JPH05339157A - Phospholipase a2 inhibitor - Google Patents

Abstract

PURPOSE: To obtain a phospholipase A2 inhibitor containing a Q12713 substance as an active ingredient, capable of suppressing the biosynthesis of a prostaglandin and a leucotriene, and useful for the treatment of an inflammation, a ischemic arterial disturbance, an ulcer, a sepsis, etc. CONSTITUTION: This phospholipase A2 inhibitor contains a Q12713 substance as an active ingredient. The properties of the Q12713; m.p.: 152-156 deg.C; molecular weight: FAB-MS-m/Z-894 (M-H); specific degrees of optical rotation: [α]D<20> =(-)49.7 deg. (C=0.1, methanol). The Q12713 is obtained by culturing a strain Actinomadura sp Q12713 (FERM 12723) belonging to the genus Actinomadua as a producing bacterium by a commonly known method and collecting the substance. The dosage is 10mg to 1g for an adult daily in an oral administration, and 1-100mg is administered in the case of a parenteral administration as divided into 2-3 portions.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a phospholipase A ₂ inhibitor containing a Q12713 substance as an active ingredient.

[0002]

2. Description of the Related Art Japanese Unexamined Patent Publication (Kokai) No. 3-218394 discloses an actinomajura velcosospora.
B. from the culture fluid of R. ver verrucosospora
It was reported that a peptide named U-3983T was isolated, and the publication describes that this compound has an antitumor activity and exists in solution as a tautomer represented by the following formula. ing.

[0003]

[Chemical 1]

[0004]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The present inventors cultivated a microorganism isolated from soil collected in Zhouzhou, People's Republic of China, and conducted research on this culture. As a result, phospholipase C inhibitory activity was found. We succeeded in purely isolating the component with and identified it.
It was found to be the same substance as BU-3983T described above. Furthermore, when various pharmacological activities were examined, it was newly found that this culture has phospholipase A ₂ inhibitory activity. Nothing is known about the fact that this compound has phospholipase A ₂ inhibitory activity, and the present inventors have studied the pharmacological action in this aspect,
The present invention has been completed.

[0005]

That is, the present invention relates to a phospholipase A ₂ inhibitor containing a Q12713 substance having the following physicochemical properties as an active ingredient. Like BU-3983T, the active ingredient of the drug of the present invention is confirmed to have tautomers by high performance liquid chromatography (hereinafter referred to as HPLC) and nuclear magnetic resonance spectrum (hereinafter referred to as NMR),
The medicament of the present invention includes these separated isomers and mixtures. The pharmacological effect (phospholipase A ₂ inhibitory action) of the pharmaceutical ingredient of the present invention is measured as follows.

Method for measuring the activity of phospholipase A ₂ The phospholipase A ₂ enzyme solution used was that derived from rabbit platelets. Phospholipase A ₂ enzyme solution 5 μl Inhibitor DMSO solution 2.5 μl Tris-HCl buffer (final concentration 50 mM, pH 9.0) 25 μl Calcium chloride (final concentration 4 mM) 25 μl [ ¹⁴ C] -phosphatidyl Ethanolamine (0.5 nM / tube) 25 μl Potassium chloride (final concentration 10 mM) 2.5 μl Distilled water 160 μl

The above reaction mixture is reacted at 37 ° C. for 20 minutes. Dole's reagent (isopropanol-heptane-1N
After 1.25 ml of sulfuric acid, 78: 20: 2, v / v) was added to stop the reaction, 0.5 ml of water and 0.75 ml of heptane were added and stirred. After centrifugation, 0.8 ml of the upper layer is transferred to another test tube, 0.75 ml of heptane is added thereto, a small amount of silica gel powder is added, and the mixture is stirred and then centrifuged.
The supernatant is placed in a vial for liquid scintillation, the radioactivity is measured, and the phospholipase A ₂ activity is calculated. Phospholipase A ₂ activity of Q12713; IC ₅₀ =
0.5 μg / ml

Next, the toxicity (L
D ₅₀ ) is 3 when administered intraperitoneally to female ICR mice.
It was mg / kg. As a result of the above pharmacological experiments, Q127
It is clear that 13 substances have a phospholipase A ₂ inhibitory action. The compound of the present invention can suppress the biosynthesis of prostaglandin and leukotriene by inhibiting the phospholipase A ₂ activity, and can be a strong anti-inflammatory agent with few side effects. Furthermore, the compounds of the present invention can be expected to be effective for the treatment of ischemic vascular disorders, ulcers, and pulmonary diseases which are said to be involved in phospholipase A ₂ . The compound of the present invention is made into tablets, capsules, powders, granules or pills by mixing the active ingredient with pharmaceutically acceptable carriers, excipients and the like known per se. The dose varies depending on the administration subject, administration route, and symptom, but usually 10 mg orally per day for adults
1g, 1-100mg for parenteral administration,
Administer in 3 divided doses.

