JPH05271145A - New physiological active substance bequionstain a and b, its production and its use - Google Patents

Abstract

PURPOSE:To obtain new physiological active substance bequionstain A and B having high specificity, inhibitory activity against glutathione transferase and immunomodulating action and its pharmaceutically acceptable salt. CONSTITUTION:A physiological active substance bequionstain A and B of formula I (R is COOH and H, respectively or its pharmaceutically acceptable salt such as bequionstain A of formula II and bequionstain B of formula III. The compound of formula I is obtained by culturing a bacterium (FERM P-1,384-OF12) in a nutritive medium and collecting from the culture mixture. A glutathione transferase inhibitor is obtained from bequionstain A or its pharmaceutically acceptable salt and a medical additive. An immunomodulator is obtained from bequionstain A or B or its pharmaceutically acceptable salt and a medical additive.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel bioactive substance, bequinostatin, which has anti-glutathione transferase activity and immunomodulatory activity.
n) A and B, their production and their use.

[0002]

Glutathione transferase is an enzyme localized in the cell membrane and cytoplasm. It has been reported that an inhibitor of a cell membrane enzyme has an immunomodulatory action (T. Aoyagi, “Protease inhi”).
bitor and biological cont
roll ”, Bioactive Metabolite
s from Microorganisms, eds
by M.M. E. Bushell and U. Gra
fe, pp. 403-418, ElsevierScie.
nce Publishers B.N. V. , Amste
rdam, 1989). Therefore, even an inhibitor of glutathione transferase is expected to have an immunomodulatory action. Furthermore, it has been reported that the membrane-bound glutathione transferase also acts as a leukotriene C ₄ synthase and is deeply involved in inflammation / allergic reaction [protein / nucleic acid / enzyme, Vol. 33, 156].
4 to 1573 (1988)]. Cytoplasmic glutathione transferase has been reported to be deeply involved in the resistance mechanism of anticancer drug-resistant tumor cells [Cancer and Chemotherapy, Vol. 16, 592-598 (1989)]. As glutathione transferase inhibitors, indomethacin and meclofenamic acid have been reported [Biochemical and Bi].
Ophysical Research Commun
ications, 112, 980-985 (1
983)].

[0003]

Glutathione transferase inhibitors such as indomethacin and meclophenanoic acid have low specificity for glutathione transferase. Therefore, an inhibitor having high specificity for glutathione transferase is desired. An object of the present invention is to provide a physiologically active substance having such highly specific glutathione transferase inhibitory activity and immunomodulatory activity, a method for producing the same, and a use thereof.

[0004]

SUMMARY OF THE INVENTION The present invention is summarized as follows. The first invention of the present invention is a novel bioactive substance, bequinostatin A.
And an invention relating to B, wherein the following formula (1):

[Chemical 2] Bequinostatin A, a physiologically active substance characterized by being a compound represented by the formula (wherein R represents COOH or H)
And B or a pharmaceutically acceptable salt thereof.

Among the compounds represented by the formula (1), bequinostatin A (R = COOH compound) is represented by the following formula.

[Chemical 3] The physicochemical properties of bequinostatin A are as follows. Physicochemical properties of bequinostatin A (1) Color and shape: red powder (2) Molecular formula: C ₂₈ H ₂₄ O ₉ (3) Molecular weight: 504 FAB-MS (Positi
ve) m / z 505 (M + H) ⁺ (4) Melting point: 312-314 ° C. (dec.) (5) Specific optical rotation: [α] ²⁵ _D −320.0 ° (C 0.01, chloroform: methanol = 1) 1) (6) Ultraviolet absorption spectrum: shown in FIG. 1 of the accompanying drawings. (7) Infrared absorption spectrum: shown in FIG. 2 of the accompanying drawings. (8) Hydrogen nuclear magnetic resonance spectrum: FIG. 3 of the accompanying drawings.
Shown in. (9) Carbon nuclear magnetic resonance spectrum: FIG. 4 of the accompanying drawings.
Shown in. (10) Solubility: dimethyl sulfoxide, methanol,
It is soluble in acetone and ethyl acetate, but insoluble in water. (11) Rf value of thin layer chromatography: 0.49 A thin layer of silica gel (Art. 5715 manufactured by Merck & Co., Inc.) was used, and chloroform-ethyl acetate-acetic acid (60: 35: 5) was used as a developing solvent.

