JPH04360855A - Catechol derivative, production thereof and use thereof - Google Patents

Abstract

PURPOSE:To provide a highly safe 4-(2-nitroethyl)-1,2-benzenediol having an excellent 5-lipoxygenase-inhibiting activity. CONSTITUTION:4-(2-Nitroethyl)-1,2-benzenediol of the formula. The compound of the formula is produced by culturing a bacterium producing the compound of the formula and belonging to the genus Streptomyces, e.g. Streptomyces SP-MA-1189 (FERM 12273) and subsequently separating the compound from the cultured product. The compound of the formula has a 5-lipoxygenase- inhibiting activity and is effective for treating and preventing allergic diseases such as bronchial asthma and various inflammations.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention has an inhibitory activity on 5-lipoxygenase, which is one of the lipoxygenase metabolic enzymes of the arachidonic acid cascade, and is capable of treating allergic diseases such as bronchial asthma and various inflammations based on this action. The present invention relates to catechol derivatives effective for prophylaxis, methods for producing the same, and pharmaceuticals containing the same as active ingredients.

0002

[Prior art] Allergic diseases such as bronchial asthma,
Many factors are involved in various types of inflammation, including histamine, serotonin, prostaglandins (PG), leukotrienes (LT), trompoxane (TX), platelet activating factors, lysolecithin, and various lymphokines. Among them, PG and LT play a particularly important role in allergic diseases and various inflammatory reactions due to their diversity in number and physiological activity. When abnormalities occur in the arachidonic acid metabolic system, overproduction or underproduction of arachidonic acid metabolites such as PG, LT, and TX occur within cells, which causes increased vascular permeability, bronchoconstriction, and promotion of platelet aggregation. Various pathological conditions such as reactions appear, and they are the cause of various inflammations and allergic diseases. Many drugs that specifically inhibit various enzymes involved in the arachidonic acid metabolic pathway have been developed as anti-inflammatory agents. For example, non-steroidal anti-inflammatory drugs such as aspirin and indomethacin are drugs that exhibit anti-inflammatory effects by inhibiting cyclooxygenase activity and, as a result, suppressing PG production. Although these are effective against inflammation involving the PG system, they do not have the effect of suppressing inflammation caused by LT. Slow reacting substances of anaphylaxis are powerful chemical mediators of bronchial asthma.
tance of anaphylaxis; SR
The existence of S-A) has been clarified. This SRS-A
is a mixture of LTC4, LTD4 and LTE4. Furthermore, LTB4 has a strong leukocyte attracting effect and a leukocyte activating effect, and its involvement in inflammation is attracting attention. Therefore, compounds that inhibit 5-lipoxygenase, which is the initial enzyme in producing these LTs from arachidonic acid, are expected to be effective in treating and preventing allergic diseases such as bronchial asthma and various types of inflammation. From this perspective, 5-
Many compounds that inhibit lipoxygenase activity have been reported.

0003

[Problem to be solved by the invention] However, the conventional 5
- Compounds reported to have lipoxygenase inhibitory activity are not fully satisfactory in terms of anti-allergic effect, anti-inflammatory effect, and safety, and are currently not commercially available as pharmaceuticals. Therefore, an object of the present invention is to provide a novel compound that has excellent 5-lipoxygenase inhibitory activity and is highly safe, and a medicine containing the same as an active ingredient.

0004

[Means for solving the problem] Therefore, the present inventors have
- We have screened a large number of microbial metabolites for lipoxygenase inhibitory activity as an indicator for screening anti-inflammatory and anti-allergic drugs, and have discovered a novel catechol derivative produced by a microorganism belonging to the genus Streptomyces. The present invention was completed based on the discovery that it has remarkable 5-lipoxygenase inhibitory, anti-inflammatory and anti-allergic effects, and is useful as a pharmaceutical.

[0005] That is, the present invention is based on the following formula (1)

0006
]

[Chemical formula 1]

4-(2-nitroethyl)- represented by
The present invention provides 1,2-benzenediol, a method for producing the same, a drug containing the same as an active ingredient, and a microorganism that produces the same.

4-(2-nitroethyl)-1 of the present invention,
2-Benzenediol [compound (1)] can be obtained by culturing a compound (1) producing bacterium belonging to the genus Streptomyces, for example.
Compound (1) can be produced by collecting it from this culture.