Next, a bacterium producing the Q12713 substance which is a medicinal component, a method for culturing the bacterium and a method for collecting the Q12713 substance will be described. Bacteriological Properties of Q12713 Substance-Producing Strain This strain is a microorganism isolated from the soil collected in Zhouzhou, People's Republic of China, and has the following mycological properties.

1. Morphology This strain grows well in various organic and inorganic media, and the color of the basic hyphae is yellowish gray to light yellow tea. Aerial mycelia are well formed on yeast / malt agar medium, nutrient agar medium, starch / inorganic salt agar medium, and tyrosine agar medium, and are grayish to light gray. 15 on spore stalk branched from aerial mycelium
~ 30 spores are linked, curving and coiling as they mature. Fragmentation of basal hypha is not observed in liquid culture.
Observation by an electron microscope shows that the spores are cylindrical and have a size of 0.7 to 0.9 μm × 0.8 to 1.2 μm, and the surface is wart-like. No special organs such as sporangia and motile spores are observed.

2. Properties on various agar media Properties on various agar media are shown in Table 2. Unless otherwise specified, the cells were cultured at 27 ° C. for 21 days and observed according to a conventional method. The description of the color tone was based on the color standard (Japan Color Research Institute).

[0012]

[Table 2]

(Note) G: degree of growth A: settlement of aerial mycelium and its hue R; hue of back surface S; soluble pigment Physiological properties

[0014]

[Table 3]

(Note) The growth temperature is at each temperature (5, 10, 1
5,20,24,27,30,33,37,40,4
The results of observation up to 7-21 days at 5,50 ° C., the effects on milk were observed at 37 ° C. for 3-21 days, and otherwise, the observation results after 2 weeks at 27 ° C. were used unless otherwise specified. Show. 4. Utilization of carbon source (Pridham-Godlieve agar medium, 27 ° C culture)

[0016]

[Table 4]

5. Chemical analysis of cell components LECHVALIER and other methods (LECHVALIE
R, MP, et al; PP277-238 in D
IETZ, A et al ed. , Actinomy
cete Taxonomy, SIM Special
Publication No. 6, 1980), the acid hydrolyzate of this strain was analyzed.
-Diaminopimelic acid was detected. In addition, majurose was detected as a characteristic whole cell sugar component (cell wall type III-B). The major bacterial menaquinone is MK-9
(H6) and MK-9 (H8).

From the above, the properties of the Q12713 strain are summarized as follows: the basic mycelia are yellow ash to pale yellow-brown and aerial mycelia are gray to white.
It is a bright ash. 15 to 30 spore chains are formed on the aerial hyphae, and the basal hyphae are not fragmented. Cell wall type III −
B, the major menaquinone is MK-9 (6H), MK-9
(H8). Bacterial species having the above-mentioned various properties are described in various documents (Bergey's Manual of System).
atic Bacteriology vol. 4,1
989), this strain is judged to belong to the genus Actinomadura . Therefore, this strain was designated as Actinomadura sp. Q12713. This strain has been registered in the Institute for Microbial Industry, Institute of Industrial Science and Technology as Microorganism Research Institute No. 12723.

Culturing Method and Collection Method of Q12713 Substance A culture containing the Q12713 compound can be obtained by inoculating Actinomadura sp. Q12713 strain into a medium containing a nutrient source and aerobically growing. As the nutrients, those known as nutrient sources for microorganisms may be used. For example, commercially available peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, soybean flour, yeast extract, NZ-amine, casein hydrolyzate,
Inorganic or organic nitrogen sources such as fish meal, sodium nitrate and ammonium nitrate, commercially available molasses, starch, dextrin, sucrose, glucose, maltose, fructose, xylose, rhamnose, mannitol, glycerol and other carbohydrates or fats and other carbon sources Can be used.