Among the compounds represented by the formula (1), bequinostatin B (compound with R = H) is represented by the following formula.

[Chemical 4] The physicochemical properties of bequinostatin B are as follows. Physicochemical properties of bequinostatin B (1) Color and shape: red powder (2) Molecular formula: C ₂₇ H ₂₄ O ₇ (3) Molecular weight: 460 FAB-MS (Negati)
ve) m / z 459 (M−H) ⁻ (4) Melting point: 274 to 276 ° C. (dec.) (5) Specific optical rotation: [α] ²³ _D −250.0 ° (C 0.01, methanol) ( 6) Ultraviolet absorption spectrum: shown in FIG. 5 of the accompanying drawings. (7) Infrared absorption spectrum: shown in FIG. 6 of the accompanying drawings. (8) Hydrogen nuclear magnetic resonance spectrum: FIG. 7 of the accompanying drawings.
Shown in. (9) Carbon nuclear magnetic resonance spectrum: FIG. 8 of the accompanying drawings.
Shown in. (10) Solubility: dimethyl sulfoxide, methanol,
It is soluble in acetone and ethyl acetate, but insoluble in water. (11) Rf value of thin layer chromatography: 0.65 A thin layer of silica gel (Art. 5715 manufactured by Merck & Co., Inc.) was used, and chloroform-ethyl acetate-acetic acid (60: 35: 5) was used as a developing solvent.

Bequinostatin A and B may be in the form of their pharmaceutically acceptable salts, such salts including alkali metals such as sodium, potassium, lithium and alkaline earth metals such as calcium. And the like.

A second invention of the present invention relates to a method for producing the novel physiologically active substances bequinostatin A and B, which comprises culturing a bequinostatin-producing bacterium belonging to the genus Streptomyces in a nutrient medium, It is characterized in that the physiologically active substances bequinostatin A and B represented by the formula (1) are separated and collected. One example of the bequinostatin-producing bacterium used in the present invention is actinomycetes isolated from the soil of Suginami Ward, Tokyo by the present inventors, and MI3
There is a strain with the strain number of 84-DF12. MI
The mycological properties of the 384-DF12 strain are as follows.

1. Morphology MI384-DF12 strain extends aerial hyphae from basal hyphae branched under a microscope. Aerial aerial hyphae are usually straight, but have helical spore chains, counting spore chains of 20 or more. Pseudosporangium with a diameter of 1.5-6 microns as a characteristic morphology
To form. No limbus or sporangia are found. The spore size is about 0.5-0.6 x 0.7-0.8 microns and its surface is smooth.

2. Regarding the description of the growth state color in various media The standard shown in [] below is the color of Continer Corporation of America.
Harmony Manual (Container Corp)
"ation of America" color
Harmony manual) was used. (1) Sucrose / nitrate agar medium (27 ° C. culture) Growth was light brown [4 ng, Lt Brown] to dark tea [3 pn, Dk Brown], and aerial hyphae were not observed. Soluble dyes have a faint brownish tinge. (2) Glucose / asparagine agar medium (27 ° C. culture) Growth is light tea [3ie, Camel] to red tea [5ui,
Rosewood Brown] and aerial hyphae are not observed. Soluble dyes are slightly pink.

(3) Glycerin / asparagine agar medium (ISP-medium 5, 27 ° C. culture) On the growth of brown ash [4 ni, Spice Brown] to dark olive ash [1 po, Ebony], scattered white aerial mycelia To live. Soluble dyes are slightly pink. Developmental tone and soluble pigment is 0.05N-Na
Adds OH to give a green color, but 0.05N-HCl
Was not changed by the addition of. (4) Starch / inorganic salt agar medium (ISP-medium 4,
Culture at 27 ° C.) Brown ash [4 ni, Spice Brown] to dark brown [4
A cotton-like aerial hyphae grow on the growth of pn, Dk Brown]. Soluble dyes have a faint brownish tinge.
The color tone of development and the soluble pigment took on a green color by the addition of 0.05N-NaOH, but did not change by the addition of 0.05N-HCl.

