JPH10313886A - Novel physiologically active substance na00226a, its production and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a novel physiologically active substance NA00226 (salt) that has phosphodiesterase inhibitory action with reduced side effect and is useful as an antiasthmatic agent and antiallergic agent. SOLUTION: This physiologically active substance NA00226A (its salt) has the following physical and chemical properties: Appearance: reddish powder; molecular weight: 403; molecular formula: C21 H25 NO7 ; solubility: soluble in lower alcohols, insoluble in hexane, petroleum ether and water, Rf value in ODS thin layer chromatography: 0.3 with the eluent of acetonitrile-water-formic acid (50:50:1); carbon nuclear magnetic resonance spectrum: δ (ppm) 188.9, 180.7, 174.7, 158.1, 149.6, 141.3, 134.8, 128.0, 119.2, 112.5, 99.3, 74.8, 71.1, 69.6, 40.7, 38.7, 35.3, 29.3, 29.2, 28.9 and 28.7; color reaction: positive to vanillin-sulfuric acid, iron chloride, iodine. The substance NA 00226A (or its salt) is obtained by culturing a microorganism such as Streptomyces sp. NA 00226 strain (FERM P16196).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a novel physiologically active substance NA00226A and its use. The compound of the present invention has a phosphodiesterase inhibitory action, for example, an anti-asthmatic drug, a bronchodilator, a therapeutic agent for bronchitis,
It is expected to be a bioactive substance used as an antiallergic drug, antiinflammatory drug, antirheumatic drug, antihypertensive drug, angina pectoris drug, arrhythmia drug, cerebral circulation metabolism improving drug, blood coagulation inhibitor, antidepressant, etc. .

[0002]

2. Description of the Related Art As a physiologically active substance, which has a phosphodiesterase inhibitory action and is used as a bronchodilator, a drug for treating angina, a drug for treating arrhythmia, a drug for improving cerebral circulation and metabolism, an antidepressant, etc. , Theophylline, amrinone and the like are known.

[0003]

However, these compounds are not satisfactory because they have strong side effects. The invention of new compounds suitable for these uses is awaited.

[0004]

Means for Solving the Problems Accordingly, the present inventors have:
As a result of various searches for metabolites of microorganisms, one strain belonging to the genus Streptomyces has a novel physiologically active substance NA00226A having a phosphodiesterase inhibitory action.
Was found to be produced. That is, the present invention relates to the following (1) to (7).

(1) Physiologically active substance NA00226A or a pharmaceutically acceptable salt thereof having the following physicochemical properties 1) Appearance; red powder 2) Molecular weight; 403 3) Molecular formula; C ₂₁ H ₂₅ NO ₇ 4) Dissolution Soluble; soluble in lower alcohols, insoluble in hexane, petroleum ether and water. 5) Rf value by ODS thin-layer chromatography; 0.3 in a developing solvent of acetonitrile-water-formic acid (50: 50: 1). 6) Ultraviolet absorption spectrum; shown in FIG. 7) Infrared absorption spectrum; FIG. 2 shows a spectrum measured with a potassium bromide tablet. 8) Hydrogen nuclear magnetic resonance spectrum; the spectrum measured in deuterated chloroform is shown in FIG. 9) Carbon nuclear magnetic resonance spectrum; FIG. 4 shows a spectrum measured in deuterated chloroform. The chemical shift values are shown below. δ (ppm) 188.9 (s), 180.7 (s), 1
74.7 (s), 158.1 (s), 149.6
(S), 141.3 (s), 134.8 (s), 12
8.0 (s), 119.2 (d), 112.5 (s),
99.3 (d), 74.8 (d), 71.1
(S), 69.6 (d), 40.7 (t), 38.
7 (t), 35.3 (t), 29.3 (q), 2
9.2 (q), 28.9 (q), 28.7 (t) 10) Color reaction; positive for vanillin-sulfuric acid, iron chloride and iodine. (2) Streptomyces
A microorganism which belongs to the genus and has the ability to produce the bioactive substance NA00226A is cultured in a medium, the bioactive substance NA00226A is produced and accumulated in the culture, and the bioactive substance NA00226A or a pharmaceutical thereof is collected. For the production of chemically acceptable salts. (3) A medicament comprising the physiologically active substance NA00226A or a pharmaceutically acceptable salt thereof as an active ingredient. (4) The medicament according to (3), which is a phosphodiesterase inhibitor.

