JPH03240495A - New physiologically active substance cyclooctatin, production and use thereof - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula having the following physical and chemical properties. Color and property: colorless powder. Molecular formula: C20H34O3. Molecular weight: 322 (FD=MS, m/z). Melting point: 183-185 deg.C. Specific rotatory power: [alpha] =+90.6 deg.C (C=0.5, methanol). Elemental analysis (%); C 74.09, H 10.63. Solubility: soluble in dimethyl sulfoxide, methanol, acetone and ethyl acetate, insoluble in water, etc. USE:A lysophospholipase inhibitor and an immunosuppressive agent. PREPARATION:A strain such as Streptomyces melanousporafaicens MI 614-43F2 (FERM P-10,431) belonging to the genus Streptomyces is cultured.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention is directed to a novel physiologically active substance Cyclo';
atin), its production method a-3, and its uses.

1 Conventional technology 1 It has been reported that inhibitors of cell membrane enzymes have immunomodulatory effects (H, E, BtlSheBioa
active Hetabolites from Hic
roorganisms, No. 4.03-418 True, El
sevierScience Publishers
B, V., msterdam, 1989). Therefore, even substances harmful to lysophospholipase are expected to have immunomodulatory effects.

Lysophospholipase was detected in bronchial asthma patients and in the dark by Chareo℃1.
It has been reported that it is present in high concentrations in inflammatory cells such as eosinophils, and is deeply involved in inflammation and anti-1,6. [ The Journal
ofImmuno+ogy, Volume 128, 1346-1
349 pages (1982)].

As lysophospholipase inhibitors, diisopropylfluorophosphate, various surfactants, acylcarnitine, etc. have been reported [Proteins/Nucleic Acids/Enzymes, Vol. 32,
1091-1096 (1987)].

[Problems to be Solved by the Invention] Lysophospholipase inhibitors such as diisopropylfluorophosphate, various surfactants, and acylcarnitine have low specificity for lysophospholipase. therefore,
A highly specific inhibitor of lysophospholipase is desired. An object of the present invention is to provide a physiologically active substance having such highly specific lysophospholipase inhibitory activity, a method for producing the same, and uses thereof.

[Means for Solving the Problems] To summarize the present invention, the first invention of the present invention relates to a novel physiologically active substance cyclooctatine, which is a compound represented by the following general formula (■): It provides the physiologically active substance cyclooctatine, which is characterized by

The physical and chemical properties of cyclooctatine are as follows.

Physical and chemical properties of cyclooctatine (1) Color and shape: Colorless powder (2) Molecular formula "20" 3403 (3) Molecule f2i + 322 FD-MS m/z (4) Melting point: 183-185℃ 7 (5) Specific rotation Degree: [α] +90.6° (C
O65, methanol) (6) Elemental analysis count: Cnea 4.09%, mouth; 10.
63% (7) Ultraviolet absorption spectrum: 1/- Shows no specific absorption between 210 and 350 nm in ethanol solution.

(8) Infrared absorption spectrum: Shown in Figure 1 of the attached drawings.

(9) Hydrogen nuclear magnetic resonance spectrum: Shown in Figure 2 of the attached drawings.

(10) Carbon nuclear nuclear magnetic resonance spectrum: 3rd figure in the attached drawing
As shown in the figure.

(11) Solubility: dimedyl sulfoxide, methanol,
Soluble in acetone and ethyl acetate, insoluble in water.

(12) Rf value of thin layer chromatography: 0.47 Using a thin layer of silica gel (RT, 5715, manufactured by Merck & Co., Ltd.), chloroform-methanol (9:
1) was used.

The second invention of the present invention relates to a method for producing a new physiologically active substance, cyclooctatine, in which a cyclooctatine-producing bacterium belonging to the genus Streptomyces is cultured in a nutrient medium, and the above-mentioned general The method is characterized by separating and collecting the physiologically active substance cyclooctatine represented by the formula (I>).

The signature [1 octatine-producing bacteria 01 used in the present invention] is an actinomycete isolated from the soil of Zentsuji, Koyo Prefecture by the present inventors, and has a strain number of MI614-43F2. be.

