JPH1129561A - New compound am6105 and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new compound useful as an immunosuppressive agent for specifically suppressing IgE production. SOLUTION: This compound shown by the formula is obtained by culturing a bacterium capable of producing AM6105 compound belonging to the genus Pseudallescheria, such as Pseudallescheria sp M6150 strain (FERM P-16, 308) separated from soil in a paddy field in Sizuoka Prefecture in a medium and separating the compound from the culture product. Preferably the medium contains glucose, sucrose, etc., as a carbon source, a yeast essence, peptone, etc., as a nitrogen source, sodium chloride, potassium chloride, etc., as an inorganic salt, a vitamin, a liquid paraffin, etc., and the culture is carried out at a temperature of 27-30 deg.C for 2-5 days by adjusting pH to 5.0-8.0. A medicine comprising the compound as an active ingredient is used as an agent for alleviating atopic bronchial asthma, an agent for alleviating atopic dermatitis, an agent for alleviating allergic rhinitis, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel compound having an inhibitory effect on IgE antibody production, its production method and use, and a producing bacterium.

[0002]

2. Description of the Related Art Allergic reactions are classified into type I allergic reactions, type II allergic reactions, type III allergic reactions and type IV allergic reactions, depending on the mechanism of onset and the difference in symptoms. A typical allergic reaction is a type I allergic reaction which is also called an immediate allergic reaction. In the type I allergic reaction, IgE is generated by an antigen that has entered the living body, and the IgE antibody fixed to the cell membrane of mast cells in tissues or basophil cells in blood reacts with the antigen to produce the cells. Releases histamine, serotonin, and various other chemical mediators. The chemical mediators cause various tissue reactions such as contraction of smooth muscle and enhancement of vascular permeability. The pathological conditions of type I allergy include atopic bronchial asthma, atopy
Dermatitis, allergic rhinitis, hay fever, anaphylaxis, food allergies and the like.

[0003] At present, as agents for suppressing the type I allergic reaction, drugs for inhibiting the release of chemical messengers from mast cells and basophils and antagonists for chemical messengers have been developed. However, there are few known chemical mediators that specifically inhibit the release or production of chemical mediators or specific antagonists, and there are a wide variety of chemical mediators. At present, it is necessary to analyze the interaction of various chemical mediators in order to suppress the activity. Furthermore, the above agents do not suppress the production of IgE, the underlying cause of the allergic reaction, and cannot be a fundamental therapeutic agent in that sense.

Attempts have also been made to suppress IgE production with an immunosuppressant such as cyclosporin A to treat allergic diseases. However, non-specific immunosuppression is accompanied by the problem of side effects. Is difficult to overcome.

[0005]

Heretofore, there has been a demand for providing novel compounds useful as immunosuppressants that specifically inhibit IgE production.

[0006]

Means for Solving the Problems The present inventors have conducted intensive searches to find novel biologically active substances that specifically inhibit IgE antibody production from microbial metabolites. as a result,
The present inventors have found that a novel physiologically active substance having an inhibitory activity against IgE antibody production is produced from a culture solution of strain M6105 belonging to the genus Pseudallescheria, and completed the present invention.

That is, the present invention provides a compound of the formula (I)

[0008]

Embedded image

(Hereinafter, the compound of the present invention may be referred to as AM6105 or AM6105 compound). The properties of the AM6105 compound of the present invention are as follows. 1) Appearance: colorless columnar crystal 2) Distinguishing between acidic, basic and neutral: neutral substance 3) Specific rotation: [α] _D ²³ + 42.3 ° (C = 1.0,
CHCl ₃ ) 4) Molecular formula: C ₁₆ H ₂₄ O ₅ 5) Molecular weight: FAB-MS m / z 297 (M + H)
⁺ 6) Elemental analysis: Calculated (%) (relative to C ₁₆ H ₂₄ O ₅₎
C, 64.84; H, 8.16 Actual value (%) C, 64.88; H, 8.24 7) Ultraviolet absorption spectrum: terminal absorption (MeOH) 8) Infrared absorption spectrum: (KBr, cm) ^-1 ) As shown in FIG.

