JP2979271B2 - Bioactive substance TAN-1515, its production method and use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to novel physiologically active substance TAN-1515 with available phospholipase A ₂ inhibitory activity as anti-inflammatory agents (hereinafter, sometimes abbreviated as "TAN-1515".) Preparation of Laws and uses.

[0002]

_2. Description of the Related Art Phospholipase A ₂ cleaves arachidonic acid from phospholipids and is considered to be a key enzyme for producing inflammatory mediators such as prostaglandins and leukotrienes. On the other hand, the mechanism of action of steroids having a strong anti-inflammatory effect has recently been investigated, and the protein produced by administration of steroids is phospholipase A
A hypothesis has been proposed to suppress the activity of ₂ . Compounds which effectively inhibit phospholipase A ₂ Therefore,
It does not have steroid drug-specific side effects such as susceptibility to infection and adrenal atrophy, and can be expected to have a strong anti-inflammatory effect.

[0003]

Compounds known to date as phospholipase A ₂ inhibitors include mepacrine and p-bromophenacyl bromide in synthetic products,
Natural products include sponge-derived manoalide, blue mold-derived plastatin, and bacterial-derived pripastatin.
However, these anti-inflammatory actions are much weaker than existing non-steroidal anti-inflammatory drugs such as indomethacin, diclofenac and the like, and have poor utility as anti-inflammatory drugs. Therefore, new phospholipase A ₂ inhibitors that may be used clinically as anti-inflammatory agents has been desired.

[0004]

Means for Solving the Problems Conventionally, phospholipase A
₍₂ ) Research on inhibitors has been performed mainly on exocrine enzymes such as pig pancreas as an enzyme source, but the present inventors have found that platelet-derived phospholipase a ₂ as an enzyme source, a result of the inhibitor was searched for the microbial metabolites, found a compound from certain fungi have inhibitory activity of platelet phospholipase a _2, referred to as a physiologically active substance TAN-1515 of this I decided that. TAN-1515 was purified and isolated from the culture solution, clarified to be a novel compound, and as a result of further intensive studies, the present invention was completed. That is, the present invention provides (1) a physiologically active substance TAN-1515 or a salt thereof,
(2) belongs to the genus Acremonium and is a physiologically active substance TAN-1
Culturing a microorganism capable of producing 515 in a medium;
Producing and accumulating the TAN-1515 in a culture, and collecting the TAN-1515; and collecting the TAN-1515.
15 preparation and (3) about the phospholipase A ₂ inhibitor comprising a physiologically active substance TAN-1515. The microorganism that can be used in the present invention may be any microorganism that belongs to the genus Acremonium and produces the physiologically active substance TAN-1515. For example, Acremonium SP FL-17559 isolated from soil in Hyogo prefecture is mentioned as a usable example. The mycological symptoms of the FL-17559 strain are as follows.