The mycological properties of MI614-43F2 strain are as follows.

1. Morphology Under a microscope, strain MI614-43F2 extends aerial hyphae from branched basal hyphae to form a spiral.

Whorl techniques and sporangia are not permitted. Although it is difficult to distinguish the individual spores of this strain by electron microscopy, there is a chain of more than 10 spores at the tip of the aerial hyphae, and each spore is 0.8 to 1. OXl, approximately 0 to 1.2 microns.

Note that the surface of the spore is scalloped.

2. Regarding the description of growth state colors in various media, the standard shown in 1 below is the Color Harmony Manual of Container Corporation of America (Conta: nerCOr
Co1 of DOratlOn of All1erica
or harmony manual) was used.

(1) Sucrose/nitrate agar medium (cultured at 27°C) Colorless growth with grayish white to light gray color [3fe.

311ver ciraV ~5 fe, ^5hes
] Aerial mycelium grows and becomes moist around the 21st day of culture.

No soluble dyes are observed.

(2) Glucose-asparagine agar medium (27℃
Culture) Growth is colorless, aerial mycelia are light gray [2dc, Natur
al] ~Gray [3ih, Beige Gray ~5i
h, Lead Gray], no soluble pigment observed.

(3) Glycerin-asparagine agar medium (l5P
-Medium 5.27℃ culture) Light yellow [2ga, Colonial 'T'0110
W] ~Yellow [2j2C.

Gold], yellowish white to bright gray [3fest
+ver Grayl aerial mycelia were attached and cultured for 10 days.
Around the day, water droplets form on the aerial mycelium, and no soluble pigment is observed.

(4) Starch/Inorganic Salt Agar Medium (ISP-Medium 4)
．． Cultivated at 27°C> Colorless to pale gray [2Jle, Mustard] and bright gray [3fe, 5ilver Grayl]
~Gray [31hBeige Gray] aerial mycelia are attached and open from about 21 days after culture. No soluble dyes are observed.

(5) Tyrosine agar medium (ISP-medium 7.27℃
Culture) Light tea [3ie, Camel ~ Yellow tea [3 praise, C
On the growth of 10VeBrOWn, yellowish ash (3ac,
B15Que] ~ Ming Rui Ash [3fe, 5ilver
It grows on Gray aerial mycelium, and the soluble pigment is slightly reddish brown.

(6) Nutrient agar medium (27℃ culture) Growth is colorless, aerial mycelium is white to gray F2cb, 'IvoryTint]
It grows thickly and evenly, and no soluble pigment is observed.

(7) Yeast Mugii Agar Medium (ISP-Medium 2.
Cultured at 27°C) Colorless to pale [2gc, Bamboo to 2j!
eMustard], yellowish gray to light gray 1
:2fe, Covert Gray ~3fe, 5
It grows aerial mycelia of silver gray. No soluble dyes are observed.

(8) Oatmeal agar medium (ISP-Medium 3.2
(Cultured at 7℃) Growth is colorless, aerial mycelia are bright gray [2fe, Cover
tGray ~3fe, 5ilver Grayl
~Ash [3ih, BcigcGrab] becomes moist and dark from about 14 days after incubation with ash [3mj! , Beaver G
It becomes rayl. No soluble dyes are observed.

(9) Glycerin/IB salt agar medium (27℃1
8) Growth is light yellow [2ea, Lt Wheat]
~Yellow [21eSquash Yellow], aerial mycelia are yellowish K [2ca, LtIvory] ~gray white, and water droplets begin to form on the aerial mycelium from around 7 days after cultivation. Soluble pigments exhibit a slight yellow tinge.

(10) Starch agar medium (cultured at 27°C).Light gray [3fe, 5ilver Gray ~5
f0. ^5heS] ~ Gray [5ih, 1ead G
ray ] aerial mycelium, but it becomes moist from around 14 days after cultivation and gradually turns black. No soluble dyes are observed.

(11) Apple IQ lime agar medium (27°C culture)
The growth is colorless, and the aerial mycelium is white to grayish-white and grows in a thin layer, but gradually becomes lighter gray (3fe, 5ilverGray).
~5fe, Ashes become wet and become moist around the 21st day after Pi cultivation. No soluble dyes are observed.