The main absorptions are as follows. 3469, 2
971, 2925, 1729, 1457, 1386, 1
209, 1149, 1112, 10399) ¹ H-NMR spectrum: (400 MHz, CDC
l ₃ ) As shown in FIG. 10) ¹³ C-NMR spectrum: (100 MHz, CD
Cl ₃ ) δ (ppm) 204.2 (s), 134.7
(S), 118.5 (d), 89.9 (d), 76.2
(S), 62.4 (s), 60.8 (s), 60.7
(Q), 58.7 (d), 49.2 (t), 36.2
(T), 28.3 (t), 27.2 (t), 25.9
(Q), 18.2 (q), 14.1 (q) 11) Solubility: chloroform, ethyl acetate, acetone,
It is soluble in methanol and insoluble in water.

12) Color test: The reaction with iodine vapor and the potassium permanganate decolorization reaction are positive, and the ferric chloride reaction and ninhydrin reaction are negative. 13) Thin layer chromatography: stationary phase developing solvent Rf value silica gel f S201 Hexane: acetone 0.37 (manufactured by Tokyo Kasei) (2: 1) 14) High performance liquid chromatography: separation column: Shodex C18-5A (column size) (φ4.5 × 150 mm, manufactured by Showa Denko KK) Solvent: 35% acetonitrile Flow rate: 1.0 ml / min Detection: UV 205 nm Retention time: 7.9 min The compound has the same molecular formula as the compound AM6105 of the present invention, and has similar physical properties. It is represented by the formula (II) as a compound exhibiting chemical properties, and is represented by Pseudourotiu
ovalis) [HPSigg et al., He
lv.Chim.Acta, 51: 1395 (1968)]

[0012]

Embedded image

Is known. However, ovalicin shows a negative optical rotation [HPSigg et al., Helv.
him.Acta, 51: 1395 (1968); Japanese Patent Application Laid-Open No. 3-4712],
Compound AM6105 of the present invention shows a positive optical rotation.
Therefore, the compound AM6105 of the present invention is clearly distinguished from ovalicin. The present inventors have examined the structure of the compound of the present invention in detail, and as a result, it has been found that the structure of the compound has an R configuration at the asymmetric carbon atom bonded to the methoxy group and is a diastereomer of ovalicin. Obaricin is chemically synthesized [EJCorey et al., J. Am. Chem. Soc, 107, 256 (1985)] and biosynthesis from precursors [DECane et al., Tetrahedron Le
tt., 28, 6545 (1987)], but there is no description of the compound AM6105 of the present invention. Therefore, the compound of the present invention was found to be a novel compound.

[0014] The present invention is also a method for producing AM6105, comprising culturing a microorganism belonging to the genus Pseudo Alesseria having the ability to produce the AM6105 compound, and collecting the AM6105 compound from the culture. Examples of microorganisms belonging to the genus Pseudo Alesseria having the ability to produce the AM6105 compound of the present invention include:
Although there is no particular limitation as long as it has the ability to produce the compound of the present invention, for example, the following Pseudallescheria sp. Strain M6105 (FERM)
P-16308).

The M6105 strain is a filamentous fungus isolated from paddy soil in Shizuoka Prefecture, and its bacteriological properties are as follows. 1. Growth state on each medium 1) East-phosphate / soluble starch (YpSs) agar medium Culture at 25 ° C for 7 days, colonies having a diameter of 37-37.
5 mm, and the surface has a white (1A1) felt shape. The back of the colony is yellow-white (1A2) to gray (1B
2). After culturing at 25 ° C. for 30 days, the colony spreads over the entire surface of a petri dish (90 mm), and the surface becomes felt-like. The color of the colony is light gray (1B1) to pastel gray (1C1). The color of the back of the colony is gray (1B
2) -Light gray (1B1) as the main body, and the ascidian capsule is scattered on the back surface. The growth of the colony at 37 ° C. is 23-24 mm in diameter for 7 days.

2) Potato glucose agar medium Cultured at 25 ° C. for 7 days, colonies having a diameter of 32 to 33 m.
m, the surface is fluffy to wool-like and the color is white (1A1)
It is. The back of the colony is white (1A1) to yellowish white (3
A2). When cultured at 25 ° C. for 30 days, the colony spreads over the entire surface of a Petri dish (90 mm), and the surface is felt-like wool-like and the color is white (1A1) to yellow-gray (4B2).
It is. Back color is white (1A1) to brown orange (6C3)
It is. Colony growth at 37 ° C was 19 days in diameter in 7 days.
２０20 mm.