(5) antiasthmatics, bronchodilators, bronchitis therapeutics, antiallergics, antiinflammatory, antirheumatics, antihypertensives, angina pectoris, arrhythmias, cerebral circulation metabolism improvers, The medicament according to (3), which is any one of an anticoagulant and an antidepressant. (6) Streptomyces
A microorganism belonging to the genus and having the ability to produce the physiologically active substance NA00226A. (7) Streptomyces sp. NA00226
(Streptomyces sp. NA00226)
Co., Ltd.
RM P-16196) and its mutants.

The bioactive substance NA00226A-producing bacterium having a phosphodiesterase inhibitory activity in the present invention belongs to the genus Streptomyces, and for example, Streptomyces isolated by the present inventors.
SP NA00226 (Streptomyces
sp. NA00226) strain (Accession No. FERM P-16196, National Institute of Biotechnology, Japan)
Is an example of a strain most effectively used in the present invention.

[0008] The strain belonging to the genus Streptomyces used in the present invention, like other strains of the genus Streptomyces, is liable to change its properties, and is easily mutated by, for example, an artificial mutating means using ultraviolet rays, X-rays and drugs. Any mutant strain having the ability to produce the physiologically active substance NA00226A of the present invention can be used in the present invention.

In order to produce NA00226A according to the present invention, the strain is first aerobically cultured in a medium containing nutrients that can be used by the strain. As the nutrient source, known ones conventionally used for culturing bacteria can be used.For example, as a carbon source, glucose, fructose, glycerin, sucrose, dextrin, galactose, organic acids, etc. are used alone or in combination. be able to.

As inorganic and organic nitrogen sources, ammonium chloride, ammonium sulfate, urea, ammonium nitrate,
Sodium nitrate, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soy flour, cottonseed oil scum, casamino acid, bactosoytone, soluble vegetable protein, oatmeal and the like can be used alone or in combination.

In addition, if necessary, inorganic salts such as salt, calcium carbonate, magnesium sulfate, copper sulfate, iron sulfate, zinc sulfate, manganese chloride, phosphate and the like can be added, and organic substances such as amino acids, vitamins, and nucleic acids can be added. And inorganic substances can be appropriately added. The most suitable culture method is a liquid culture method, particularly a submerged stirring culture method.
It is desirable that the culturing is performed at a culturing temperature of 20 ° C. to 45 ° C. and a pH of slightly acidic to slightly alkaline.

[0012] In liquid culture, NA00226A substance is usually produced and accumulated in the culture solution after culturing for 3 to 5 days. When the production amount in the culture solution reaches the maximum, the culture is stopped, the cells are separated from the culture solution, and the target substance is purified and isolated from the filtrate.
The purification and isolation of the substance from the filtrate generally employs the separation and purification method used to isolate the microbial metabolite from the culture filtrate.

That is, the culture solution is separated into a filtrate and cells by a usual filtration method. The obtained filtrate is used as Diaion HP-20.
(Trade name; Mitsubishi Kasei) The solution is passed through a column to adsorb the target substance, washed with water and eluted with 50% acetone water. The eluted fraction is concentrated and extracted with ethyl acetate under hydrochloric acid.

[0014] Concentration of the ethyl acetate layer under reduced pressure
Octadecylsilyl (hereinafter abbreviated as ODS) column chromatography developed with acetonitrile-formic acid is performed. About the obtained active fraction, Sephadex LH-
20 (trade name; Pharmacia Biotech) column chromatography (mobile layer: ethanol), silica gel column chromatography (mobile layer: chloroform-methanol), and ODS column chromatography (mobile layer: water-acetonitrile-formic acid) ),
To obtain NA00226A.