(12) Se, I+/[1-su0! ! Paper strip addition base M, 2
7°Ct8 cultivation> Growth is colorless, aerial mycelia are grayish white to light gray to gray, and no soluble pigments are observed.

(13) Gelatin puncture culture In 15% simple gelatin medium (20'C culture), after culture
It starts to grow from about 1 day onwards, and thin white aerial mycelia are attached to the colorless growth. Soluble pigments have a slight brown tinge.

In glucose-peptone-gelatin medium (cultivated at 207°C), no aerial mycelia were attached to the colorless growth, and no soluble pigment was observed.

(14) Skimmed milk (cultured at 37°C) Colorless to tinge-like growth, with no aerial mycelia attached and no soluble pigments observed.

3. Physiological properties (1) Growth temperature range Glucose-asparagine agar (glucose 1%, asparagine 0.05%, K2OPO40.05%, string agar 3.0%, pH 7.0) was used at 20°C and 24°C. C1
As a result of tests at each temperature of 27°C, 30°C, 37°C, and 50'C, it grew at all temperatures except 50°C, but the optimum growth temperature seems to be around 27°C to 37°C.

(2) Liquefaction of gelatin (15% simple gelatin medium,
Culture at 20°C; glucose-peptone-gelatin medium, 2
7°C culture> In simple gelatin medium, slight liquefaction is observed at around 1'', 14 days after culture. Its effect is weak. In the glucose-peptone-gelatin 1 medium, liquefaction started around 1'' on the 5th day after culture, and even after 2 weeks, about 2/3 of the test tube was liquefied during culture. The effect is moderate to strong.

(3) The effect of starch hydrolysis (starch/inorganic salt agar medium and starch agar medium, both cultured at 27°C) is moderate.

(4) Coagulation and peptonization of skimmed milk (skimmed milk, 3
7°C culture> Coagulation is observed from about the 7th day after culture, and it is almost completed at about the 100th day, and peptonization begins. Peptonization was completed approximately 3 weeks after culture. The effect is moderate to strong.

(5) Production of melanin-like pigment (tryptone yeast broth, l5P-medium 1: peptone yeast iron agar medium, l5P-medium 6: tyrosine agar medium, l5P
-Medium 7; all cultured at 27°C) 2 The production of melanin-like pigments was negative in the above three types of media.

(6) Utilization of carbon sources (Pridham-Gotlieb agar medium, 15P-medium 9; 27°C culture) D-glucose, arabinose, D-xylose, D-fructose, sucrose, inositol, rhamnose, raffinose, D Grows using mannitol and lactose.

(7) Dissolution of malic acid lime (malic acid lime agar medium, cultured at 27°C) Dissolution of malic acid lime was observed from around the 5th day after culturing, and its effect was strong.

(8) Nitrate reduction reaction (o, peptone water containing 1% potassium nitrate, 15P-medium 8.27°C culture) is positive.

(9) Decomposition of cellulose (synthetic solution with filter paper pieces added, 27
℃ culture) is negative.

To summarize the above properties, the aerial mycelium of strain MI614-43F2 forms a spiral, and no whorls or spores are observed. The surface of the spores is warty.

In various media, the plant grows colorless or pale and grows bright gray, abundant aerial mycelia on the growing plants. From around the 21st day of culture, the water temperature of the aerial mycelia was observed to increase, and a pale ash appeared.

Soluble dyes are not found in most media. Production of melanin-like pigments is negative.

The ice-melting ability and proteolytic ability of starch are both moderate to strong. The 2,6-diatautopimelic acid contained in the cell wall was of the 1-1-1 type.

Based on these properties, the MI614-43F2 strain is a stove [
It is thought to belong to the genus Streptomyces. When searching for closely related known bacterial species, Streptomyces melanosborofascens (streptomyces
ces melanosporofactcnsInt
National Journal of Sy
StemeticBacter+olooV, vol. 19, p. 452, 1969; Bcrgey's Manua
l of Determinative Bacteri
ology, 8th edition, 772 pages.