(I) Morphological characteristics Mycelium: Mycelium is colorless and has partition walls, and the surface is smooth. The diameter is not constant at 1-3 μm. Fialide: A large number of phialides having a partition at the base stand almost upright on the mycelium. Hardly branches. Gently taper from base to tip. Length is 30-75 μm. The base diameter is 1.4-1.6 μm. Colorless and smooth surface. Conidia: Fiaro-type conidia. Spindle type or banana type. 1.2-1.5 ×
6.0-10.0 μm. Colorless and smooth surface. A spherical conidium mass is formed at the tip of the phialide, and its diameter is 10-30.
μm, size is not fixed. (II) Growth state on various media 1) Malt extract agar medium Growth was poor or moderate, colonies spread slowly at 24 ° C, and after 2 weeks the diameter was about 20-22 mm. The surface consists of a flat, thin mycelium and the outer edge is slightly irregularly bordered. The central part is grayish white, and the middle part and the outer peripheral part are yellowish white. The development of aerial mycelium is poor, and a faint gray-white mycelium is slightly observed in the middle part. Conidium formation is good.
The back has a pale yellow gray color. No soluble dye formation is observed. 2) Potato / Glucose agar medium Growth is moderate and the diameter of colonies after 24 weeks at 24 ° C is 3
It was 0-33 mm. The surface consists of a flat and thin mycelium, and wrinkles are seen radially from the center. The outer edge is regularly bordered. The central part is grayish white, and the middle part and the outer peripheral part are yellowish gray. The aerial hyphae are poorly developed, but the middle part is dominated by rope-shaped hyphae. Conidium formation is good. The back surface is yellow-grey to dark yellow-gray. No soluble dye formation is observed. 3) Tzapek agar medium Growth is poor, colony diameter after 2 weeks at 24 ° C is 40
-42 mm. The surface is flat and extremely thin mycelia elongate radially. The outer edge is regularly bordered. Both front and back are colorless. Conidium formation is good. No soluble dye formation is observed. 4) Oatmeal agar medium Growth is moderate and the diameter of colonies after 24 weeks at 24 ° C is 5
It was 0 mm. The surface is composed of thin mycelium and the outer edge is regularly bordered. Both front and back are colorless. Conidium formation is good. No soluble dye formation is observed.
From the above mycological properties, it is clear that the present strain belongs to the genus Acremonium described in the following document, and Acremonium sp. FL-175
59 (Acremonium sp. FL-17559). [WALTER GAMS; Cephalospori
um-artage Schimmelpilze (Hyphomycetes), Gustav F
ischer Stuttgart, (1971)] The above Acremonium sp. FL-17559 strain has been deposited with the Fermentation Research Institute from June 26, 1991 under the deposit number IFO-32400.
The microorganism has been deposited with the Ministry of International Trade and Industry of Japan, the Institute of Microbial Technology, from the deposit number FERM BP- based on the Budapest Treaty on July 18, 1991.
Deposited as 3487.

Acremonium can be mutated naturally or by a mutagen as a general property of microorganisms. For example, X-rays, gamma rays, irradiation of radiation such as ultraviolet rays, further isolation of single spores, treatment with various drugs or culturing on a medium containing drugs, many mutant strains obtained by mutating by other means, Alternatively, even a naturally-occurring mutant strain or the like having the property of producing TAN-1515 can be used in the method of the present invention.

[0007] The medium used for the culture of the present invention may be liquid or solid as long as it contains a nutrient that can be used by the strain used. However, when processing a large amount, it is more appropriate to use a liquid medium. The medium is appropriately blended with an assimilable carbon source, a digestible nitrogen source, inorganic substances, and micronutrients. Examples of the carbon source include glucose, lactose, sucrose, maltose, dextrin, starch, glycerin, mannitol, sorbitol, fats and oils (eg, soybean oil, olive oil, nuka oil, sesame oil, lard oil, chicken oil, etc.), nitrogen source For example, meat extract, yeast extract, dried yeast, soybean flour, corn steep liquor, peptone, cottonseed flour, urea, ammonium salts (eg, ammonium sulfate, ammonium acetate, etc.) and the like are used. Further, salts including sodium, potassium, calcium, magnesium, etc., metal salts such as iron, manganese, zinc, cobalt, nickel, phosphoric acid, salts such as boric acid and acetic acid,
Salts of organic acids such as propionic acid are appropriately used. In addition, amino acids (eg, glutamic acid, aspartic acid, alanine, lysine, valine, methionine, proline, etc.), peptides (eg, ヂ peptide, tripeptide, etc.), vitamins (eg, B ₁ , B ₂ , nicotinic acid, B ₁₂ ,
C), nucleic acids (eg, purine, pyridine and derivatives thereof) and the like. Of course, an inorganic or organic acid, alkali, buffer or the like is added for the purpose of adjusting the pH of the medium, or an appropriate amount of an oil or fat, a surfactant or the like is added for the purpose of defoaming.