An example of a strain producing compound (1) is strain MA-1189, which was isolated from soil in Sendai City. M
The mycological properties of strain A-1189 are shown below.

(a) Form Cultures cultured on glucose-asparagine agar, yeast-malt agar, and oatmeal agar at 25° C. for 14 days were observed using a microscope and an electron microscope. As a result, mature spores occurred in linear chains of more than 30 spores. According to electron microscopy, the spores are cylindrical and have a size of 0.9 to 1.4×0.
It was 6-0.9 μm. The spore surface was smooth.

(b) Growth status in various media (1)
White aerial hyphae on sucrose/nitrate agar; good aerial hyphae growth; colony surface slightly purplish pink; white base medium hyphae (2) White aerial hyphae on glucose/asparagine agar; conidia are gray; surface is light brown (3) ) Pale yellow shiny colonies on glycerol-asparagine agar;
Surface white powdery or pale pink; reverse side yellowish light brown to blackish brown (4) Starch/inorganic salt agar medium White, raised colonies; surface pale pink to white; reverse side pale yellow with bluish black center (5) Tyrosine agar medium Center bluish black; peripheral pale yellow flattened colony;
The reverse side is also the same color (6) Nutrient agar medium pale yellow Colony with glossy surface; aerial mycelium is white; reverse side is pale yellow (7) Yeast/malt agar medium entirely white to purplish pink; reverse side light brown (8) Oatmeal agar medium The surface of the colony is white to purplish pink; the back surface is pale yellow. In all of the above-mentioned media, no production of diffusible pigment into the medium was observed.

(c) Physiological properties (1) It grows well in the growth temperature range of 10 to 37°C, particularly well in the range of 20 to 32°C. (2) Liquefaction of gelatin Slight liquefaction (
3) Starch hydrolysis + (4)
Litmus milk coagulation/peptonization No coagulation, slight peptonization (5) Melanin-like pigment-producing tyrosine agar medium No production Peptone/yeast iron agar medium No production (6) Sugar-assimilating L-arabinose + D-xylose + D-glucose +D -Fructose
- Sucrose + Inositol - L-Rhamnose - Raffinose
- D-Mannitol - (7) Cell wall analysis Analysis of the whole cell hydrolyzate of this strain revealed that it contained LL-diaminopimelic acid. Therefore, the cell wall of this strain was considered to be type I.

Based on the above characteristics, strain MA-1189 was identified as a bacterium belonging to the genus Streptomyces, and was designated as Microbiological Research Institute No. 12273 (FERM P-12273) by the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry.
Deposited as.

The MA-1189 strain, like other actinomycete strains, is susceptible to changes in its properties, and mutant strains can be easily obtained by artificial mutation methods using, for example, ultraviolet rays, X-rays, or various chemicals. Although possible, mutant strains obtained in this way can also be used in the present invention as long as they produce compound (1).

In order to produce compound (1) according to the present invention, the above-mentioned bacterial strain is first aerobically cultured in a medium containing a nutrient source in which a normal strain belonging to the genus Streptomyces can grow. As the nutrient source, any known nutrient source that has been conventionally used for culturing strains of the genus Streptomyces can be used. As the medium, a liquid medium such as a malt extract medium, a medium consisting of malt extract, peptone, and glucose known as a malt extract medium, or a yeast malt (YM) medium is particularly preferably used. As the culture method, various known aerobic culture methods can be used, but the shaking culture method using a liquid medium is most preferable from the viewpoint of mass production. Culture temperature is approximately 10-37
℃, and the pH of the medium is preferably neutral or slightly acidic. Compound (1) accumulates in the medium 2 to 7 days after the start of culture, so the culture is stopped and the culture solution is filtered to obtain a culture supernatant. In order to collect compound (1) from the culture supernatant thus obtained, the culture supernatant is subjected to adsorption column chromatography and then eluted with methanol. The methanol elution fraction in which 5-lipoxygenase inhibitory activity is observed is concentrated under reduced pressure and extracted with ethyl acetate. The extract is concentrated under reduced pressure and subjected to silica gel column chromatography. Next, methanol-containing chloroform is eluted with increasing methanol concentration, and the eluted fraction in which 5-lipoxygenase inhibitory activity is observed is concentrated under reduced pressure, and finally purified by high performance liquid chromatography (HPLC).