(5) Tyrosine agar medium (ISP-medium 7, culture at 27 ° C.) The growth is grayish green tea [3 ni, Clove Brown] to dark tea [4 nl, Dk Brown], and aerial hyphae are not observed. Soluble dyes have a brownish tinge. (6) Nutrient agar medium (27 ° C. culture) Growth of light tea [3 ng, Yellow Maple]
No settling of gray taste tea [3 ni, Clove Brown] and aerial hyphae was observed. Soluble dyes have a brownish tinge. (7) Yeast-malt agar medium (ISP-medium 2, 2
Cultivation at 7 ° C. White cotton-like aerial hyphae are grown on the development of light yellow tea [2 pg, Must Gold] to light tea [2 ni, Must Brown]. No soluble dye is found.

(8) Oatmeal agar medium (ISP-
Medium 3, culture at 27 ° C) Light gray [13cb, Pearl Gray] aerial hyphae grow slightly and sparsely on the development of light pink [5ba, Shell, Pink] to light yellow tea [21g, Must Tan]. . Soluble dyes are light pink. (9) Glycerin / nitrate agar medium (27 ° C. culture) Growth is pink [6ie, Redwood] to grayish red [6]
1 g, Dk Redwood], no aerial hyphae were observed. Soluble dyes are slightly pale pink. (10) Starch agar medium (27 ° C. culture) Growth light tea [3 ng, Yellow Maple]-
Dark brown [4Pn, DkBrown] and aerial hyphae are not observed. Soluble dyes are slightly pale pink. (11) Lime malate agar medium (27 ° C. culture) Growing thin olive [1le, Olive Yellow
w], aerial hyphae do not settle, and soluble dyes are not recognized.

(12) Cellulose (synthetic solution containing filter paper pieces, cultivated at 27 ° C.) After culturing for 3 weeks, no growth was observed. (13) Gelatin stab culture (15% simple gelatin medium, 20 ° C. culture; glucose / peptone / gelatin medium, 24 ° C. culture) In the case of simple gelatin medium, the growth of light tea and aerial mycelium does not settle, and soluble dye Has a brownish tinge. In the glucose-peptone-gelatin medium, the growth is pale yellow tea, aerial hyphae do not grow, and the soluble pigment has a brownish tinge. (14) Skim milk (cultured at 37 ° C.) Growth is light tea, aerial hyphae do not grow, and soluble dyes are brown.

3. Physiological properties (1) Growth temperature range Yeast-starch agar medium (soluble starch 1.0
%, Yeast extract 0.2%, string agar 3.0%, p
H7.0), 20 ° C, 24 ° C, 27 ° C, 30 ° C,
Tested at 37 ℃ and 50 ℃, 20 ℃ and 24 ℃
It grew at ℃, 27 ℃, 30 ℃, 37 ℃, but the growth at 20 ℃ is very poor, it does not grow at 50 ℃. The optimum temperature for growth seems to be around 27 ° C to 30 ° C. (3) Liquefaction of gelatin (15% simple gelatin medium, 2
Culture at 0 ° C; glucose / peptone / gelatin medium, 27
C. Culturing) 15% simple gelatin medium did not show liquefaction, and glucose-peptone-gelatin medium showed weak liquefaction on the 21st day after culturing. Its action is weak.

(3) Hydrolysis of starch (starch
Inorganic salt agar and starch agar, both at 27 ° C
Cultivation) In any medium, water-decomposability is recognized on the 3rd day after culturing, and its action is moderate. (4) Coagulation and peptone conversion of skim milk (Skim milk, 37
C. Cultivation) Peptone formation begins about 6 days after the culture and completes around 13 days. Its action is moderate to strong. Coagulation
unacceptable.

(5) Formation of melanin-like pigment (tryptone yeast broth, ISP-medium 1; peptone yeast iron agar medium, ISP-medium 6; tyrosine agar medium, ISP-medium 7; both cultures at 27 ° C. ) Tryptone yeast broth (ISP-medium 1) and peptone yeast iron agar medium (ISP-medium 6) are positive, and tyrosine agar medium (ISP-medium 7) produces a slight melanin-like pigment. (6) Utilization of carbon source (Pridham-Gotrieve agar medium, ISP-medium 9, 27 ° C culture) L-arabinose, D-glucose, D-fructose, sucrose, inositol, and D-mannitol are used for growth. . Probably also utilizes D-xylose. Rhamnose, raffinose and lactose are not used.

(7) Dissolution of lime malate (lime malate agar medium, 27 ° C. culture) Negative. (8) Nitrate reduction reaction (0.1% potassium nitrate-containing peptone water, ISP-medium 8, 27 ° C. culture) Positive. (9) Decomposition of cellulose (synthesis solution with filter paper pieces added, 27 ° C)
Culture) Does not grow.

