JPH05255305A - New bioactive substance k-18, its use and its production - Google Patents

Abstract

PURPOSE:To provide the subject new compound K-18 or its base adduct salt exhibiting an inhibitory effect on calcium overload, an active oxygen-removing effect and an inhibitory effect on generation of a lipoperoxide. CONSTITUTION:The compound K-18 represented by the formula or its base adduct salt. The compound K-18 of the formula can be produce by aerobically culturing a K-18-producing cell strain [Bikoken stipulation No. 3741 (FERM- BP-3741)] belonging to Hyphomycetes, plexogenic Fungi Imperfecti in a suitable culture medium and collecting the above-mentioned compound from the cultured material. The above-mentioned new compound is useful as a medicine for preventing or improving various kinds of diseases or various kinds of symptoms such as inflammation or edema caused, e.g. by disturbance of circulation in the brain, the heart or the periphery.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to a novel substance, more specifically to a novel compound k-18 having a calcium overload inhibitory action, an active oxygen scavenging action and a lipid peroxide production inhibiting action.

[0002]

Accumulation of calcium in brain cells (calcium overload) is found, for example, after convulsions, migraine, anoxia and ischemia. While intracellular calcium levels are crucial for the regulation of cell function, high levels of intracellular calcium that cannot be regulated in cells lead directly or indirectly to the degenerative changes associated with the above diseases. It is believed to cause. In addition, excessive accumulation of calcium ions (Ca ²⁺ ) in myocardium or vascular smooth muscle cells leads to myocardial damage, abnormal cardiac conduction disorders, abnormal vascular disorders, etc., and causes cardiovascular diseases. Therefore, the calcium overload inhibitor is not only useful for anoxia, ischemic brain disease, migraine, and epilepsy, but also a useful preventive and therapeutic drug for ischemic heart disease, heart failure, hypertension or arrhythmia. sell. On the other hand, in the pathological state of the living body, the active oxygen radical (active oxygen radical)
s) is generated in large quantities, and the locally generated reactive oxygen species form a strong hydroxyl radical (OH.) by forming a complex with endogenous iron.
As a result, a large amount of lipid peroxide is generated in the tissue. It is known that this lipid peroxide damages blood vessels and other normal organs / tissues, and causes secondary lesions such as vascular disorders, inflammation, and edema. Therefore, substances that eliminate reactive oxygen species and suppress lipid peroxide production are
It can be used as a preventive and therapeutic agent for various disorders and diseases such as cerebrovascular disorder, arteriosclerosis, inflammation, renal disease, cancer and peptic ulcer. Conventionally, flunarizine hydrochloride having a brain metabolism improving action has been known as a drug having both a calcium overload inhibitory action and an action of eliminating active oxygen species to suppress lipid peroxide production. It can be said that there is a constant desire for substances.

[Outline of the Invention]

DISCLOSURE OF THE INVENTION The present invention provides a novel compound that meets the above-mentioned needs, that is, a compound having both a calcium overload inhibitory action and an action of scavenging reactive oxygen species to suppress lipid peroxide production. The purpose is that.

[0004]

[Means for Solving the Problems]
As a result of extensive studies using a calcium overload inhibitory action test using T3 cells and a lipid peroxide production inhibition test using rat brain homogenate as an index, a soil microorganism k18 strain producing a compound suitable for the purpose was found, and this finding was found. Based on the above, the inventors have invented a novel compound k-18 represented by the following formula (I) or a base addition salt thereof. That is, the novel compound according to the present invention is the compound k-18 represented by the following formula (I) or a base addition salt thereof.

[Chemical 2] The compound k-18 represented by the above formula (I) according to the present invention
Is preferably an aerobically cultivated strain belonging to the incomplete filamentous fungus, incomplete colonization order and capable of producing k-18, in a suitable medium, and the compound k-18 is obtained from the culture. It can be manufactured.