The culturing may be performed by static culturing, shaking culturing, aeration stirring culturing, or the like. It is needless to say that for a large amount of treatment, a treatment by so-called deep aeration stirring culture is desirable. Naturally, the conditions for cultivation are not fixed depending on the state of the medium, the composition, the type of strain, and the means of culturing, but they are usually at a temperature of about 15 ° C to 32 ° C.
The initial pH is preferably selected around 5-9. In particular, the temperature during the middle stage of the culture is about 20 ° C to 30 ° C, and the initial pH is about 6 ° C.
The condition of −8 is desirable. The cultivation time is not fixed depending on the above conditions, but it is preferable to culture until the concentrations of various physiologically active substances are maximized. The time required for this is generally about 1 to 10 days in the case of shaking culture or aeration and stirring culture using a liquid medium. The generated physiologically active substance TAN-1515
Is present in the culture filtrate and cells, so the culture can be separated into a supernatant and cells by centrifugation or filtration, and the culture can be purified from the supernatant and cells, respectively. In some cases, it may be advantageous to purify from an extract obtained by directly adding an organic solvent such as methanol, acetone or ethyl acetate to the product. In order to collect TAN-1515 from a culture solution, since the substance is a fat-soluble acidic substance, a separation / purification means usually used for collecting such a microbial metabolite is appropriately used. For example, a method utilizing the difference in solubility with contaminants, chromatography using various carriers such as activated carbon, a nonionic high-porous resin, silica gel, alumina, dextran gel, etc. are used alone or in combination.

The method of collecting TAN-1515 produced in the culture is specifically described. First, the culture is extracted with an organic solvent which is acidic or neutral and is immiscible with water, such as ethyl acetate. Examples of the organic solvent used for the extraction include esters such as ethyl acetate and butyl acetate, halogenated hydrocarbons such as chloroform and methylene chloride, and alcohols such as n-butanol and i-butanol. Alternatively, a water-miscible organic solvent such as methanol or acetone is added to the culture solution, and the mixture is stirred and extracted. After the organic solvent in the extract is distilled off, the water-immiscible organic solvent (ethyl acetate) is used. Etc.) to extract the substance. The extract thus obtained is washed successively with acidic water and water, and the crude powder obtained by concentration is subjected to silica gel column chromatography.
As a developing solvent, for example, a mixed solvent such as chloroform-methanol or hexane-ethyl acetate, if necessary, a system to which an acid such as acetic acid or oxalic acid is added is used. In each solvent system, methanol or ethyl acetate or the like is sequentially used. By increasing the ratio of the polar solvent, it can be separated and eluted from other contaminants. The fraction containing TAN-1515 eluted in this manner is concentrated, and n-hexane is added to obtain a powder of TAN-1515. This is redissolved in an organic solvent such as chloroform or ethyl acetate, washed successively with dilute hydrochloric acid and water, concentrated, and crystallized from hydrated methanol to obtain TAN-1515 as colorless crystals.

The thus obtained physiologically active substance TA
The physicochemical properties of N-1515 are as follows. 1) Shape: colorless crystals 2) Melting point: 102-104 ° C 3) Mass spec: (SIMS): m / z 543 (M +
H) 4) Molecular formula: C ₃₂ H ₄₆ O ₇ 5) Elemental analysis: Calculated: C, 70.82; H, 8.54 Found: C, 70.99; H, 8.91 6) Ultraviolet absorption spectrum: MeOH 1% λ (E): 210 (960), 269 (384), 302 (24)
6) max 1cm 7) Infrared absorption spectrum (KBr tablet method): 332
5, 3075, 2920, 2850, 1665, 1610, 1595,1460, 1415,
1385, 1350, 1310, 1245, 1200, 1170, 1140, 1070, 105
5, 995, 850, 800, 720, 685, 630, 605 (cm ⁻¹ , see FIG. 2) 8) ¹ H nuclear magnetic resonance spectrum (300 MHz, d ₆ -DM
Chemical shift in SO, δ ppm): 0.85 (3H, br.t),
1.15-1.35 (28H, m), 1.53 (2H, m), 2.38 (3H, s), 2.61 (2H, m
m), 6.18 (1H, d), 6.22 (1H, d), 6.52 (1H, br.d), 6.57 (1
H, d), 9.85 (1H, br.s), 10.13 (1H, s). 9) ¹³ C nuclear magnetic resonance spectrum (75 MHz, d ₆ -DM
Chemical shift in SO, δ ppm): 13.9 (q), 21.3
(q), 22.0 (t), 28.7 (t), 28.8 (t), 29.0 × 10 (t), 31.0
(t), 31.2 (t), 33.9 (t), 100.4 (d), 107.3 (d), 108.5
(d), 109.0 (s), 114.6 (d), 115.7 (s), 139.8 (s), 144.2
(s), 152.6 (s), 158.9 (s), 159.4 (s), 160.6 (s), 166.8
(s), 170.8 (s), where s indicates a singlet, d indicates a doublet, t indicates a triplet, q indicates a quartet, and br indicates a broad. 10) Color reaction positive: Barton, iodine, phosphomolybdate, potassium permanganate Negative: ninhydrin, dragendorff 11) Thin layer chromatography (carrier, silica gel glass plate 60F ₂₅₄ , 0.25 mm, manufactured by Merck, Germany) : Developing solvent Rf chloroform-methanol-acetic acid (100: 5: 1) 0.52 toluene-methanol-acetic acid (20: 2: 1) 0.49 ethyl acetate-acetic acid (100: 1) 0.591 12) acidic, Neutral, basic: acidic From the above physicochemical properties, TAN-1515 is a novel compound having the following structural formula.