3) Oatmeal agar medium Cultured at 25 ° C. for 7 days, the colony diameter is 37 to 39 m.
m, and the surface is felt-like and white (1A1).
The back of the colony is white (1A1). 30 at 25 ° C
When cultured for one day, the colonies spread over the entire surface of a Petri dish (90 mm), and the surface was white (1A1) to dull green (30D
4) to dark green (30F4). Ascocarps are scattered in particles over the entire back surface. The growth of the colonies at 37 ° C. is 20-21 mm in diameter for 7 days. 2. Physiological properties When cultured in a potato-glucose liquid medium, the range of pH that can grow is 4.0 to 8.0, and the optimum growth pH
Is 4.0 to 8.0. When cultivated on a potato glucose agar medium, the temperature range at which the cells can grow is 16 to 3
7 ° C, and the optimal growth temperature is 28-31 ° C.

3. Morphological characteristics under a microscope The bacterium forms sexual and asexual generations. In the sexual generation, it forms dark brown (7F6), spherical, 130-170 μm diameter amycetes. The shell wall of the ascocarp is 5-6 μm thick and is composed of dark brown (7F6), polygonal cells. The ascus is evanescent, 13-16 × 10-12 μm in size, spherical or subspherical in shape and 8-spores. The ascospores are unicellular oval and have a size of 5.5-6 × 3.5-4 μm. Some spores have germination holes at both ends, but the germination holes are unclear. The surface of the ascospores is smooth and the mature one is pale yellow (4B4) to pale orange (5C5). Asexual generation is transparent to yellowish white (3A2) and has a smooth Sedospor.
Form ium-type conidia. Conidia size is 5
8 × 2 to 4 μm. The conidium is unicellular and has various shapes such as spherical, subspherical, oval, oval, and rod-like, but a trace like a cut at the base is commonly observed.

4. Identification This fungus belongs to the ascomycetes because of the formation of the ascocarp in the sexual generation. In addition, the ascospores are single cells, smooth and small, the color is pale yellow (4B4) to pale orange (5C5), and the germination holes at both ends of the ascospores are often unclear. Belongs to the Microascus family
¹⁾ do. The morphological characteristics of the fungus are 130 sized ascocarp
~ 170 μm, size of the ascus 13-16 × 10-12
μm, ascospores are elliptical and 5.5 to 6 × 3.5 in size
４4 μm. In addition, there is a Scedosporium-type asexual generation (conidia), and the size is 5-8 × 2-4.
μm. From these characteristics, Pseudolesc
belongs to the genus heria. ^{1) 2)} Therefore, this strain was transformed into Pseudallscheria sp.
6105. In addition, this strain is FERM P-16308 by the National Institute of Biotechnology and Industrial Technology.
Has been deposited as

The color of the growth state in each medium is indicated by Ko
rnerup A. and Wanscher JH1978 "Methuen Handbook o
f Color 3rd ed. "Eyre Methuen, according to London. References 1). von Arx, JA1973. The Genera petriellidium an
d Pithoascus (Microascaceae). Persoonia 7 : 365-375 2). McGinnis, MR, Padhye, AAand Ajello, L.1970.P
seudallescheria Negroni et Fischer, 1943 and itslat
er synonym petriellidium Malloch.1970.Mycotaxon 1
4 : 94-102. A typical and preferred method for producing AM6105 according to the present invention is exemplified by a method of culturing the compound-producing bacterium belonging to the genus Pseudoalceria (for example, Pseudallescheria sp. M6105) in a suitable medium and isolating it from the culture. .

The medium used for producing the substance of the present invention is most preferably, but not limited to, shaking culture or aeration-agitation culture using a liquid medium. The medium is AM610
No particular limitation is imposed as long as the 5 producing bacteria grow and accumulate the compound in the medium. Examples of the carbon source include glucose, sucrose, dextrin, starch, glycerin, molasses, and organic acids. Etc. can be used. Examples of the nitrogen source include yeast extract, peptone, meat extract, soybean powder, cottonseed powder, gluten meal, wheat germ, corn plica, amino acids, ammonium salts, nitrates, and various other organic or inorganic substances. Nitrogen compounds are used. As the inorganic salt, sodium chloride, potassium chloride, calcium carbonate, various phosphates, magnesium salts, copper salts, cobalt salts and the like may be added. In addition, growth of bacteria and AM610
(5) Vitamins, coenzymes, etc. which promote production may be added. In particular, if the medium foams strongly, liquid paraffin, animal oil, vegetable oil, mineral oil, silicon, etc. may be added when necessary.

