apiosordaria is cultured and the objective compound is collected and obtained from the cultured material. The above-mentioned apiodionen-producing bacterium belonging to the genus Apiosordaria is Apiosordaria effusa SANK 15083 strain (Bikouken kinki no.11524, FERMP-11524) collected and isolated from soil in Ishigaki island using a conventional method. For obtaining the objective com pound, a culture medium containing a carbon source, a nitrogen source and inorganic salts fermentable by microorganisms is used. Preferable pH of the culture medium is 5.0-7.0 and bacterial growth temperature is preferably 15-37 deg.C, especially 22-28 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention provides a new compound, apiodione, which has inhibitory activity against topoisomerase I and ■, H,N-ATPase inhibitory activity, and chemiluminescence inhibitory activity.
Apiodionen) and its production method.

(Prior Art) Conventionally, as a metabolite of Penicillium italicum, Penicillium italicum has a skeleton in which a tetrahydrofuran ring and a dihydrofuran ring are bonded with a double bond.
cid has been isolated (Y, Yamamoto et al.
Chem, Pharm, Bull.

, 37(12), 3229-3235 (1989))
However, its effect is not known.

Until now, no compound has been known that has a skeleton in which a pyrrolidine ring and a dihydropyran ring are bonded via a double bond.

(Problems to be Solved by the Invention) The present inventors discovered SAN belonging to the genus Apiosordaria isolated from the soil of Ishigaki Island.
The present invention was completed by discovering that a newly synthesized compound apiodionene, which has inhibitory activity against topoisomerase I and ■, H, -ATPase inhibitory activity, and chemiluminescence inhibitory activity, is produced from a culture of strain K15083. .

(Means for Solving the Problems) Apiodioene of the present invention has the following structural formula and physical and chemical properties.

1) Structural formula 2) Properties of substance: Neutral fat-soluble 3) Melting point: 190-192°C 4) Specific optical rotation: [α] F +155.4°C (c
1.0, chloroform) 5) Molecular formula: C, 3H,, NO3 6) Molecular weight: 233 (measured by FAB-MS method) 7) Elemental analysis: (%) Actual value C67,24H6,65N 6.08 Calculated value
C66,94H6,48N 6.008) Ultraviolet absorption spectrum: λ, , axnnI (E) The ultraviolet absorption spectra measured in methanol and acidic methanol are as shown below.

222 (3700), 327 (18600), the ultraviolet absorption spectra measured in alkaline methanol are:
It is as shown below.

245 (12300), 277 (8700), 9)
Infrared absorption spectrum. ,,cm-' The infrared absorption spectrum measured by the potassium bromide (KBr) tablet method is as shown below.

3196.1715.1674.1632.1615.
1564.1452.1392.1306.1249.
1127.1097.1062.1036.1003.
809, 71910)'H-Nuclear Magnetic Resonance Svector:
(δ: ppm) The nuclear magnetic resonance spectrum (270 MHz) measured in deuterated dimethyl sulfoxide using tetramethylsilane as an internal standard is as shown below.

0.91 (3H, d, J = 7.0 Hz), 1.74 (
3Hd, J=6.5Hz), 2.72(IHm), 3
．． 62 (2H, s), 4.84 (IHt, J=5.0
Hz), 5.59 (IHdd, J=16.0 and 5.
0 Hz), 5.99 (IHm), 6.98 (11-1dd, J=10.0 and 6.0
Hz), 7.52 (IHd, J=10.0 Hz), 7
．． 75 (IH, s), 11)'''C-Nuclear magnetic resonance vector = (δ: ppm) Nuclear magnetic resonance spectrum (67,8 MHz) measured in deuterated dimethyl sulfoxide using tetramethylsilane as an internal standard is as shown below.

12.0 (q), 17.5 (q), 32.1 (q)
, 50.4 (t), 102.7 (s), 118.7 (
d), 125.7(d), 129.9(d), 119.
4(d), 166.2(s), 167.9(s), 19
7.4(s), 12) Solubility: Soluble in alcohols such as methanol, ethanol, butanol, acetone, methyl ethyl ketone, chloroform, dichloromethane, ethyl acetate, dimethyl sulfoxide, dimethyl formamide, insoluble in n-hexane and water.