Embedded image Further, since TAN-1515 is an acidic substance, a salt with a metal salt such as a sodium salt, a potassium salt or a calcium salt, an ammonium salt, or a salt with an organic amine such as a triethylamine salt or an ethanolamine salt can be obtained by a method known per se. For example, a physiologically acceptable salt can be formed.

[0011]

The following is a description of an example of the activity of the bioactive substance TAN-
1515 shows rabbit platelet phospholipase A ₂ inhibitory activity along with the test method.

Experimental Example 1 Rabbit platelet phospholipase A
_2. Preparation of Crude Enzyme Solution Platelets are separated according to a conventional method (Information transmission and cell response, lowering and semantic chemistry course 7), and the obtained platelets are crushed by freeze-thawing method to prepare a phospholipase A ₂ crude enzyme solution did. Blood collected from rabbit (Japanese white species) carotid artery using ethylenediaminetetraacetic acid as an anticoagulant was centrifuged at 140 xg for 15 minutes to obtain a platelet-rich fraction. . This fraction is centrifuged at 940 × g for 15 minutes to precipitate platelets, and then the supernatant plasma is removed. Platelets were prepared using 1.2 mM EDTA and 0.14 M Na.
Suspended in 10 mM Tris buffer (pH 7.4) containing Cl;
The platelets are sedimented by centrifugation at 940 × g for 15 minutes, and the supernatant is removed. The platelets were then washed with 0.14M NaC.
The suspension is suspended in 10 mM Tris buffer (pH 7.4) containing the same, and the same operation is repeated twice. 5 × 10 ⁹ platelets / m
and suspended in 10 mM Tris buffer (pH 7.4) containing 0.14 M NaCl and frozen at -80 ° C. After thawing frozen platelets at 37 ° C., 10,000 × g
And centrifuged at 105,000 × g for 60 minutes to obtain a supernatant rich in phospholipase A ₂ . This preparation was aliquoted, stored at -20 ° C, and diluted for use with a 10 mM Tris buffer (pH 7.4) containing 0.1% bovine serum albumin and 0.14 M NaCl.

[0013] Measurement phospholipase A ₂ activity of Example 2 Phospholipase A ₂ activity, 1-palmitoyl-2
The release of free [1- ¹⁴ C] arachidonic acid was measured using (1- ¹⁴ C) arachidonyl-L-α-phosphatidylcholine as a substrate. The substrate was 1-palmitoyl-2-
(1- ¹⁴ C) arachidonyl -L-α- phosphatidylcholine (New England Nuclear, Inc., manufactured by the United States)
Unlabeled 1-palmitoyl at [50 mCi / mmole]
It was used after being diluted to 16 mCi / mmole by adding 2-arachidonyl-L-α-phosphatidylcholine (Avanti Polar Lipids, USA). 1.5ml
The following additives in a capacity polypropylene tube: 0.9 nmo
le (15nCi) 1- palmitoyl -2- (1- ¹⁴ C) arachidonyl - phosphatidylcholine, dimethyl sulfoxide 12.5 [mu] l, 10 [mu] l of 500mM Tris buffer (pH9.0), 80mM CaCl ₂ of 2.5 [mu] l, 1
0 μl of phospholipase A ₂ crude enzyme solution (amount that gives 20-30% hydrolysis) and drug are added and the final volume is adjusted to 50 μl with water. The mixture was stirred and kept in a constant temperature water bath for 3 hours.
Incubate at 0 ° C for 60 minutes. The reaction is stopped by adding 10 μl of acetic acid to each tube. Next, 100 mg of silica gel 60 (Merck, Germany) and 1 ml of ethyl acetate are added, and the mixture is stirred for 3 minutes using a vial mixer (Taiyo Service Center, Japan). After stirring, the mixture was centrifuged at 10,000 × g for 1 minute, the silica gel was sedimented, 700 μl of the supernatant was placed in a vial, 3 ml of scintillator A (Wako Pure Chemical Industries, Japan) was added, and the mixture was stirred. After stirring, 2200CA liquid scintillation counter ( The radioactivity was measured by Packard (USA). IC of phospholipase A ₂ inhibitory activity of manoalide used as compound of the present invention and standard drug measured by this method
_{The 50} values are shown in [Table 1].