Conditions such as culturing temperature, culturing time, stirring speed, aeration rate, and pH of the culture solution in culturing the AM6105-producing bacteria are appropriately selected and adjusted so that the accumulated amount of AM6105 is maximized. For example, in the case of ordinary aeration and stirring culture, culture is preferably performed at a culture temperature of 27 to 30 ° C. for 2 to 5 days, and the pH of the culture solution is preferably adjusted to pH 5.0 to 8.0.

The time-dependent change in the amount of AM6105 accumulated in the culture solution over the course of the culture can be measured by the below-described production inhibitory activity or reverse phase HPLC. Usually, the production reaches its maximum in culture for 48 to 96 hours. After completion of the culture, AM6105, which accumulates mainly in the liquid part, is preferably removed from the filtrate or supernatant by removing the cells and other solid parts by filtration or centrifugation. The compound can be separated from the culture solution without removing the compound. Various methods based on its physicochemical properties can be used for separating and purifying AM6105 from the culture solution. For example, AM610 present in the filtrate or supernatant
5 can be extracted and purified by an organic solvent immiscible with water under neutral pH conditions, for example, ethyl acetate, butyl acetate, butanol, chloroform, dichloromethane, methylene chloride, ethylene chloride, etc. alone or in combination thereof. . Alternatively, for example, Diaion HP-20 (manufactured by Mitsubishi Kasei) or the like is used as an adsorbent. AM61
05 is passed through the adsorbent layer to adsorb impurities to remove it, or after adsorbing AM6105, eluted with methanol, acetone or the like to elute the compound. Can be obtained. Further, adsorption column chromatography using a carrier such as silica gel, alumina, and florisil, Sephadex LH-
Purification of AM6105 by distribution column chromatography using 20 (manufactured by Pharmacia), Toyopar HW-40 (manufactured by Tohso Corporation), and high-performance liquid chromatography using normal-phase and reverse-phase columns. Can be.

[0024] The present invention is also a medicament comprising an AM6105 compound as an active ingredient. The medicament of the present invention is usually used for AM
In addition to the 6105 compound, it can comprise a pharmaceutically acceptable carrier. As the carrier, known carriers can be used, and depending on their properties, they can be classified as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizing agents, suspending agents, coating agents, and the like. Among them, for example, lactose, corn starch, magnesium stearate and the like can be mentioned.

The medicament of the present invention is, for example, an immunosuppressant, and more specifically, an IgE production inhibitor or I.
a preventive or therapeutic agent for an immune disease based on abnormal production of gE,
Examples thereof include anti-atopic bronchial asthma agents, anti-atopic dermatitis agents, anti-allergic rhinitis agents, anti-hay fever agents, anti-anaphylaxis agents or anti-food allergic agents. Although the medicament of the present invention can be administered in various forms, for example, oral administration by tablets, capsules, granules, syrups and the like or injections (intravenous, intramuscular, subcutaneous), eye drops, suppositories, ointments, Parenteral administration by spray, lotion and the like can be mentioned.

These medicaments vary depending on symptoms, age, body weight, administration method, dosage form and the like, but usually 20 mg to 1000 mg per day can be administered to an adult.

[0027]

Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[0028]

Example 1 (1) Fermentative production of AM6105 compound Glucose 2%, soluble starch 2%, soybean powder 2%, yeast extract 0.5%, NaCl 0.25% and CaCO 2
₃ Aqueous medium (pH 6.5) 100 containing 0.35%
was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 20 minutes. M6105 strain (FERM P
-16308) was inoculated with one loopful of the slant and cultured at 28 ° C. for 3 days on a rotary shaker (200 rpm / min) to obtain a primary seed culture solution. The resulting primary seed culture was inoculated into 20 l of the same sterile medium described above in a 30 l jar fermenter. The fermenter was aerated at 20 l / min and aerated with stirring at 200 rpm and 28 ° C. for 48 hours to obtain a secondary seed culture solution.