13) Color reaction: Positive for sulfuric acid, iodine, and ninhydrin.

14) Thin layer chromatography m: Rf value; 0.5 Adsorbent: Silica gel plate (manufactured by Merck & Co., Ltd., Art,
5715) Developing solvent; dichloromethane:methanol = 90:10 15) High performance liquid chromatography m: Separation column; Senshu Pack ODS H-2151 (
Column size, φ6 x 150 mm, manufactured by Senshu Kagaku ■) Solvent: 30% acetonitrile-water Flow rate: 1 ml/min Wavelength: 325 nm Retention time: 7.17 minutes and 7.75 minutes The apiodionene of the present invention has species and species 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.

The SANK 1 of the present invention producing apiodionene
Strain 5083 was collected and isolated from the soil of Ishigaki Island according to conventional methods.

The mycological properties of SANK 15083 strain, which is an apiodionene producing bacterium, are as follows.

Colonies on PCA medium are widely spread and flat, with thin and buried vegetative hyphae. The color of the aerial mycelia is pale to mouse-colored, and the back side of the colony and the agar are bright flesh-colored.

The hyphae are transparent to pale yellow-gray and branched.

The diameter is 2-7 μm, and hyphae with smooth surfaces often form hyphal bundles.

There are two types of conidia. One of them is Cladorrhin
Um-shaped, monophyllidically formed, conidiophores absent or indistinct. Conidiogenic cells are cylindrical and variable in length, most commonly 6-1
2×2-3.5 μm. The conidia are transparent, unicellular, oval in shape, and their size is 2-3 x 1.5-2 μm. The walls are smooth and aggregate into a viscous spherical mass (diameter 1
2-18 μm).

The other conidia are formed singly on the holoplast, scattered on the hyphae, and have no conidiophores. It is pear-shaped, its size is 3-4 x 2.5-3 μm, and the base is road-like.
The upper part is round and the walls are smooth.

Hyphae grow better at 37°C than at 23°C, but ascus shells are not formed.

Cleistocysts are thinly covered with downy aerial hyphae on the substrate, and may be half-buried in the substrate, superficial and scattered, or in small clusters. The shape is spherical to subspherical, and the diameter is 320-
It is 560 μm in length, grayish-black in color, and covered with numerous mycelium-like hairs. The shell wall is thin, 12-20 μm, initially translucent, but later becomes dark olive-colored due to the deposition of carbonaceous material. The surface of the outer layer resembles intertwined bacterial tissue, and the cell composition is unclear. The inner layer is transparent and consists of polygonal cells with a diameter of 6-24 μm. The hairs of cleistoclastic fruits are transparent to pale yellow-gray, straight and 1.5-3.5 μm in diameter.

The ascus is four-celled and cylindrical in shape, and its size is 90-1.
00 x 14-16 μm. The upper part is wide and circular;
The apex has a thick, ill-defined ring structure and is non-amyloid.

The side threads are numerous, transparent, and consist of swollen cells 3-7 μm in diameter.

Ascospores are arranged in diagonal lines and are oval to oval in shape.
Initially 1 cell, later divided into 2 cells by the basal transverse septum.

The cells above the ascospores are broadly oval in shape, with a tractate base, measuring 18-22 x 11-17 μm, olive brown to dark olive brown in color, uniformly shaped into small spines of about 0.5 μIll. covered in.

There is one germination pore for each ascospore. It is present at the apical or apical end of the upper cell and has a diameter of 1.5 μm. The lower cells are transparent, small, and triangular. Its size is 4
-6×5-6 μm, and the surface is smooth to somewhat rough. Often collapses when mature.

As a result of comparing the above thin shapes with those of known bacterial strains, we found that Morinaga, Minoura, Uda Yo, "Japan Dutch Society Bulletin" Vol. 19,
pp. 135-148, (1978) and J, C, Kr.
ug, S., L.I., and R.S., Jeng.
Author rMYCOTAXONJ Volume 17, 533-549
It was in good agreement with Apiosoldaria ehusa (Morinaga, Minouraetoudagawa) Frug and Udagawaetjengu, described in P., (1983). Therefore,
This bacterium is known as Apiosordaria effusa, Apiosordaria effusa, Flug, Apiosordaria effusa, and Apiosordaria effusa.
(Morinaga, Minoura et tld
agaua) Krug, Udagawaet Je
ng), and the preservation number was Apiosoldaria.
Ehusa SANK 15083 (Feikoken Bacteria 11th
No. 524, FERM P-11524).