[Table 1] The compounds of the present invention the value of LD ₅₀ by intraperitoneal administration in an acute toxicity test using mice was 100 mg / kg or more.
The compound of the present invention has phospholipase A ₂ inhibitory activity, and various diseases caused by oxidized metabolites of arachidonic acid, such as allergic diseases, bronchial asthma, rheumatoid arthritis, osteoarthritis, various inflammatory diseases including pancreatitis It can also be applied as a treatment and prevention of thrombosis and arteriosclerosis, and as an analgesic. This compound is used for oral administration, parenteral injection or topical application to skin, mucous membrane, rectum, etc.
The dose may vary depending on the disease, administration route and the like of the subject. For example, when used for the treatment of inflammatory diseases, it is usually 0.01 to 100 mg / kg, especially 0.1 per adult.
-20 mg / kg is orally or parenterally administered about 1-3 times a day. For oral administration, capsules, tablets,
Granules, syrups, powders and other dosage forms, which contain, together with the compound of the present invention, additives such as excipients, binders, disintegrants, actives, coloring agents, crosslinking agents, stabilizers and the like. Is also good. Among them are starch, sucrose, fructose, lactose, glucose, mannitol, sorbitol precipitated calcium carbonate, crystalline cellulose, carboxymethyl cellulose,
Dextrin, gelatin, gum arabic, magnesium stearate, talc, hydroxypropyl methylcellulose and the like can be mentioned. For parenteral administration, the active ingredient can be dissolved or suspended in a conventional diluent (aqueous or non-aqueous carrier). As a diluent,
Examples include physiological saline, Ringer's solution, aqueous glucose solution, alcohols, fatty acid esters, glycols, glycerin fatty acid glycerides, oil sources derived from plants and animals, paraffins and the like. In addition, pharmaceutical preparations include, as additives, emulsifiers, suspending agents, solubilizers, stabilizers, preservatives, soothing agents, isotonic agents, buffers, pH adjusters, coloring agents, coating agents, etc. May be included. In addition, these preparations can be manufactured by a usual method.

[0014]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. In addition, in the following examples, the composition percentage of the medium indicates the weight / volume percentage.

Example 1 Bacto potato dextrose agar (manufactured by Difco Co., Ltd.)
A platinum loop of Acremonium sp. FL-17559, which had been sufficiently grown in advance on a slant culture medium (US), was prepared using glucose 2.0%, maltose 3.0%, raw soy flour 1.5%, and corn steep. 40 ml of a seed culture medium consisting of Liquor 1.0%, polypeptone 0.5%, yeast extract 0.3% and pH 6.0 was inoculated into a dispensed and sterilized 200 ml Erlenmeyer flask, and placed on a rotary shaker. C. for 2 days. Two milliliters of this culture was used with 1.0% glucose, 4.0% dextrin, 0.5% raw soy flour, 0.5% malt extract.
%, Polypeptone 0.5%, yeast extract 0.2%, iron sulfate heptahydrate 0.05%, magnesium sulfate heptahydrate 0.05%
Manganese sulfate heptahydrate 0.05%, monopotassium phosphate 0.5
% And sedimented calcium carbonate 0.5%, pH 7.5, were transferred to a disinfected 200 ml Erlenmeyer flask, cultured on a rotary shaker at 24 ° C. for 4 days, and then TAN-1515. Was generated and accumulated.