Summarizing the above properties, MI384-D
The F12 strain, by its morphology, elongates aerial hyphae with helix formation and a characteristic pseudosporangium. 20 spore chains
Counting more than one, the surface is smooth. In various media,
Growth is light to dark brown with pink color on certain media. Although aerial hyphae are not often observed, white aerial hyphae are established in ISP-mediums 2, 3, 4, and 5. The soluble pigment may be pinkish or brownish depending on the medium. The formation of melanin-like pigments is positive, the proteolytic activity is moderate, and the water degradability of starch is also moderate.

The 2,6-diaminopimelic acid contained in the cell wall was LL-type. From these properties, M
The I384-DF12 strain is Streptomyces ( Strep)
It is considered to belong to the group of Searching for closely related known strains revealed the following two as having the pseudospores characteristic of the MI384-DF12 strain. That is, the Streptomyces paradox ( Str
eptomyces paradocus , Inter
national Journal of System
magic Bacteriology, 36, 57
Pp. 3-576, 1986; Systematic a
nd Applied Microbiology; 8
, Pp. 61-64, 1986) and Streptomyces Vita Mino Filth (Streptomyces
Vitaminophilus , Internet
onal Journal of System
cBacterology, 36 volumes, 573-576
P., 1986; Systematic and Ap.
plied Microbiology, 8 volumes, 61-
64, 1986; International J.
individual of Systematic Bacte
riology, 33, 557-564, 1983.
Year). Among them, it was distinguished from Streptomyces vitaminophilus in vitamin requirement. M
I384-DF12 to Streptomyces sp. ( S
treptomyces sp. ) MI384-DF1
It was identified as 2.

The MI384-DF12 strain was applied for deposit to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, and on February 8, 1990, it was entrusted as Microorganism Research Institute No. 11270. 1991
Transferred to the international deposit under the Budapest Treaty on 18th of March, and the Micromachine Research Institute No. 3228 (FERM BP-322
The accession number of 8) is assigned.

The MI384-DF12 strain is likely to change its properties as seen in the case of other actinomycetes. Therefore, for example, even if it is a mutant strain (spontaneous or inducible) derived from the MI384-DF12 strain, a transfusion or a gene recombinant, all Streptomyces strains having the ability to produce bequinostatin are of the present invention. Can be used in the method.

In the method of the present invention, the above-mentioned bacterium is cultivated in a medium containing nutrients that can be utilized by ordinary microorganisms. As the carbon source, glucose, starch syrup, dextrin, sucrose, starch, molasses, animal / vegetable oil and the like can be used.
As the nitrogen source, soybean flour, wheat, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used. In addition, if necessary, sodium, cobalt,
It is useful to add inorganic salts capable of producing chlorine, sulfuric acid, phosphoric acid, and other ions. Also,
Organic and inorganic substances that support the growth of the bacterium and promote the production of the physiologically active substance bequinostatin can be appropriately added.

As the culture method, a culture method under aerobic conditions,
The submerged culture method is particularly suitable. The appropriate temperature for culturing is 15
The temperature is ˜37 ° C., but in most cases, the culture is carried out at around 26˜30 ° C. The production of the physiologically active substance bequinostatin varies depending on the medium and culture conditions, but the maximum accumulation is usually reached within 1 to 10 days in both shaking culture and tank culture. When the amount of the bioactive substance bequinostatin accumulated in the culture reaches the maximum, the culture is stopped and the target substance is isolated and purified from the culture medium.

When collecting the bequinostatin obtained from the present invention from the culture broth, it can be extracted and purified by appropriately combining ordinary separation means utilizing its properties. Bequinostatin A and B are present in both culture filtrate and cells. The culture filtrate can be extracted with a water-immiscible organic solvent such as ethyl acetate. The cells can be extracted with an organic solvent such as methanol or acetone, the extract is concentrated under reduced pressure, and solvent extraction can be performed in the same manner as for the culture filtrate.

In addition to the above-mentioned methods, known methods used for collecting fat-soluble substances, such as adsorption chromatography,
Bequinostatin A or B can be purely isolated by appropriately combining or repeating gel filtration chromatography, scraping from thin layer chromatography, high performance liquid chromatography and the like.