As the metal salt, Na, K, Mg, C
a, Zn, Fe and other sulfates, hydrochlorides, nitrates, phosphates,
Carbonate etc. are added as needed. If necessary, valine, leucine, isoleucine, threonine, phenylalanine, tryptophan, methionine, lysine,
In addition to arginine, cysteine, cystine, etc., commonly known amino acids, and Q12713 substance production-promoting substances such as methyl oleate, lard oil, silicone oil, and surfactants, or defoaming agents are appropriately used. Other than these, any one can be used as long as it can be used by the producing bacterium and is useful for the production of the Q12713 substance. The culturing method may be the same as the method for producing a general radiobacterium, and the culturing method may be solid culture or liquid culture. In the case of liquid culture, any of static culture, stirring culture, shaking culture and the like may be carried out, but aeration stirring culture is particularly preferable.
The culturing temperature can be appropriately changed within the range of 20 ° C to 45 ° C, that is, the temperature at which the producing bacterium grows to produce the Q12713 substance, which is preferably 28 ° C. PH of medium is 4
The pH can be appropriately changed within the range of 9 to 9, but pH 6 to 8 is preferable if possible. The culture time varies depending on various conditions.
The time is from 168 hours to 168 hours, but usually, the target compound accumulated in the culture solution reaches the highest titer in about 24 hours to 120 hours.

In order to collect the desired compound from the culture, the usual means for extraction, separation and purification used for metabolites produced by microorganisms are appropriately used. The target compound in the culture solution is an organic compound that is immiscible with water, such as ethyl acetate, chloroform, benzene, and toluene, in the culture filtrate obtained by filtering the culture solution as it is or by centrifuging or adding a filter aid to the culture solution. Extract by adding a solvent. Alternatively, the target compound can be extracted by bringing the culture filtrate into contact with an appropriate carrier to adsorb the target compound in the filtrate and then eluting with a suitable solvent. More specifically, the target compound is adsorbed by contacting it with a porous adsorption resin such as Amberlite XAD-2, Diaion HP20, Diaion CHP20P or Diaion SP900. Then, the target substance is eluted with a mixed solution of water with an organic solvent such as methanol, ethanol, acetone, or acetonitrile.

Needless to say, the mixing ratio of the solvent at this time is set to a value at which the target compound can be eluted most efficiently.
That is, by increasing the solvent ratio stepwise or continuously from a low concentration to a high concentration, it is possible to obtain a fraction having a higher ratio of the target compound. When extracting with an organic solvent such as ethyl acetate or chloroform, add these solvents to the culture filtrate and shake well to extract the target compound. Next, the fraction containing the target compound obtained by each of the above-mentioned operation methods is subjected to column chromatography using a conventional adsorption carrier such as activated carbon, alumina, silica gel, cellulose, or a silica gel-based ODS reverse phase carrier. Further pure separation and purification can be carried out by a conventional method such as high performance liquid chromatography using a column. Above, Q
Although the method for producing the 12713 substance has been described, it will be described more specifically below with reference to Examples.

[0023]

Example: Glucose 1.0%, potato starch 2.0
%, Yeast extract 0.5%, polypeptone 0.5%, calcium carbonate 0.4%, a medium (pH 7.0) was prepared, and 60 ml of each was dispensed into a 500 ml Erlenmeyer flask at 120 ° C. What was sterilized for 20 minutes was used as a seed medium, and the mycelia of Actinomadura sp. Q12713 strain well grown on Bennett's agar were scraped and inoculated, and shake culture was performed at 28 ° C for 96 hours to obtain a seed culture solution.

Next, the main culture was carried out with potato starch 1.0.
%, Polypeptone 1.0%, roasted wheat germ 0.5%, copra meal 1.0%, carrot meal 0.5%, calcium carbonate 0.15% (pH)
7.0) was prepared, and this was added to a 500 ml Erlenmeyer flask 5
100 ml of each was dispensed into 0 bottles and sterilized at 120 ° C. for 20 minutes. 4 ml of the seed culture solution was inoculated into each flask and cultured at 28 ° C. for 120 hours.

The bacterial cells were removed from the culture solution by filtration to obtain 3.7.
1 liter of filtrate was obtained. The filtrate was adjusted to pH 7.0, extracted twice with 4 liters of ethyl acetate, the ethyl acetate layer was dehydrated with anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure to obtain 261 mg of a brown oily substance. Thin layer silica gel chromatography {Kieselgel 60F
₂₅₄ (Merck). Elute and elute with ethyl acetate: methanol = 9: 1. The active fractions are collected and concentrated under reduced pressure to obtain 65.3 mg of active substance (i).