20 l of the secondary seed culture thus obtained was further added to glucose 2%, peptone 1%, C.I. S. L. 1
%, KH ₂ PO ₄ 0.2%, MgSO ₄ .7H ₂ O.
1% and Antiform FS-028 (Dow Co.
running K. 300 containing 0.03%
Sterile medium 250 in 0 l stainless steel fermenter
0 l was inoculated. 2000l / min ventilation and 150r
The culture was performed at 28 ° C. for 72 hours under stirring at pm.

(2) Purification of AM6105 Compound Diatomaceous earth (50 kg) was added to 2500 l of the broth obtained by the above culture method, followed by filtration to obtain a filtrate (1600 l). After the obtained filtrate was adjusted to pH 7.0 with hydrochloric acid, butyl acetate (900 l) was added, and an extraction operation was performed. The butyl acetate layer was concentrated under reduced pressure to obtain about 500 ml of a syrup. Add about 5 benzene to this syrup.
00 ml was added, and about 1 liter of hexane was further added. Insolubles were removed by filtration, and the filtrate was concentrated under reduced pressure. Silica gel column (3.5 l) prepared from hexane in advance of the concentrate
After elution with 2 l of the same solvent, elution was carried out with a mixed solvent of hexane and acetone (10: 3), and 500 ml of each fraction was fractionated. For detection of the active substance in the purification process, silica gel thin layer chromatography [TLC: silica gel fS201, manufactured by Tokyo Kasei Co., Ltd .; developing system: benzene-ethyl acetate = 2: 1, Rf value 0.31 substance] was used. Using.

Fraction No. 7-No. In No. 11, the AM6105 component mainly showing an Rf value of 0.31 was eluted. Fractions containing the AM6105 component were collected and concentrated under reduced pressure. The concentrate containing the AM6105 component was further purified by a silica gel column (1.5 l) previously prepared with benzene.
The mixture was eluted with 8.2 l of the same solvent, and then eluted with 4 l of ethyl acetate. The AM6105 component was eluted in the ethyl acetate eluted fraction, and the ethyl acetate fraction was concentrated under reduced pressure. AM
The concentrate containing the 6105 component was further applied to a silica gel column (1 l), and chloroform-hexane (10: 3) was used.
Elution was carried out with a mixed solvent. Fraction No. 10-No. AM610 at 80
Five components were eluted. The fraction containing the AM6105 component was collected and concentrated under reduced pressure, and the concentrate was further purified by preparative liquid chromatography. That is, column: YM
Chromatography was performed using C-Pack ODS-A, mobile phase: 25% acetonitrile, flow rate: 50 ml / min, and fractionation was performed in 500 ml increments. Fraction No. 1 to
No. In 6 the AM6105 component was eluted. AM610
Fractions containing the five components were collected and concentrated under reduced pressure, and the concentrate was further purified by preparative liquid chromatography. That is, chromatography was performed using a column: Shodex C18-5F, a mobile phase: 20% acetonitrile, and a flow rate: 5 ml / min, and fractionation was performed in 10 ml portions. Fraction No. 130-No. The AM6105 component was eluted at 230. The fraction containing the AM6105 component was collected and concentrated under reduced pressure, and the concentrate was further purified by preparative liquid chromatography. That is, column: YMC-P
ack ODS-A, mobile phase: 20% acetonitrile,
Chromatography was performed at a flow rate of 50 ml / min.
After elution, fractionation was performed in 50 ml portions. Fraction No.
21-No. The AM6105 component was eluted in 29. A
The fraction containing the M6105 component was collected, concentrated under reduced pressure, dissolved in about 2 ml of chloroform, and then dissolved in about 20 ml of hexane.
Then, the mixture was allowed to stand at room temperature, and AM6105 was obtained as columnar crystals. The crystals are filtered through a glass filter.
A, a colorless columnar crystal purified by collecting on top and drying
M6105 (2.2 g) was obtained.

[0032]