DETAILED DESCRIPTION OF THE INVENTION Novel Substance k-18 1) Chemical Structure The novel compound k-18 according to the present invention has the chemical structure represented by the above formula (I). The chemical structure of k-18 is the ultraviolet absorption spectrum (FIG. 1), infrared absorption spectrum (FIG. 2), proton nuclear magnetic resonance spectrum (FIG. 3), carbon-13 nuclear magnetic resonance spectrum (FIG. 4), and mass spectrometry. It was determined as described above by examining the spectrum in detail. 2) Physicochemical properties (1) Appearance: Yellow powder (2) Melting point: 115-117 ° C (3) Specific optical rotation [α] _D ²⁵ = + 34.8 ° (4) Solubility Methanol, dimethyl sulfoxide , Soluble in chloroform and ethyl acetate. Insoluble in water and hexane. (5) Rf value (using "Silica gel 60F ₂₅₄ " manufactured by Merck & Co., Inc.): Chloroform-methanol-acetic acid (100: 2: 1) 0.33 (6) Mass spectrum: ( m / z ) Measured value 572 (M) ⁺ (7) Molecular formula: C ₃₁ H ₂₄ O ₁₁ (8) UV absorption spectrum: shown in FIG. (9) Red external absorption spectrum (KBr disk method): Fig. 2
Is shown in. (10) Proton nuclear magnetic resonance spectrum (500 MHz, in deuterated chloroform): shown in FIG. (11) Carbon-13 nuclear magnetic resonance spectrum (125 MHz, in deuterated chloroform): shown in FIG. (12) Elemental analysis: C H O analysis value (%) 67.06 4.50 28.44 calculated value (%) 65.04 4.23 30.73 The compound k-18 represented by the formula (I) is There may be base addition salts at the acidic hydroxyl positions. The compound of the present invention is
These addition salts are also included. As the base addition salt, for example, a salt with an alkali metal compound (eg sodium hydroxide, potassium hydroxide etc.), a salt with an alkaline earth metal compound (eg calcium hydroxide, magnesium hydroxide etc.), an ammonium salt, an organic salt Salts with bases (eg triethylamine, ethanolamine, etc.) can be mentioned. Needless to say, when the base addition salt is used as a medicine, the base must be pharmaceutically acceptable.

Production of k-18 1) Overview Although k-18 has so far been obtained only by culturing microorganisms, it can be produced by synthetic chemical or microbiological modification of related compounds, or total synthesis. It could be manufactured chemically. It could also be done by genetic engineering techniques. That is, it would be possible to incorporate the gene involved in the production of the compound k-18 into an appropriate microorganism, culture the resulting transformant, and obtain it from this culture. As a strain for culturing a microorganism, for example, a strain having the ability to produce k-18 belonging to the incomplete filamentous fungi and the incomplete order of the crowded bacteria is used. Specifically, it has been revealed by the present inventors that the k18 strain isolated by the present inventors produces k-18, but for other strains, conventional methods for isolation of antibiotic-producing strains are used. It is possible to separate suitable ones from the natural world. Also, k18
There is still room for increasing the k-18 production ability by subjecting the k-18 producing strains including the strain to irradiation and other mutation treatments. As mentioned above, it is possible to use a genetic engineering method.

2) k18 strain The k18 strain found by the present inventors as a strain belonging to the incomplete filamentous fungi, the order of incomplete fungi, which has the ability to produce k-18.
The strain has the following contents. (1) Origin and deposit number The k18 strain was isolated from soil collected in Matsuida-cho, Usui-gun, Gunma Prefecture, and was deposited on February 7, 1992 at the Institute of Microbial Science and Technology, Institute of Industrial Science and Technology. Has obtained the number of "Microtechnical Laboratory Article No. 3741" (FERM BP-3741). (2) Mycological properties The mycological properties of the k18 strain are as follows. (i) Morphology The k18 strain has a fungus-like fungus having a colony tissue, its colors are black and gray, and mycelium has a septum. The hyphae are smooth and linear, and their thickness is about 1 to 3 μm. Unbranched or branched near the base and conidia spherical to oval at the tip. Conidia are Aleuro type, colorless to black, and have a diameter of about 20 μm. In addition, the surface of conidia is smooth single cells, which are detached from conidia-forming cells and become olive-brown to form a double circular color. (ii) Growth condition on various media a. Malt extract agar medium The surface of the colony is dark yellow tea covered with hyphae of grayish yellow tea, and the back surface has wavy circles and radial black stripes.
A wide black color and a narrow light yellowish brown form a striped pattern. Growth is rapid and widespread. On day 14 of culture, a colony with a diameter of about 8 cm is formed. Many conidia are recognized. b. Czapek Agar Growth is not good. Spread from the center of the village in the shape of a peony flower. The color of aerial hyphae is light olive ash, and the aerial hyphae are dark red brown to dark yellow brown. Conidia are recognized. c. Potato dextrose agar medium The diameter of the colony is 7.5 cm and it has a light yellow-brown color.
The circle with aerial hyphae is smaller than the size of aerial hyphae. Black powder is scattered in the center of the village. Mycelium is not long. The back surface of the village is black. Conidia are recognized. d. The color tone of the YpSs agar medium is based on brown ash, and the mycelia of slightly bright brown ash are partially present on the spots. Growth is rapid and diffuse. The color of the back side is black. Conidia are recognized. (iii) Physiological properties The range of pH and temperature at which this strain can grow and the optimum growth p
H and temperature are as shown below. pH Temperature (° C) Growth range 3 to 110 to 35 Optimal growth conditions 5 to 9 15 to 35 For pH, potato dextrose liquid medium is used, and for temperature, potato dextrose agar medium is used, and each culture is performed for 7 days. I did. Based on the above mycological properties, Shunichi Udagawa, Keisuke Tsubaki et al .: "Fungal Encyclopedia" (1978) Kodansha, when searching with reference to this strain, from the morphology and color of the conidium and mycelium, It seems to be similar to Monotosporella sphaerocephala and Gilmaniella humicola , which belong to the incomplete filamentous fungi. Therefore, a comparison test was carried out between the above-mentioned two strains and k18, but none of them was in agreement. Then k
18 is a fungus belonging to the group of imperfect filamentous fungi, imperfect fungi
Name it 18.