1974), and Streptomyces hygroscopicus
s4 International Journal o
f Systematic Bacter+ology
, vol. 22, p. 307, 1972: S8, Wakem
Written by an The 八CtinOm17COte
8, vol. 2, p. 230, 1961;
Bergey's Manual of Deter
Minative Bacteriology 7
editon, p. 796, 1957).

Therefore, the M I 614-43 F 2 strain and the above two strains were actually compared and studied, and the results are shown in Table 1.

As is clear from Table 1, the MI611-43F2 strain exhibited properties extremely similar to Stryp1-Mr. melanosborofascens and Stryp1-Mrs.

However, the milk coagulation, inositol and raffinose utilization, l
5P-Soluble pigment of reddish brown produced in medium 7, yellowish ash~
Light gray aerial hyphae, these characteristics are clearly different from Strip1-Mrs. hygroscopicopinos.

Although the MI614-43F2 strain uses sucrose, which is contrary to both strains, it was considered to be extremely closely related to Streptomyces melanosborofascens. Therefore, the MI614-43F2 strain was used as Streptomyces melanosborofascens (streptomyces
ycesmelansporofaciens) M
It was identified as I 614-43 F 2.

An application was made for depositing strain MI61/4-4.3F2 to the Institute of Microbial Technology, Agency of Industrial Science and Technology, and it was deposited on February 2, 1988, as Microbiology Research Institute No. 104.31 @.

As seen in the case of other actinomycetes, the MI614-43F2 strain is susceptible to changes in its properties. For example, MI61
All strains of the genus Streptocyces that have the ability to produce cyclooctatine can be used in the method of the present invention, even if they are mutant strains (naturally occurring or induced) derived from the 4-43F2 strain, fusion products, or genetically modified products. It can be used for.

In the method of the present invention, the above-mentioned bacteria are cultured in a medium containing nutrients that are commonly used by microorganisms. As a carbon source,
Glucose, starch syrup, dex1-phosphorus, sucrose, starch, molasses, animal/vegetable oils, etc. can be used. Further, as a nitrogen source, soybean flour, wheat, wheat germ, cornstap liquor, cottonseed waste, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, etc. can be used.

In addition, it is effective to add, if necessary, inorganic salts capable of producing sodium, cobalt, chlorine, sulfuric acid, phosphoric acid, and other ions. In addition, organic and inorganic substances that aid the growth of bacteria and promote the production of the physiologically active substance cyclooctatine can be appropriately added.

As a culture method, a culture method under aerobic conditions, particularly a deep culture method is suitable. The appropriate temperature for culturing is 15 to 37°C, but in many cases, culture is carried out at around 26 to 30°C. The production of the physiologically active substance cyclooctatine varies depending on the medium and culture conditions, but it is usually 1 to 10 minutes for both shaking culture and tank culture.
Its accumulation reaches its maximum during the day. When the amount of physiologically active substance cyclooctatine accumulated in the culture reached its maximum,
The culture is stopped, and the target substance is isolated and purified from the culture solution.

When collecting cyclooctatine obtained from a culture solution according to the present invention, it can be extracted and purified using an appropriate combination of conventional separation means that take advantage of its properties. Cyclooctatine is present in both the culture solution and the bacterial cells. The culture filtrate can be extracted with a water-immiscible core solvent such as butyl acetate, or it can be eluted with aqueous methanol, aqueous acetone, etc. after being adsorbed on an organic 9 adsorbent such as P-2MG. After extracting the bacterial cells with an organic solvent such as methanol or acetone, the extract can be concentrated under reduced pressure, and further solvent extraction can be carried out in the same manner as the culture solution.

In addition to the above-mentioned methods, known methods used for collecting fat-soluble substances, such as adsorption chromatography, glue filtration chromatography, scraping from thin layer chromatography, high-performance liquid chromatography, etc., can be appropriately combined or repeated. Octatine can be isolated in pure form.

The third invention of the present invention is a lysophospholipid inhibitor and an immunosuppressant containing cyclooctatine as an active ingredient.

As shown in the test examples below, cyclooctatine strongly inhibits lysophospholipase, a cell membrane enzyme, but does not exhibit toxicity. Therefore, cyclooctatine is extremely useful as a phospholipase inhibitor and immunosuppressant.