Although the SANK 15083 strain has been described above, it is well known that the properties of fungi of the genus Apiosoldaria are not constant and can easily change naturally or artificially, and thus can be used in the present invention. The bacterial strain includes all strains that produce apiodionene belonging to the genus Apiosoldaria.

In order to obtain the novel compound apiodionene of the invention, the cultivation of these microorganisms is carried out in a medium like that 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 powder, cottonseed oil, cottonseed flour, casein hydrolyzate, firmamine, fishmeal, corn steep liquor, peptone, meat extract, yeast, yeast extract, malt extract, sodium nitrate, ammonium nitrate, ammonium sulfate, etc.

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.

Apiosordaria ehusa
iaeffusa) The pH of the medium for culturing SANK 15083 strain and producing apiodionene is 5.0-7.
．． It can be changed to 0.

The growth temperature of the fungus ranges from 15°C to 37°C, but growth is good in the range of 22°C to 35°C, and 22°C to 28°C is suitable for producing apiodionene.

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

For small-scale cultivation, it is best to culture with shaking at 26°C for several days.

Cultivation is started by the seed growth step (1)-2 in an Erlenmeyer flask equipped with a baffle (water flow control wall). 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 26° C. for 7 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 26°C with aeration and stirring. This method is suitable for obtaining large amounts of compounds.

Changes over time in the amount of apiodionene produced over the course of culture can be measured using high performance liquid chromatography. Usually, apiodionene production reaches its maximum value after 72 to 150 hours of culture.

After the cultivation is completed, the liquid part of the culture solution and the apiodionene present in the bacterial cells are separated from the bacterial cells and other solid parts by filtration or centrifugation using diatomaceous earth as a filtering agent, and the filtrate or supernatant is separated. It is obtained by extracting and purifying the apiodionene present in the bacteria and the bacterial cells using its physicochemical properties. For example, apiodionene present in the filtrate or supernatant is extracted and purified using a water-immiscible organic solvent such as ethyl acetate, chloroform, ethylene chloride, methylene chloride, etc. alone or in combination under acidic pH conditions. be able to. Alternatively, as an adsorbent, for example, activated carbon or adsorption resin Amberlite XAD-2, XAD-4 (manufactured by Rohm and Haas), Diaion HP-10, HP-
20, CHP-20, HP-50 (manufactured by Mitsubishi Kasei Corporation)
etc. are used. By passing a liquid containing apiodionene through a layer of adsorbent as described above to adsorb and remove impurities, or by adsorbing apiodionene and eluting it with methanol water, acetone water, n-butanol water, etc. can get. Moreover, apiodionene present in the bacterial cells can be obtained by extracting with 50-90% aqueous acetone or aqueous methanol, removing the organic solvent, and then performing the same extraction and purification operation as the filtrate.

The apiodionene thus obtained is further subjected to adsorption column chromatography using a carrier such as silica gel or magnesium-silica gel-based Florisil, and distribution column chromatography using Sephadex LH-20 (manufactured by Pharmacia). It can also be purified by high performance liquid chromatography using normal phase or reversed phase columns.

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

The apiodionene of the present invention is a novel compound that has not been described in any literature, and exhibits inhibitory activity against topoisomerase 1 and H in animals (e.g., humans, dogs, cats, rabbits, etc.).
It is useful as an antitumor agent.

It also exhibits chemiluminescence inhibitory activity and is useful as an anti-inflammatory agent.

When the apiodione of the present invention is used as a medicine, it is prepared by itself or mixed with appropriate pharmaceutically acceptable carriers, excipients, and diluents according to a conventional method, and is prepared in the form of powder, granules, tablets, capsules, injections, etc. It can be safely administered orally or parenterally. The dosage varies depending on the target disease, route of administration, frequency of administration, etc., but for adults, for example, it is preferable to administer 10 mg to 2000 mg per day, once or in divided doses, depending on the symptoms.