Example 2 (1) IgE Antibody Production Inhibitory Activity of AM6105 The measurement of the IgE antibody production inhibitory activity of AM6105 obtained in Example 1 was performed as follows, and the activity was confirmed. The spleen of a 6-week-old female BALB / c mouse was aseptically removed and passed through a stainless steel mesh to remove large cell clumps, and then 10% fetal calf serum (Hyclone).
2 mM glutamine, 5 × 10 ⁻⁵ M2-mercaptoethanol, 0.2% NaHCO ₃ , penicillin G (5
0 IU / ml) and streptomycin (50 μg / m
l) containing RPMI-1640 medium (GIBCO BR
L company) to obtain a spleen cell suspension. This solution was centrifuged at 1200 rpm for 10 minutes, and then 140 mM NH ₄
Erythrocytes are destroyed with a Cl-15 mM Tris solution, PBS
_{(-) (2.7mMKCl, 1.5mMKH 2} PO 4,
0.14MNaCl, 8.1mMNa ₂ HPO ₄ · 7H
After washing twice with a solution (containing ₂ O), the anti-Thy-
T cells were killed using antibody 1 and rabbit complement and used as the source of B cells. This B cell source was washed twice with a PBS (−) solution, and adjusted to 5 × 10 ⁶ cells / ml with the above RPMI-1640 medium to obtain a B cell suspension. LPS (Difco; E.C.) is used as a B cell stimulant in this B cell suspension.
E. coli) to a concentration of 50 μg / ml,
Preculture was performed for 24 hours at 37 ° C. under 5% CO ₂ . Thereafter, the cells were collected and washed twice with a PBS (-) solution.
The cells were again cultured in the above RPMI-1640 medium at 5 × 10 ^6.
Cells / ml and add 100 μl of this cell suspension to 96
Each well (5 × 10 ⁵ cells / well) of the well plate was added thereto, and 5 μg of the above LPS and commercially available mouse IL-4 were added thereto.
(Genzyme) 100 U was added. Subsequently, 50 μl of various concentrations of AM6105 compound were added, and 200 μl
/ Well and 3% using a 5% CO ₂ incubator.
After culturing at 7 ° C. for 7 days, the amount of IgE in the medium was measured. Since the AM6105 compound is insoluble in water,
After dissolving in SO, it was diluted with the above medium. As a control, the above operation was repeated except that the AM6105 compound was not added.

The IgE level in the culture supernatant was measured by the following method. Commercially available rat anti-mouse IgE as primary antibody
2 µg / m of monoclonal antibody (Yamasa Shoyu Co., Ltd.)
and dissolved in a PBS (-) solution to obtain a 96-well EL.
The mixture was dispensed into an ISA plate (Dynatech) at a rate of 50 μl / well and kept at room temperature for 1.5 hours. Thereafter, the PBS (−) solution was discarded, and each well was washed by adding a PBS (−) solution containing 0.05% Tween 20 at a rate of 300 μl / well. This washing operation is performed 4 times.
Repeated times. After washing, a blocking solution [1% BSA (Miles; fr;
actionV) -containing PBS (-) solution]
For one day and night to perform blocking. The blocking solution was then discarded and the previously described 0.05% Tween
The washing operation with a PBS (-) solution containing 20 was repeated four times. Next, a series of sample diluents for activity measurement prepared in advance are added at a rate of 50 μl / well, and 1
Hold for hours. PBS containing 0.05% Tween 20
(−) The washing operation with the solution was repeated four times. Next, a commercially available biotin-labeled rat anti-mouse IgE monoclonal antibody (Yamasa Shoyu Co., Ltd.) was used as a secondary antibody at 2 μg / ml.
Was dissolved in a PBS (-) solution, added at 50 μl / well, and kept at room temperature for 1 hour. 0.05% Tween
After the washing operation with the n (20) -containing PBS (−) solution was repeated four times, the Horderadish Peroxidase diluted 2000-fold with the PBS (−) solution was used.
5 Avidin D (Vector, A2004)
0 μl / well was added and kept at room temperature for 1 hour. 0.05
After the washing operation with a PBS (-) solution containing 20% Tween 20 was repeated four times, an ABTS solution [0.129 g citric acid; 0.276 g Na ₂ HPO ₄ .12H ₂ O;
% H ₂ O ₂ 1 μl; 3 mg 2,2-Azino-bis
(3-ethylbenzothiazoline-6
-Sulfonic acid) diamonium
salt (Wako Pure Chemical Industries, Ltd .; 10 ml distilled water)
Add 0 μl / well, keep at room temperature for 10 minutes, and add 5% (w
(V) 50 μl / well of oxalic acid was added to stop the reaction. Then, the IgE amount was measured by measuring the absorbance at 415 nm.