The physiologically active substance N obtained as described above
The physical and chemical properties of A00226A are shown below.

1) Appearance; red powder 2) Molecular weight: 403 3) Molecular formula; C ₂₁ H ₂₅ NO ₇ 4) Solubility; soluble in lower alcohol, dimethyl sulfoxide, insoluble in hexane, petroleum ether, water 5) ODS thin Rf value by layer chromatography; water-
A developing solvent of acetonitrile-formic acid (50: 50: 1) shows 0.3. 6) Ultraviolet absorption spectrum; shown in FIG. 7) Infrared absorption spectrum; FIG. 2 shows a spectrum measured with a potassium bromide tablet. 8) Hydrogen nuclear magnetic resonance spectrum; the spectrum measured in deuterated chloroform is shown in FIG. 9) Carbon nuclear magnetic resonance spectrum; FIG. 4 shows a spectrum measured in deuterated chloroform. The chemical shift values are shown below. δ (ppm) 188.9 (s), 180.7 (s), 1
74.7 (s), 158.1 (s), 149.6
(S), 141.3 (s), 134.8 (s), 12
8.0 (s), 119.2 (d), 112.5 (s),
99.3 (d), 74.8 (d), 71.1
(S), 69.6 (d), 40.7 (t), 38.
7 (t), 35.3 (t), 29.3 (q), 2
9.2 (q), 28.9 (q), 28.7 (t) 10) Color reaction; positive for vanillin sulfate, iron chloride and iodine. NK00226A and a pharmaceutically acceptable salt thereof can be produced by a known method, for example, by treating with a solution containing sodium hydroxide, potassium hydroxide, hydrochloric acid, sulfuric acid and the like.

Various methods known in the art can be applied to the preparation and administration of the composition when used as a pharmaceutical. That is, injection, oral, rectal administration and the like can be used as an administration method. The preparation may take the form of injections, powders, granules, tablets, suppositories and the like.

As long as the preparation does not adversely affect NA00226A, various adjuvants used for medicine, ie, carriers and other auxiliaries, such as stabilizers, preservatives, soothing agents, emulsifiers, etc. are required. May be used. In the preparation, the content of NA00226A can be widely varied depending on the preparation form and the like.
6A in an amount of 0.01 to 100% (weight), preferably 0.1
７０70% (by weight), with the balance consisting of carriers and other auxiliaries normally used for medicine.

The dose of NA00226A varies depending on symptoms and the like, but is about 0.01 to 800 mg per adult per day. When continuous throwing is required, it is preferable to reduce the daily consumption.

[0020]

The following describes the phosphodiesterase inhibitory action of NA00226A with reference to experimental examples. Experimental example

Preparation of bovine phosphodiesterase 50 g of bovine airway smooth muscle after sacrifice was cut into small pieces with scissors and a scalpel, suspended in a 20 mM Tris buffer (pH 7.4) containing 5 mM EDTA 2 mM, and then polytron homogenizer. To prepare a crude phosphodiesterase enzyme solution. After centrifugation at 10,000 × g for 20 minutes, the supernatant is used as a soluble phosphodiesterase crude enzyme solution. Furthermore, Q-Sepharose Fast Flow Column (trade name; Pharmacia Biotech) (100 m
To 1), a soluble phosphodiesterase crude enzyme solution is added, washed with a buffer, and eluted with a concentration gradient of 50 mM to 1 M sodium acetate. The phosphodiesterase activity fraction eluted with 0.7 M sodium acetate was used as a phosphodiesterase enzyme solution.