Example 2 Bacto potato dextrose agar (manufactured by Difco Co., Ltd.)
Five platinum loops of Acremonium sp. FL-17559 previously grown sufficiently on a slant medium consisting of US 2.0), glucose 2.0%, maltose 3.0%, raw soy flour 1.5%, corn steep Inoculate 500 ml of a seed culture medium consisting of 1.0% liquor, 0.5% polypeptone, 0.3% yeast extract and pH 6.0 into a 2 liter sterile dispensed Sakaguchi flask. The cells were cultured at 28 ° C. for 2 days. One liter of this culture was added to glucose 1.
0%, dextrin 4.0%, raw soy flour 0.5%, malt extract 0.5%, polypeptone 0.5%, yeast extract 0.2
%, Iron sulfate heptahydrate 0.05%, magnesium sulfate heptahydrate 0.05% manganese sulfate heptahydrate 0.05%, monopotassium phosphate 0.5% and precipitated calcium carbonate 0.5%, pH7 .
120 liters of main culture medium consisting of 5
It was transplanted to a 00-liter fermenter. This main fermentation is 24
° C, aeration (120 l / min), stirring (140 r.p.
m.) and culture under an internal pressure of 1.0 kg / cm ² for 114 hours,
TAN-1515 was produced and accumulated.

Example 3 The culture solution (2.2 liters) obtained in Example 1 was subjected to centrifugation to separate a supernatant and cells. The supernatant (1.8 l) was extracted twice with ethyl acetate (0.9 l) at pH 3.0. The ethyl acetate layer was washed (1.7 L) with water (1 L) and concentrated to give an oily residue (1.25 g). The cells were added with 80% aqueous acetone (1.8 L), extracted with stirring for 30 minutes, and then filtered using Hyflo Super Cell. The obtained filtrate (1.5 liter) was concentrated to 200 ml, and water (8
Then, the mixture was extracted with ethyl acetate (0.5 l) twice at a pH of 3.0. Ethyl acetate layer (0.
9 liters) is washed with water (1 liter) and concentrated.
An oily residue (1.13 g) was obtained. The supernatant and the oily residue obtained from the cells were combined (2.38 g), dissolved in chloroform (10 ml) and subjected to silica gel (50 g) column chromatography. The column was charged with chloroform (200 ml) and chloroform-methanol (49:
1600 ml), chloroform-methanol (19:
1,400 ml). The eluate was fractionated in 100 ml portions, and fractions 5 to 11 were collected and concentrated, and hexane was added to obtain 570 mg of TAN-1515 powder. Dissolve powdered TAN-1515 (150 mg) in ethyl acetate (30 ml) and add 0.5N hydrochloric acid (10 ml).
And water (10 ml). The ethyl acetate layer was dried over anhydrous sodium sulfate, concentrated, and crystallized from aqueous methanol to give colorless crystals of TAN-1515 (88 m 2).
g) was obtained.

Example 4 100 liters of the culture solution of Example 2 was treated in the same manner as in Example 3 to obtain 4 g of TAN-1515 colorless crystals.

[0019]

The novel physiologically active substance TAN-15 of the present invention
15 has strong phospholipase A ₂ inhibitory activity, can be used as an anti-inflammatory agent, and is also useful as an intermediate for the synthesis of novel derivatives.

[0020]

[Brief description of the drawings]

FIG. 1. UV spectrum of TAN-1515

FIG. 2 IR spectrum of TAN-1515

FIG. 3 ¹ HNMR spectrum of TAN-1515

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ identification symbol FI // (C12P 7/42 C12R 1: 645) (58) Investigated field (Int.Cl. ⁶ , DB name) C07C 69/773 C12N 9/99 C12P 7/42 CA (STN) REGISTRY (STN)

Claims (3)

(57) [Claims] (1) Formula (1) Or a salt thereof. 2. A compound belonging to the genus Acremonium and having the formula: A method for producing a compound represented by the above formula, which comprises culturing a microorganism having the ability to produce the compound represented by the formula (1) in a medium, producing and accumulating the compound in a culture, and collecting the compound. 3. A phospholipase A ₂ inhibitor comprising the compound according to claim 1 or a salt thereof.