The compound (1) of the present invention exhibits a remarkable inhibitory effect on 5-lipoxygenase in the arachidonic acid cascade, and its metabolites 5(S)-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene , 5(S)-hydroxyeicosatetraenoic acid (5
-HETE). Furthermore, since the compound (1) of the present invention has extremely low toxicity in toxicity tests, pharmaceuticals containing the compound (1) of the present invention as an active ingredient can be used to treat allergic diseases such as bronchial asthma, rheumatic diseases, etc.
It is useful in the treatment and prevention of psoriasis and various other inflammations.

Pharmaceutical products containing the compound (1) of the present invention as an active ingredient can be prepared as tablets, capsules, liquids, injections, or suppositories using the compound (1) of the present invention as is or with known carriers and excipients. It can be administered orally or parenterally in the following dosage form. Although the dosage varies depending on the subject, route, symptoms, etc., for example, when administering to adults with bronchial asthma, it is usually recommended to administer about 0.1 to 50 mg/kg body weight.

[0018]

EXAMPLES Next, the present invention will be explained in detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 (Production of compound (1)) (Culture) Medium composition (YM medium) Glucose
10g peptone (Difc
o)
5 yeast extract (Difco)
3 Malt extract (Di
fco)
3 Purified water
1,000m
lpH
6.2 100 ml of YM medium having the above composition was dispensed into a 500 ml Erlenmeyer flask and sterilized in an autoclave at 121° C. for 15 minutes. A loopful of strain MA-1189 was inoculated into this sterilized medium, and cultured with shaking at 25° C. and 250 revolutions per minute for 4 days to obtain a seed culture. In order to produce compound (1), 1.5 liters of YM medium was newly dispensed into six Erlenmeyer flasks each having a capacity of 3 liters, and sterilized in an autoclave at 121°C for 15 minutes to obtain a production medium. 15 ml of the prepared seed culture solution was inoculated into each Erlenmeyer flask, and cultured with shaking at 25° C. and 250 revolutions per minute for 4 days. The above culture was carried out five times to finally obtain 45 liters of culture solution. The culture solution thus obtained was filtered by suction to separate the culture supernatant and the bacterial cells. (Purification) After applying the culture supernatant obtained above to a 7.5 liter adsorption chromatography column HP-20,
% methanol aqueous solution 25l, 50% methanol aqueous solution 2
5 liters and 25 liters of 100% methanol were eluted in this order. 5
-Lipoxygenase inhibitory activity was observed only in the 50% methanol elution group and the 100% methanol elution group, so
These eluted sections were combined and concentrated under reduced pressure to completely distill off methanol. The remaining aqueous solution was adjusted to pH 3 with hydrochloric acid, extracted three times with 5 liters of ethyl acetate, and the extracts were combined. This ethyl acetate extract was then diluted with saturated NaHCO3.
It was washed three times with 5 liters of an aqueous solution and further dehydrated by adding anhydrous Na2SO4. After dehydration, Na2SO4 was removed and ethyl acetate was distilled off under reduced pressure to obtain an oily substance. This oily substance was applied to a 500 g silica gel column and eluted by increasing the concentration of methanol in chloroform stepwise. That is, elution was performed with 5 liters each of 0%, 2%, 4%, and 6% methanol solutions. methanol 2
Since 5-lipoxygenase inhibitory activity was observed only in the % elution area, only this elution area was concentrated under reduced pressure. The obtained concentrate was purified by HPLC under the following conditions. Elution conditions; 0 minutes A=100%,
B=0% → 50 minutes A=70%,
Elute at 10 ml/min with a concentration gradient of B = 30%. As a result, 19-2
Activity was observed in the 1 minute elution area. About this fraction
Purity testing using PLC and gas chromatography confirmed that the product was almost pure, so structural analysis was performed using instrumental analysis. The results are shown below.