(Examples) Next, the present invention will be explained in more detail with reference to Examples and Test Examples, but the present invention is not limited thereto.

Example 1. Apiodioene (small amount culture) A) Culture Apiosoldaria ehusa SANK 15083
100 m of a culture medium with the composition described below, in which the strain was aseptically sterilized]
A platinum loop was inoculated into two 500 ml Erlenmeyer flasks with baffles containing the following, and cultured for 7 days at 26° C. in a rotary shaker incubator at 200 rpm (7 Cm radius of rotation).

Using the culture solution thus obtained as a seed culture solution, a 500 ml bottle with baffles containing 100 ml of a medium with the same composition was prepared.
Inoculate 50 Erlenmeyer flasks with 3% seed culture,
Cultures were incubated at 6° C. for 7 days in a rotary shaker at 200 rpm (7 cm radius of rotation).

Medium composition Glycerin 50 g Raw potato
50 g yeast extract
5g malt extract 5.

Deionized water 1000 ml pH 6,0 B) Add Celite 545 (
(manufactured by Jones Manville Project Corporation, USA) was added and filtered, and the filtrate 4.
Obtained 5L. The obtained filtrate was adjusted to pH 2.0 with hydrochloric acid, and then extracted twice with ethyl acetate. The obtained ethyl acetate layer was washed with water, further dried over anhydrous sodium sulfate, and then concentrated to dryness using a rotary evaporator under reduced pressure to obtain 112 mg of an oily substance.

The obtained oil was dissolved in the same solvent and adsorbed onto a column filled with 10 g of silica gel filled with methylene chloride. Then, after developing with the same solvent, elution was sequentially performed with methylene chloride-ethyl acetate (1:1) and ethyl acetate. 1 eluate
Fractionate into 5 ml portions, collect fractions Nα51 to Nα70 containing apiodionene, and concentrate to dryness under reduced pressure to obtain a crude powder of 39.
．． 0 mg was obtained.

39.0 mg of the obtained crude powder was further fractionated using high performance liquid chromatography. Preparation was performed under the following preparative conditions (column, Senshu Pack 005 H-4251 (manufactured by Senshu Kagaku Co., Ltd.; eluent, 25% acetonitrile; flow rate, 4 ml/min; measurement wavelength 325 mμ). Minute peaks were collected.

The peak portion was concentrated and freeze-dried to obtain 12 mg of crystalline apiodionene.

Example 2. Apiodioene (large scale culture) A) Culture Example 1. 500 ml of medium with the same composition as
A platinum loop was inoculated into a 2 L Erlenmeyer flask with a baffle containing the following, and cultured at 26°C for 7 days in a rotary shaker incubator at 200 rpm (7 cm radius of rotation).

In two 3OL stainless steel jar fermenters,
Add 15 L each of medium with the same composition as the seed culture,
This was heat sterilized at 120°C for 30 minutes. Next, 450 ml of the above seed culture solution was added to this, and the stirring speed was automatically controlled in the range of 100-300 rpm to maintain the dissolved oxygen concentration at 5 ppm at 26°C for 3 days with an air flow rate of 15 L/min. The cells were cultured with stirring.

B) Celite 54 was added to 30 L of the isolated culture solution as a filtration medium.
5 (manufactured by Jones Manville Project Corporation, USA) was added, filtered, and separated into a filtrate and a bacterial cell section. The obtained filtrate has a pH of 1 with hydrochloric acid.
, 9, and extracted twice with 30 L of ethyl acetate. The obtained ethyl acetate layer was sequentially washed with water and saturated brine, further dried over anhydrous sodium sulfate, and then concentrated to dryness using a rotary evaporator under reduced pressure to obtain 13 g of an oil.