The pharmaceutically acceptable salt of bequinostatin A or B can be prepared by a known method, for example, a solution containing sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide and the like. It can be obtained by treating bequinostatin.

A third invention of the present invention is bequinostatin A.
Alternatively, it is a glutathiotransferase inhibitor consisting of a pharmaceutically acceptable salt thereof and a pharmaceutical additive. The fourth invention of the present invention relates to an immunomodulator comprising bequinostatin A or B or a pharmaceutically acceptable salt thereof and a pharmaceutical additive.

The pharmaceutical additives are not particularly limited, and commonly used ones can be used. The ratio of the active ingredient (bequinostatin A or B) in the composition is different depending on its dosage form and so cannot be generally stated, but it can be used in a wide range of about 0.05 to 99%, and is usually 0 for injections. .1
~ 50%, other formulations 1% ~ 60%
It is a degree. The rest is pharmaceutical additives.

Bequinostatin A strongly inhibits glutathione transferase, which is an enzyme localized in the cell membrane and cytoplasm as shown in the following test example, but it does not show toxicity. Bequinostatin A exhibits an immunostimulatory action, and bequinostatin B tends to stimulate immunity at a low concentration, and exhibits an immunosuppressive action at a certain concentration or higher. Therefore, bequinostatin A is extremely useful as a glutathione transferase inhibitor and an immunostimulant. Bequinostatin B is an immunomodulator
It is useful as (mainly an immunostimulant).

Usually, bequinostatin A or B is orally administered to warm-blooded animals including humans, or an effective amount thereof is administered parenterally such as intravenously, intracutaneously or intramuscularly to administer glutathione transferase in the living body. Inhibition and immunomodulation (immunostimulation or immunosuppression) can be performed.
Accordingly, the present invention optionally provides glutathione transferase inhibition methods and immunomodulatory methods. The dose varies depending on the administration subject, administration route, etc.
0.05-150 mg / Kg / day, 0.5-100 mg
/ Kg / day, preferably 1 to 50 mg / Kg / day.

The formulations for administration include conventional dosage forms. In the case of oral administration, tablets, granules, capsules and the like, which are molded together with pharmaceutical additives, for example, ordinary excipients such as starch, are used. In the case of parenteral administration, usual injections and the like formulated using pharmaceutical additives such as physiological saline and solubilizers are used.

[0033]

INDUSTRIAL APPLICABILITY As described above in detail, the present invention provides novel physiologically active substances bequinostatin A and B, and bequinostatin A strongly inhibits glutathione transferase. Therefore, it is extremely useful as a glutathione transferase inhibitor. Since bequinostatin A has an immunostimulatory action, as an immunostimulant, bequinostatin B stimulates immunity at a low concentration (low dose) and suppresses immunity at a certain concentration or more,
It is useful as an immunomodulator, but can usually be used as an immunostimulant.

The present invention will be described in more detail below with reference to examples and test examples.

[Examples] Next, production examples and formulation examples of bequinostatin A and B of the present invention will be shown by Examples. Example 1 As a seed medium, galactose 2.0%, dextrin 2.0%, Bacto Soyton (registered trademark) (manufactured by Difco) 1.0%, corn steep liquor (manufactured by Iwaki) 0.5%, ammonium sulfate 0 .2%, calcium carbonate 0.2%, silicon KM-70 (manufactured by Shin-Etsu Chemical Co., Ltd.) as an antifoaming agent, and soybean oil (local method) (mixing ratio 1:
1) A medium containing 0.05% was used. The medium before sterilization was adjusted to pH 7.4 before use.

The seed medium in which 110 ml was dispensed in a 500 ml Erlenmeyer flask was sterilized at 120 ° C. for 20 minutes, and 1-2 platinum loops of the slant culture of Streptomyces sp. MI384-DF12 strain (FERM P-11270) were added thereto. Was inoculated and cultivated for 3 days on a rotary shaker at 180 ° C./min at 30 ° C. for seed culture. Then, glycerin 2.0% as a production medium, Essan meat (manufactured by Ajinomoto Co., Inc.) 1.
A medium containing 5%, dipotassium hydrogen phosphate 0.1%, and cobalt chloride hexahydrate 0.0005% (pH 6.2, adjusted with IM monopotassium phosphate) was added to a 500 ml Erlenmeyer flask.
The solution was dispensed in an amount of ml, sterilized at 120 ° C. for 20 minutes, 2 ml each of the seed culture solution was transplanted, and shake-cultured at 27 ° C. for 4 days. After completion of the culture, the culture solution was filtered to separate the culture filtrate and the cells.