Cyclooctatine can usually be administered orally or parenterally, such as intravenously, intravenously, or intramuscularly.

The dosage varies depending on the subject to be administered, the route of administration, etc., but it is usually 0.5 to 100m97/9/day, preferably 1/day.
~50■/Ky/day.

Preparations for administration include commonly used dosage forms. In the case of oral administration, tablets, granules, capsules, etc. molded together with common excipients such as starch are used.

In the case of parenteral administration, ordinary injections formed using physiological negative saline, solubilizers, etc. are used.

"Effects of the Invention" As explained in detail above, the present invention provides a novel physiologically active substance, cyclooctatine, which strongly inhibits lysophospholipase, and therefore can be used as a lysophospholipase inhibitor and immunosuppressant. Extremely useful.

[Example] Next, production examples and formulation examples of cyclooctatine of the present invention will be shown in Examples.

1 Example 1 As a seed medium and production site, 2.0% galactose,
Dex 1 Herin 2.0%, Glycerin 1.0%, Bacto Soyton■ (manufactured by Difuco) 1.0%, Cornstape Liquor (manufactured by Iwaki) 0.5%, Ammonium sulfate 0.2%, Calcium carbonate 0 .2%, silicone KM70 (manufactured by Shin-Etsu Chemical Co., Ltd.) Futamaura (local law) as an antifoaming agent
A J8 base containing 0.05% of a mixture of (1:1) was used. The culture medium before sterilization was adjusted to p) 17.4 before use.

The above medium containing 110- was dispensed into a 500 d square flask was sterilized at 120°C for 20 minutes, and added to Streptomyces melanosborofascens Ml 614,-43.
One to two platinum loops of slant culture of F2 strain (FERM P-10431) were inoculated and cultured for 30 days at 27°C in a rotary shaker at 180 rpm. This seed culture solution 2d
The cells were transplanted into a 500-sized Erlenmeyer flask prepared by dispensing and sterilizing the 8-110-ml sample, and cultured with shaking under the same conditions described above for 4 days.

After the culture was completed, the culture solution was filtered and separated into culture filtrate and bacterial cells.

2 Butyl acetate 39.i! was added to the culture filtrate 39 (39.i!), stirred well to extract the active ingredient, and this was S-condensed to obtain 3.28 g of brown mucilage. This mucilage was dissolved in methanol 20. Dissolve and add silica gel 60 (Merck, Art, 7734)
) and concentrated to dryness under reduced pressure. Next, this was suspended in chloroform, applied to a silica gel 60 400- column filled with chloroform in advance, and washed with chloroform. Subsequently, the active ingredient was eluted with 1 coroform:methanol (95:5) and concentrated to dryness to obtain 2.099 g of brown sticky substance. This sticky substance is mixed with methanol
Silica gel 60''159 was added and concentrated to dryness.Next, this was dissolved in chloroform:ethyl acetate:
After suspending with acetic acid (60:35:5), it was applied to a silica gel 60285d column packed with the same mixed solvent in advance,
The active ingredient was eluted with the same mixed solvent and concentrated to dryness to obtain brown powder 1.139. Next, this powder is cut in advance.
The active fraction obtained by passing through a column for high performance liquid chromatography (PLC) equilibrated with % acetonitrile (manufactured by Shiseido, Capsule 3 Pack C, 20 Φ x 250 m, flow rate 8 d/min) 8 and eluting with the above equilibrium solution. was concentrated to dryness to obtain 23.3 mg of light brown powder. Furthermore, after dissolving this powder in methanol, it was applied to a column of 200-Cef/Ftx L-Rho 20 (manufactured by Pharmacia Fine Chemicals) which had been filled with methanol in advance, and gel filtration chromatography was performed using methanol as a solvent. By concentrating the fractions to dryness, 13.6111 g of pure cyclooctatine colorless powder was obtained. Infrared absorption spectra, hydrogen nuclear magnetic resonance spectra, and carbon nuclear magnetic resonance spectra were measured using pure cyclooctatine.

Cyclooctatine was tracked during the first stage of culture and in purified culms based on the measurement of anti-lysophospholipase activity. The measurement was carried out using BiOChimiCaet Bi
ophysica ACta, Volume 369, 50~
This was carried out using a modified method of the method described on page 63 (1974).