Measurement of phosphodiesterase activity Phosphodiesterase activity is determined by using 3 ', 5'-cyclic adenosine monophosphate (Sigma, USA, hereinafter referred to as cAMP) as a substrate and remaining after the reaction. The cAMP thus obtained was measured by high performance liquid chromatography. That is, 0.02 mg / ml cAMP, 2.5 mM dithiothreitol, 6 mM magnesium chloride, 50 mM Tris buffer (pH 8.0), 1 μl of a phosphodiesterase enzyme solution and an evaluation sample were added to a 0.3 ml plastic tube. 20 final volume with water
The volume was adjusted to 0 μl to obtain tubes having each evaluation sample concentration shown in the table below. The mixture is agitated and incubated at 37 ° C. for 60 minutes. The reaction is stopped by adding 20 μl of a 200 mM aqueous solution of tetraethylenediamine to each tube. High-performance liquid chromatography (ODS column: 3.9 mmφ × 150 mm, WA
(TERS, USA) to measure residual cAMP. Table 1 shows the phosphodiesterase inhibitory activity of the compound of the present invention measured by this method.

[0023]

[Table 1] Phosphodiesterase inhibitory activity Thus, NA00226A has a strong inhibitory effect on phosphodiesterase, and its IC ₅₀ value is 2.2.
× 10 ^-5 M.

[0024]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be described below, but these are merely examples and do not limit the present invention at all, and various modifications are possible.

Production Method In a 500 ml Erlenmeyer flask for rotary shaker, glycerin 2.0%, glucose 1.0%, soybean powder 0.5
%, Peptone 0.3%, yeast extract 0.5%, calcium carbonate 0.2%, dipotassium phosphate 0.05%, magnesium sulfate 0.05%, 100 ml of a medium (pH 7.0) was dispensed. The solution was autoclaved at 20 ° C. for 20 minutes.
This was inoculated with one platinum loop of NA00226 strain (FERM-16196, National Institute of Bioscience and Biotechnology).
Shaking culture was performed for 2 days at 220 ° C. at 220 ° C. to obtain a seed culture. 500ml for rotary shaker
1.0% glucose, galactose in an Erlenmeyer flask.
0%, dextrin 2.0%, corn steep liquor 1.0%, pharmaceutical media 1.0%, magnesium sulfate 0.05% cobalt chloride 0.0001%, calcium carbonate 0.2% medium (pH 7.0) 100 ml was dispensed, and 1 ml of the above seed culture solution was transplanted into a flask autoclaved at 120 ° C. for 20 minutes and cultured with shaking at 25 ° C. and 220 rpm for 5 days.

The obtained culture (40 L) was separated into a filtrate and cells by a usual filtration method. The obtained filtrate was adsorbed on a Diaion HP-20 column (6 L), and water (9 L) was used.
It was washed and eluted with 50% acetone water (8 L). After the eluted fraction was distilled off with acetone, it was extracted with ethyl acetate under acidic conditions of hydrochloric acid (pH 3). The vacuum concentrate of the ethyl acetate layer (1
1.28 g), water-acetonitrile-formic acid (2
ODS column chromatography (φ5 × 25 cm) developed at 5: 75: 1 to 75: 25: 1) was performed. The active fraction eluted at an acetonitrile concentration of 70 to 75% was concentrated to obtain a concentrated dried product (1.82 g). This concentrated and dried product is subjected to LH-20 column chromatography (φ3.5).
× 45 cm, mobile phase: ethanol) to obtain an active fraction (0.519 g), which was further subjected to silica gel column chromatography (mobile phase: chloroform-methanol (1
00: 1 to 100: 40)) and the active fraction (0.08
2 g) were obtained. Furthermore, purification was performed by ODS column chromatography (φ2.5 × 42 cm, water-acetonitrile-formic acid (30: 70: 1 to 60: 40: 1)) to obtain NA00226A (67 mg). Using purified NA00226A, appearance, molecular weight, solubility, O
The Rf value, ultraviolet absorption spectrum, infrared absorption spectrum, 1H-NMR spectrum, and 13C-NMR spectrum were measured by DS thin-layer chromatography. NA00
The physicochemical properties of 226A were as described above.