13 liters of ethyl acetate was added to 13 liters of the culture filtrate, well stirred to extract the active ingredient, and this was concentrated to obtain 3.29 g of a red crude substance. This crude material was dissolved in 20 ml of methanol, 20 g of silanized silica gel (manufactured by Merck & Co., Art7719) was added, and the mixture was concentrated to dryness under reduced pressure. Next, after suspending this in 40% methanol, it was applied to a column of 300 ml of silanized silica gel pre-filled with 40% methanol, and 4
Elute by a linear concentration gradient elution method from 0% methanol to 100% methanol, concentrate to dryness and red crude material 1.3.
5 g was obtained. This crude material was dissolved in 10 ml of methanol, and YMC-GEL (ODS-A60-200 / 60,
Yamamura Chemical Laboratory Co., Ltd.) was added and concentrated to dryness.

Next, after suspending this with 40% methanol,
YMC-GEL pre-filled with 40% methanol
The mixture was applied to a 200 ml column, and the active ingredient was eluted by a linear concentration gradient elution method of 40% methanol to 100% methanol and concentrated to dryness to obtain 566.5 mg of a red powder. A column for high performance liquid chromatography (HPLC) in which this powder was equilibrated with 58% acetonitrile containing 1% acetic acid in advance (Shiseido Co., Capsule Pack C)
₁₈ , 20Φ × 250 mm, flow rate 8 ml / min), eluted with the equilibrium solution, and concentrated and dried the active fraction to give 16.76 mg of red powder of pure bequinostatin A, and 5.50 mg of bequinostatin B red powder was obtained. Using pure bequinostatin A and B, ultraviolet absorption spectrum, infrared absorption spectrum, hydrogen nuclear magnetic resonance spectrum and carbon nuclear magnetic resonance spectrum were measured. These spectra are shown in Figure 1.
This is as shown in FIGS. 2, 3, 4, 5, 6, 7 and 8. Follow-up during the culture and purification steps of bequinostatin A and B was based on measurements of anti-glutathione transferase activity and immunomodulatory activity. As the method, the same methods as the methods for measuring glutathione transferase inhibitory activity and immunoregulatory activity shown in Test Examples described later were used.

Example 2 30 parts by weight of bequinostatin, 120 parts of crystalline lactose, 147 parts of crystalline cellulose and 3 parts of magnesium stearate were tabletted with a V-type mixer to give tablets of 300 mg each.

[0039]

[Test Example] The test examples below show that bequinostatin A has glutathione transferase inhibitory activity, and that bekinostatin A and B have an immunomodulatory action and show no toxicity.

Test Example 1 Bequinostatin A glutathione transferase inhibitory activity Glutathione transferase inhibitory activity was measured by Bioc.
chemical and Biophysical R
essearch Communications, 1st
Volume 12, pages 980-985 (1983). That is, in a test tube, 0.1 ml of 90 mM glutathione (reduced type), 0.05 ml of 20 mM 3,4-dichloronitrobenzene, 1.7 ml of 0.1 M potassium phosphate buffer (pH 7.4), and 0.1 m of an aqueous solution containing a sample.
After the mixed solution containing 1 was heated at 37 ° C. for 3 minutes, a DEAE-cellulose column from a rat liver homogenate and 0.05 ml of a glutathione transferase solution partially purified by ammonium sulfate salting out were added, and
The reaction was carried out at 30 ° C for 30 minutes.

After the reaction, the absorbance at 345 nm (a)
Was measured. At the same time, the blind absorbance (b) using only the buffer solution containing no sample was measured, and the glutathione transferase inhibition rate was calculated by [(ba) / b] × 100. The concentration of the sample showing a 50% inhibition rate was taken as the value of IC ₅₀ . Pure bequinostatin A was 1.40 in this assay.
Glutathione transferase was inhibited by 50% at a concentration of μg / ml.

Test Example 2 Immunomodulatory action of bequinostatin A and B The test method for the regulating effect of bequinostatin on the lymphocyte blastogenic reaction is as follows. 20% fetal bovine serum, 25 mM Hepes buffer, 100 μg for culture
RPMI 1640 medium supplemented with 1 / ml streptomycin and 100 units / ml penicillin G was used. Culture was performed on 96-well flat bottom microplates (COSTA
R). As mitogens, lipopolysaccharide (LPS) and concanavalin A (Con A) were used at final concentrations of 100 and 5 μg / ml, respectively.