That is, in a test tube, 4mH1-palm-1-ylglyceride [1
-3-phosphorylcholine 50μ! , 1-[14 14C Kobalumi! -yl-Glysurose 3-phosphorylcholine 2X 104 dpm, 40n+H potassium phosphate buffer <1) H7,5) 250 μA A mixed solution containing 180 μl of an aqueous solution containing one specimen was heated at 37°C for 3 minutes, and then a mixture of beef liver homogenate was added. 1 tanol extraction, DF
Partial purification was carried out using a 150 column to AE-Sephadex, and 20 μl of the phosphorylase solution was added, and the mixture was incubated at 37°C for 1 hour.
Allowed time to react.

After the reaction, isopropyl alcohol: hebutane: 0.5 N in (80: 20: 2 v/v) mixture 1-
Add and stir to stop the reaction. Then, centrifuge (30
[114C
Radioactivity ■ of cobalmitic acid was measured.

At the same time, 0 radioactivity was measured in a blind manner using only a buffer solution containing no analyte, and the lysophospholipase inhibition rate was determined by [(b-a
)/b] Calculated using Xi 00.

The 50% inhibition rate was defined as the concentration of the sample as the IC50 value.

With this quantitative method, pure cyclooctatine is 1.57 μ9
/lll1! It inhibited lysophospholipase by 550% at a concentration of 5.5%.

Example 2 30 parts by weight of cyclooctatine, 120 parts of crystalline lactose, 147 parts of crystalline cellulose and 3 parts of magnesium sulfate were tableted using a ■-type mixer to obtain tablets each weighing 300 mg.

[Test Example] Next, a test example shows that cyclooctatine has an immunosuppressive effect and does not exhibit toxicity.

Test Example 1 This example illustrates the inhibitory effect of cyclooctatine on lymphocyte blastogenesis. The test method is as follows.

For culture, 20% tallow serum, 25 mHflepesb
buffer, RPMI supplemented with 100 μg/- streptomycin and 100 units/- penicillin G
1640 medium was used. Culture was performed in a 96-well flat bottom microplate (C03T^R). For my1-diene, ribopolyzaracalide (lPS) and canavalin A (ConA') were used at a final concentration of 1600.5 μ9/d, respectively.

For the spleen cells, remove the spleen from a BALB/c mouse (ill, 25 years old), make a single cell suspension, and hyper
Red blood cells were removed and adjusted using 5hock. 2 in each well
×105 tile cells and each diluted concentration of the test compound were added to make a total of 10.2 cells, and this was cultured for 72 hours.

37 KBq [30
]-Thymidine was added and the amount taken into the cells was measured. The effectiveness was determined based on the ratio of [30]-thymidine uptake in each test compound addition group to the control (Japan Immunology Complete Edition, Immunology Experimental Procedures, Vol. 226).
7-2276).

Cyclooctatine is L, PS addition group, con
The lymphocyte priming reaction was suppressed depending on the A addition group and the 6 concentration. 50% inhibition of [31-1]-thymidine incorporation l11 degree (I05o) is 351J in LPS,
m1, Con A was 51 μg/cell.

Test Example 2 When nycrooctarine was administered intraperitoneally to mice and its toxicity was examined, it showed no toxicity even after administration of 100 my and ly.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of cyclooctatine in a potassium bromide tablet. Figure 2 shows a 400M11z hydrogen nuclear magnetic resonance spectrum of icrooctatin measured in heavy methanol. FIG. 3 shows a 100 MHz carbon nuclear magnetic resonance spectrum of cyclooctatine measured in heavy methanol.

Claims (3)

[Claims] (1) Cyclooctatine is a physiologically active substance characterized by being a compound represented by the following general formula I: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I). (2) A method for producing the physiologically active substance cyclooctatine, which comprises culturing a cyclooctatine-producing bacterium belonging to the genus Streptomyces in a nutrient medium, and collecting cyclooctatine from the culture. (3) A lysophospholipase inhibitor and immunosuppressant containing cyclooctatine as an active ingredient.