The IgE antibody production inhibitory activity is determined by measuring the concentration at which the IgE antibody production is reduced to 50% of the control by the IC
The activity of AM6105 was calculated as ₅₀ (μg / ml). (2) Cytotoxicity of AM6105 compound In this study, mouse lymphoma P388D1 cells (AT
CC CCL-46) was used. Cell viability, Mic
hael C, Alley et al. [Cancer Res., 48, 5].
89-601 (1988)] and MTT [3- (4,5-dim)
ethylthiazol-2-yl) -2,5-di
[Phenyltetrazolium bromide] reagent (SIGMA Co., Ltd.) was used for the measurement. That is, 10% fetal calf serum (Hyclone), 2 mM glutamine, 5 μM 2-mercaptoethane-l, 0.2%
Sodium bicarbonate, penicillin G (50 IU / ml)
Mouse lymphoma P388D1 cells at 1 × 10 ⁵ cells / m 2 in RPMI 1640 medium (GIBCO BRL) containing streptomycin and streptomycin (50 μg / ml).
l. 100 μl of the cell suspension was placed in each well of a 96-well plate (1 × 10 ⁴ cells / well).
In addition, various concentrations of AM610 prepared using the above medium
100 μl / well of a 5 compound solution was added, and the cells were cultured at 37 ° C. for 3 days using a 5% CO ₂ incubator. Thereafter, the above MTT reagent was added to a PBS (-) solution at 1 mg / m 2.
and add 50 μl to each well,
The cells were cultured at 37 ° C. for 4 hours using a 5% CO ₂ incubator. After completion of the culture, 5
Measured at 70 nm. Since the AM6105 compound was insoluble in water, it was dissolved in DMSO and then diluted with the above medium. As a control, the above operation was repeated except that the AM6105 compound was not added. The concentration at which the cell number was reduced to 50% of the control was determined as IC ₅₀ (μg / ml).
IC ₅₀ values AM6105 were determined as. Table 1 shows the above results.

[0035]

[Table 1]

As is clear from the above results, AM61
Compound 05 was a compound with low cytotoxicity and was confirmed to have an IgE antibody production inhibitory effect at a low concentration. Therefore, the compound of the present invention is an immunosuppressant,
It is a prophylactic / therapeutic agent for an immune disease based on abnormal production of IgE, for example, an anti-atopic bronchial asthma agent, an anti-atopic dermatitis agent, an anti-allergic rhinitis agent, an anti-hay fever agent, an anti-anaphylaxis agent or It was confirmed that it could be used as an anti-food allergic agent.

(3) Acute toxicity of AM6105 compound After dissolving the AM6105 compound in DMSO, it was diluted with a physiological saline solution, and 30 mg / kg was administered once a day for 5 days.
Five CR mice (female, 5 weeks old) were intraperitoneally administered.
As a result, no abnormal symptoms were observed in the mice, and it was confirmed that the present compound was highly safe. Example 3 Oral Capsule 30 mg of AM6105 produced in Example 1 was mixed well with 170 mg of lactose, 140 mg of Cornstarch and 2 mg of magnesium stearate, and the powder 350 was mixed.
mg was placed in a gelatin capsule to make a capsule.

[0038]

Industrial Applicability The present invention provides novel compounds, and these compounds do not show general immunosuppressive action but show excellent IgE antibody production inhibitory action. Therefore, in particular, diseases caused by abnormal production of IgE, such as atopic bronchial asthma, atopic dermatitis, allergic disease
It is very useful as a therapeutic or prophylactic agent for rhinitis, hay fever, anaphylaxis, food allergies and the like, or a material for conversion to them.

[Brief description of the drawings]

FIG. 1 shows the infrared absorption spectrum of AM6105 in a potassium bromide tablet.

FIG. 2 shows a 400 MHz ¹ H-NMR spectrum of AM6105 in a chloroform solution.

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12P 17/16 C12P 17/16 // (C12P 17/16 C12R 1: 645)

Claims (5)

[Claims] 1. A compound of the formula (I) A compound represented by the formula: 2. A method for producing a compound according to claim 1, comprising culturing a microorganism belonging to the genus Pseudo-Arceria having the ability to produce the compound according to claim 1, and collecting the compound from the culture. 3. A pharmaceutical comprising the compound according to claim 1 as an active ingredient. 4. The method according to claim 1, wherein the pharmaceutical is an IgE production inhibitor or Ig.
The medicament according to claim 3, which is an agent for preventing or treating an immune disease based on abnormal production of E. 5. Pseudo Alesseria SP M
6105 strain (FERM P-16308).