[0027]

As is clear from the above, the physiologically active substance NA00226A of the present invention or a pharmaceutically acceptable salt thereof has a phosphodiesterase inhibitory action, and is used for, for example, an anti-asthmatic drug, a bronchodilator, a treatment for bronchitis. It can be expected as an active ingredient such as a drug, an antiallergic drug, an antiinflammatory drug, an antirheumatic drug, an antihypertensive drug, a drug for treating angina pectoris, a drug for treating arrhythmia, a drug for treating cerebral circulation and metabolism, an anticoagulant, and an antidepressant.

[Brief description of the drawings]

FIG. 1. Ultraviolet absorption spectrum of NA00226A measured in methanol

FIG. 2 Infrared absorption spectrum measured with potassium bromide tablet of NA00226A

FIG. 3. Hydrogen nuclear magnetic resonance spectrum of NA00226A measured in deuterated chloroform

FIG. 4. Carbon nuclear magnetic resonance spectrum of NA00226A measured in deuterated chloroform

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 35/74 ABG A61K 35/74 ABGF ABQ ABQF ABS ABSF ABU ABUF ACB ACBF ACD ACDF ACF ACFF AED 17/00/0007 C C12N 1/20 C12N 1/20 A // (C12P 1/06 C12R 1: 465) (C12N 1/20 C12R 1: 465)

Claims (7)

[Claims] 1. A physiologically active substance N having the following physicochemical properties:
A00226A or pharmaceutically acceptable salt thereof 1) Appearance; red powder 2) Molecular weight; 403 3) Molecular formula; C ₂₁ H ₂₅ NO ₇ 4) Solubility; soluble in lower alcohol, insoluble in hexane, petroleum ether, water . 5) Rf value by ODS thin-layer chromatography; 0.3 in a developing solvent of acetonitrile-water-formic acid (50: 50: 1). 6) Ultraviolet absorption spectrum; shown in FIG. 7) Infrared absorption spectrum; FIG. 2 shows a spectrum measured with a potassium bromide tablet. 8) Hydrogen nuclear magnetic resonance spectrum; the spectrum measured in deuterated chloroform is shown in FIG. 9) Carbon nuclear magnetic resonance spectrum; FIG. 4 shows a spectrum measured in deuterated chloroform. The chemical shift values are shown below. δ (ppm) 188.9 (s), 180.7 (s), 1
74.7 (s), 158.1 (s), 149.6
(S), 141.3 (s), 134.8 (s), 12
8.0 (s), 119.2 (d), 112.5 (s),
99.3 (d), 74.8 (d), 71.1
(S), 69.6 (d), 40.7 (t), 38.
7 (t), 35.3 (t), 29.3 (q), 2
9.2 (q), 28.9 (q), 28.7 (t) 10) Color reaction; positive for vanillin-sulfuric acid, iron chloride and iodine. 2. Streptomyces (Streptomyc)
es) A bioactive substance NA00226A characterized by culturing a microorganism belonging to the genus and capable of producing the bioactive substance NA00226A in a medium, producing and accumulating the bioactive substance NA00226A in the culture, and collecting the bioactive substance NA00226A.
Or a method for producing a pharmaceutically acceptable salt thereof. 3. A medicament comprising a physiologically active substance NA00226A or a pharmaceutically acceptable salt thereof as an active ingredient. 4. The medicament according to claim 3, which is a phosphodiesterase inhibitor. 5. An anti-asthmatic drug, a bronchodilator, a therapeutic agent for bronchitis, an anti-allergic drug, an anti-inflammatory drug, an anti-rheumatic drug, a hypotensive drug, a therapeutic drug for angina, a therapeutic drug for arrhythmia, a drug for improving cerebral circulation and metabolism,
4. The drug according to claim 3, which is one of an anticoagulant and an antidepressant.
The medicament according to claim. 6. Streptomyces (Streptomyc)
es) A microorganism belonging to the genus and capable of producing the biologically active substance NA00226A. 7. Streptomyces sp. NA 00
226 (Streptomyces sp. NA002)
26) Strain (Accession No. FERM P-16196, National Institute of Bioscience and Human Technology) and its mutants.