Spleen cells were used in BALB / c mice (female,
> 20 weeks old) and remove the spleen to make a single cell suspension,
Red blood cells with hyper shock
Was removed and used for LPS as it was,
Nylon Fiber for T cell isolation for Con A
(Wako Pure Chemicals). 2 x 10 in each well ^Five
Add splenocytes and test compound at each dilution
The total volume was 0.2 ml, and this was cultured for 72 hours. End of culture
8 hours prior to completion, add 37 KBq / well [³H] -chi
Add a midine and measure its uptake into cells.
It was The effect is determined by the control of each test compound addition group.
Against [³[H] -Thymidine uptake ratio
(Edited by the Japanese Society of Immunology, immunological experimental procedure, 2267-2)
276).

Table 1 shows the effects of bequinostatin A and B on the lymphocyte blast transformation reaction in mice. Bequinostatin A strongly promoted the lymphocyte rejuvenation reaction by LPS. Bequinostatin B strongly stimulated lymphocyte blastogenesis by LPS at low concentrations. Also, 12.5μ
At g / ml or more, the lymphocyte blastogenic reaction by LPS and Con A was strongly suppressed.

[Table 1] Table 1 Effects of bequinostatin A and B on mouse lymphocyte blastogenesis by LPS and Con A ──────────────────────── ────── Sample Concentration index (%) (μg / ml) LPS Con A ───────────────────────────── -Control 100 100 Bequinostatin A 0.39 88.6 76.6 0.78 88.8 28.0 1.56 96.8 84.2 3.13 137.7 94.3 6.25 144.3 129 .3 12.5 222.3 115.0 Bequinostatin B 0.39 124.0 110.1 0.78 151.9 101.1 1.56 171.4 108.1 3.13 206.0 96.3 6 0.25 47.8 107.1 12.5 0.8 4.2 ────────────────────────────────

Test Example 3 Toxicity of Bequinostatin A and B Bequinostatin A and B were intraperitoneally administered to mice to examine their toxicity.
No toxicity was shown even at the mg / kg dose.

[Brief description of drawings]

FIG. 1 shows an ultraviolet absorption spectrum of a 10 μg / ml methanol solution of bequinostatin A.

FIG. 2 shows an infrared absorption spectrum of bequinostatin A in a potassium bromide tablet.

FIG. 3 shows a 400 MHz hydrogen nuclear magnetic resonance spectrum of bequinostatin A measured in heavy dimethyl sulfoxide.

FIG. 4 shows a 100 MHz nuclear magnetic resonance spectrum of bequinostatin A measured in heavy dimethyl sulfoxide.

FIG. 5 shows an ultraviolet absorption spectrum of a 10 μg / ml methanol solution of bequinostatin B.

FIG. 6 shows an infrared absorption spectrum of bequinostatin B in a potassium bromide tablet.

FIG. 7 shows a 400 MHz hydrogen nuclear magnetic resonance spectrum of bequinostatin B measured in heavy dimethyl sulfoxide.

FIG. 8 shows a 100 MHz carbon nuclear nuclear magnetic resonance spectrum of bequinostatin B measured in heavy dimethyl sulfoxide.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location // C12N 9/99 (C12P 15/00 C12R 1: 465) (72) Inventor Yasuhiko Muraoka Tokyo 3-11-2-1107 Takashimadaira, Itabashi-ku (72) Inventor Takayuki Aoyama 3-17-3, Shimo 3-chome, Kita-ku, Tokyo

Claims (4)

[Claims] 1. The following formula (1): Bequinostatin A, a physiologically active substance characterized by being a compound represented by the formula (wherein R represents COOH or H)
And B or a pharmaceutically acceptable salt thereof. 2. A method for producing the physiologically active substances bequinostatin A and B, which comprises culturing a bequinostatin-producing bacterium belonging to the genus Streptomyces in a nutrient medium and collecting bequinostatin A or B from the culture. 3. A glutathione transferase inhibitor comprising bequinostatin A or a pharmaceutically acceptable salt thereof and a pharmaceutical additive. 4. An immunomodulator comprising bequinostatin A or B or a pharmaceutically acceptable salt thereof and a pharmaceutical additive.