(1) Molecular weight mass spectrometry: 183 High resolution mass spectrometry: 183.0457 (2) Ultraviolet absorption spectrum (solvent: methanol) Figure 1 (3) Infrared absorption spectrum (coated on NaCl) Figure 2 (4) 1H- Nuclear magnetic resonance spectrum [400MHz
, solvent CD3OD, internal standard (CH3)4Si] Figure 3 (5) Soluble methanol Easily soluble acetone Easily soluble chloroform
Slightly soluble ethyl acetate Soluble n-hexane Insoluble water
Insoluble (6) From the structure and physical properties (1) to (5), the obtained compound is 4-
It was identified as (2-nitroethyl)-1,2-benzenediol.

Example 2 (5-lipoxygenase inhibitory effect) RBL-1 cells (rat basophilic
eukemia, purchased from Dainippon Pharmaceutical) 1.0 x 108
Cells were washed with 0.1M Tris-HCl buffer (pH 7.5).
Wash twice and add 0.1M Tris-HCl buffer to 1m
1, then disrupt the cells with ultrasound. The obtained cell lysate is subjected to ultracentrifugation at 100,000 xg for 90 minutes, and the supernatant is used as a 5-lipoxygenase enzyme solution. 250μl of this enzyme solution and 0.1M Tris-HCl buffer (pH 7.5)
) 1.75ml, arachidonic acid 100μM, CaCl
2 1mM 1ml, ATP (adenosine triphosphate) 1mM
and the compound of the present invention (final concentration 10 μM, 3.0 μM,
A reaction solution consisting of 1.0 μM, 0.3 μM, and 0.1 μM) was reacted at 37° C. for 10 minutes. The reaction was stopped by adding 50 μl of 1N HCl to the reaction solution, and the mixture was extracted with 6 ml of ethyl acetate. This extract was concentrated under reduced pressure, and this concentrated solution was used by the method of Amano et al. (vitamin, 59, 21
1-219 (1985)) and subjected to HPLC according to U.
Quantify 5-HETE with a V detector. The concentration of the compound (1) of the present invention that inhibits 5-lipoxygenase by 50% (IC
50) compared the production of 5-HETE with a control group,
It is expressed as the concentration of the compound (1) of the present invention when inhibiting by 50%. As a result, the IC50 of the 5-lipoxygenase inhibitory activity of compound (1) was 3.7 x 10-6M.

Example 3 (Single-dose toxicity test in mice) Compound (1) was intravenously administered once to 5-week-old ICR male mice (purchased from Japan SLC).
Death cases were observed during the day. As a result, compound (1) has 1
No deaths were observed at doses of 0 mg/kg to 200 mg/kg, and no abnormalities were observed at autopsy 14 days later. Therefore, the lethal dose of a single intravenous administration of compound (1) to mice is 200 mg or more.

[0023]

Effects of the Invention The compound (1) of the present invention has a remarkable 5-lipoxygenase inhibitory effect, is highly safe, and is effective against allergic diseases such as bronchial asthma, rheumatic diseases, psoriasis, and various other inflammations. It is useful as a treatment and prevention drug.

[Brief explanation of the drawing]

FIG. 1 is a drawing showing the ultraviolet absorption spectrum of compound (1).

FIG. 2 is a diagram showing an infrared absorption spectrum of compound (1).

FIG. 3 is a diagram showing a 1H-nuclear magnetic resonance spectrum of compound (1).

Claims (7)

[Claims] [Claim 1] 4-(2-nitroethyl)-1,2-
Benzenediol. Claim 2: 4-(2) belonging to the genus Streptomyces
-Nitroethyl)-1,2-benzenediol-producing bacteria were cultured, and 4-(2-nitroethyl)-1 was extracted from the culture.
, characterized by collecting 2-benzenediol,
Method for producing 4-(2-nitroethyl)-1,2-benzenediol. [Claim 3] 4-(2-nitroethyl)-1,2-
A 5-lipoxygenase inhibitor containing benzenediol as an active ingredient. [Claim 4] 4-(2-nitroethyl)-1,2-
An anti-inflammatory agent containing benzenediol as an active ingredient. [Claim 5] 4-(2-nitroethyl)-1,2-
An anti-allergy agent containing benzenediol as an active ingredient. Claim 6: 4-(2) belonging to the genus Streptomyces
-Nitroethyl)-1,2-benzenediol producing bacteria. [Claim 7] Streptomyces sp. M.A.
7. The 4-(2-nitroethyl)-1,2-benzenediol-producing bacterium according to claim 6, which is 4-(2-nitroethyl)-1,2-benzenediol.