13 g of the obtained oil was dissolved in the same solvent and adsorbed in a column filled with 170 g of silica gel filled with methylene chloride. Next, the mixture was developed and eluted with 2 L of the same solvent, methylene chloride-ethyl acetate (1:1), and ethyl acetate in this order. The eluate was monitored by thin layer chromatography, and fractions containing apiodionene were collected and concentrated to dryness under reduced pressure to obtain 3.6 g of a crude powder. The obtained coarse powder was dissolved in a mixed solvent of methylene chloride and ethyl acetate (1:1), and the Sephadex LH-2030 was filled with the same mixed solvent.
It was adsorbed to 0g. Then, it was developed and eluted with the same solvent.

The eluate was fractionated into 15ml portions and monitored by thin layer chromatography to determine the fraction N containing apiodionene.
027-50 were collected and concentrated to dryness under reduced pressure to obtain 2.3 g of crude powder of apiodionene. The obtained crude powder was further crystallized in a mixed solvent of ethyl acetate and methanol to obtain 600 mg of apiodionene in the form of colorless needle-like crystals.

(Effects of Examples) Test Example 1. Topoisomerase enzyme activity inhibition effect Topoisomerase enzyme activity inhibition was measured according to the following conventional method.

50mM Tris-HC for topoisomerase I
I(p)17.5), 120mM KCI, 10mM
MgCL, 0.5mM DTT, 0.5mM EDT
A, 30gg/ml BSA, 1, 25 μg/m
1 unit of topoisomerase 1 (Calftby+nus) and apiodione dissolved in dimethyl sulfoxide were added to the PBR322 (DNA) reaction solution, and the mixture was allowed to react at 37°C for 15 minutes, and the electrophoresis pattern was observed using 1% agarose. . As a result, 83μg
Inhibition was observed at a concentration of /ml.

For topoisomerase ■, 50mM Tris-HC
I (pH 7,7), 120mM MCI, 10mM
MgC1z, 0.5mM DTT, 0.5+nM E
DTA, 30gg/ml BSA, 1mM ATP, l
Add 1 unit of topoisomerase (derived from HL60 cells) and apiodionene dissolved in dimethyl sulfoxide to the μg/m1P4 phage DNA reaction solution and incubate at 37°C.
After reacting for 300 minutes, the electrophoresis pattern on 0.7% agarose was observed. As a result, 250gg/ml
Inhibition was observed at a concentration of .

Test example 2. Chemiluminescence inhibition activity Leukocytes are activated by various stimuli and release mediators of inflammation, one of which is active oxygen, and drugs that suppress or eliminate the production of this are expected to have anti-inflammatory effects. be done. Active oxygen was measured using a chemiluminescence method using a Kato mat (BertholdLab).
method (Kato T. et al., K11n,
Wochenschr, 59.203 (198
1)) with some improvements.

Namely, Hartley guinea pig (male, weight 300
g), 30 ml of physiological saline added with 1% casein was injected intraperitoneally, and 17 hours later, 30 ml of physiological saline added with 10 U heparin was injected intraperitoneally again, and ascites was collected. After staining this and confirming the neutrophil content, the neutrophils were suspended in Eagle-MEM solution and adjusted to a concentration of 5×10 6 /ml. 0.2 ml of this neutrophil suspension was placed in a Piormat test tube, and then 0.1 ml of E
Add MEM solution and 0.1 ml of test drug solution and
Preincubation was performed at ℃ for 5 minutes. Next, set this on a Piormat and immediately add 0.05 ml of FMLP (5
X10-8M) and 0.05 ml of 800 gg/ml luminol solution were added, and chemiluminescence was measured for 4 minutes. In addition, the medicinal efficacy was determined by chemiluminescence (CPM/4m1n) in the control group.
) was calculated from the inhibition percentage of that in the drug-added group and expressed as ■5°.

As a result, ■5° of apiodionene is 4.0 gg/m
It was l.

(Effects of the Invention) From the above, the novel compound apiodionene of the present invention is useful as an antitumor agent or an antiinflammatory agent.

Claims (1)

[Claims] 1. Apiodionene having the following formula. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ 2. A method for producing apiodionene, which is characterized by culturing an apiodionene-producing bacterium belonging to the genus Apiosoldaria and collecting apiodionene from the culture. 3. Claim 2. In , an apiodionene-producing bacterium belonging to the genus Apiosoldaria was Apiosoldaria ehusa SANK15083 strain (Feikoken Bacterial Serial No. 11524,
FERMP-11524).