This is subjected to silica gel column chromatography. First, 20 ml of chloroform and then a gradient elution of chloroform: methanol = 99: 1 to chloroform: methanol = 90: 10 are carried out to collect active fractions and concentrate under reduced pressure to obtain 29 mg of active substance (ii). This active substance (ii) was treated with a 0.5 M phosphate buffer (pH 4.2) on a silica gel plate (Kieselgel 60F ₂₅₄ manufactured by Merck) in advance, and the plate was activated (120 ° C., 30 minutes), and then applied to the plate.
Chloroform: methanol = 9: 1 developed with saturated water solution,
Elute. 20.2m when the active fractions are collected and concentrated under reduced pressure
g of active substance (iii) was obtained.

This active substance (iii) was analyzed by HPLC (Inertsil Prep O manufactured by GL Sciences Inc.).
DS 20 mmφ × 250 mm). Elute at a flow rate of 10 ml / min with 80% acetonitrile as the mobile phase. The activity peaks at 9.9 minutes and 12.3 minutes were collected and concentrated under reduced pressure to obtain Q12713A (9.9
Min) 0.15 mg and Q12713B (12.3 min)
2.13 mg was obtained. These materials are HPLC and N
From the behavior of MR, it is considered to be a tautomer. The physicochemical properties of the compound of the present invention are shown below.

Melting point: 152-156 ° C. [α] ²⁰ _D : -49.7 ° (C = 0.1, methanol) Molecular weight: FAB MS m / z 894 (M-
H) ^- IR absorption spectrum: FIG. ¹ 1 H-NMR: FIG 2 ¹³ C-NMR: FIG

[Brief description of drawings]

FIG. 1 shows an infrared absorption spectrum of Q12713 substance.

FIG. 2 shows a ¹ H-NMR spectrum of Q12713 substance.

FIG. 3 shows a ¹³ C-NMR spectrum of Q12713 substance.

FIG. 4 shows an elution pattern of Q12713 substance by HPLC.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location // C12P 21/02 A 8214-4B (C12P 21/02 C12R 1:03) (72) Inventor Hiroshi Ogawara 2-33-9 Yushima, Bunkyo-ku, Tokyo (72) Kyoichiro Higashi Inventor Kyoichiro Higashi 84 Futako, Takatsu-ku, Kawasaki-shi, Kanagawa No. 205, Estate Tama 205 (72) Keizo Inoue 1-3-17-605 Etchujima, Koto-ku, Tokyo ( 72) Inventor Koichi Tanaka, 5-7 Shiui Building No. 3-7-11 Shimura, Itabashi-ku, Tokyo (72) Inventor Koji Nagai 2-19-5 Kizawa, Toda City, Saitama Prefecture No. 207 Revere Pal (72) Inventor Yasuyo Shimizu 2-1-30 Fujimidai, Kunitachi, Tokyo No. 2-102, National House (72) Inventor, Liang Sufan, People's Republic of China, Shanghai 200031 Yu Yang Road, 319 Shanghai Institut of Materia Medica Academia Sinica (72) Inventor Wang Fu Qin Shanghai, China 2000 31 Yu Yang Road 319 Shanghai Institute of Materia Medica Academia Sinica (72) Inventor Tsang Wei Wen Yang 31 China 2000 Shanghai 319 Shanghai Institut of Materia Medica Academia in Cinica (72) Inventor, Leucha Way, People's Republic of China Shanghai City 200031 Yu Yang Road 319 Shanghai Institut of Materia Medica in Academia Sinica

Claims (1)

[Claims] 1. A phospholipase A ₂ inhibitor containing, as an active ingredient, a Q12713 substance characterized by the following physicochemical properties: Melting point: 152-156 ° C. Molecular weight: FAB MS m / z 894 (MH)
^- Specific rotation: [α] _{^{20 D = -49.7 ° (C =}} 0.1, methanol) Infrared absorption spectrum: Tori ¹ H- nuclear magnetic resonance spectrum of FIG. 1: Toh of FIG 2 ¹³ C- Nuclear Magnetic Resonance Spectra: Figure 3