3) Culture / Production of k-18 Compound k-18 is preferably aerobically cultivated in a suitable medium with a k-18 producing bacterium belonging to the incomplete filamentous fungi, incomplete order of phyla. , Can be produced by collecting the target substance from the culture. The medium may contain any nutrient source available to the k-18 producing bacterium. Specifically, for example, soluble starch, glucose, galactose, glycerol, fats and oils can be used as a carbon source, soybean flour, fish meal, dry yeast as a nitrogen source,
Organic substances such as yeast extract and inorganic substances such as ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride can be used. Further, if necessary, inorganic salts such as sodium chloride, potassium chloride, calcium carbonate, phosphate, heavy metal salt (eg magnesium sulfate) can be added. In order to suppress foaming during fermentation, a suitable antifoaming agent according to a conventional method,
For example, silicone oil can be added. As the culture method, the aerobic liquid culture method is most suitable as in the generally used antibiotic production method. The culture temperature is suitably 0 to 35 ° C, preferably 20 to 30 ° C. With this method, the production amount of k-18 reaches the maximum in 7 days of culture in both shaking culture and aeration-agitation culture. In this way, a culture with accumulated k-18 is obtained. In the culture, k-18 is partially present in the culture filtrate, but most of it is present in the bacterial cells. From such a culture
Any purposeful method can be used to collect -18. One of the methods is based on the principle of extraction. Specifically, k-18 in the culture filtrate is extracted with a water-immiscible solvent for k-18, such as ethyl acetate. Alternatively, for k-18 in the microbial cells, the microbial cell assembly obtained by filtration, centrifugation, or the like is added with methanol,
There is a method of recovering by treating with ethanol or acetone. The culture itself may be subjected to the above extraction operation without separating the cells. Countercurrent partitioning with an appropriate solvent can also be included in the extraction category. K- from culture
Another method for collecting 18 is based on the principle of adsorption, in which the k-18 content already in liquid form,
For example, a suitable adsorbent such as silica gel, activated carbon, "Diaion H" is used for the culture filtrate or the extract obtained by performing the extraction operation as described above.
P-20 ”(manufactured by Mitsubishi Kasei Co., Ltd.)
Can be adsorbed and then eluted with a suitable solvent to obtain k-18. The k-18 solution thus obtained is concentrated under reduced pressure to dryness to obtain a crude k-18 sample. In order to further purify the crude k-18 preparation thus obtained, the extraction method and adsorption method described above may be combined with a gel filtration method, high performance liquid chromatography, etc., as necessary, and carried out a required number of times. .. For example, an adsorbent such as silica gel, "Sephadex LH-
20 "(manufactured by Pharmacia) or the like, column chromatography using a gel filtration agent such as" YMC Pack "(manufactured by Yamamura Scientific Co., Ltd.), and countercurrent distribution may be appropriately combined. it can. Specifically, for example, the k-18 crude sample was dissolved in a small amount of chloroform: methanol (1: 1), and the active fraction was added to a “Sephadex LH-20” column with chloroform: methanol (1: 1). Elute the fractions and concentrate to dryness to obtain a pure product of k-18. The resulting compound k-1
8 can be converted into a base addition salt as described above by a conventional method.

Use of k-18 The compound k-18 according to the present invention has a calcium overload inhibitory action and an excellent lipid peroxide production inhibitory action by eliminating activated carbon species, as shown in Experimental Examples described later. It is useful in that it has an inhibitory effect on calcium accumulation and lipid peroxide production, specifically, a prophylactic / therapeutic agent for various diseases caused by circulatory disorders in the brain, heart and periphery, and various pathological conditions such as inflammation and edema. Can be expected as.

[Experimental Example] The following experimental example is for more specifically explaining the present invention, and the present invention is not limited thereto.

Examples Example 1: Preparation of k-18 The medium used was prepared by dissolving the components having the following composition in 1 liter of water and adjusting the pH to 7.0. Soluble starch 50.0 g Dry yeast 20.0 g Monopotassium phosphate 0.5 g Magnesium sulphate 0.5 g Calcium carbonate 5.0 g Agar 1.0 g 10 pieces of the above medium 100 ml were dispensed into a 500 ml conical flask with warts. And sterilized, k18 strain (FE
RM BP-3741) was inoculated from the slant into each flask by one platinum loop, and cultured with shaking at 27 ° C. for 7 days. After the completion of the culture, the culture solution was filtered with a paper filter to separate the cells. The cells were extracted with 500 ml of methanol, the extract was concentrated and then extracted twice with ethyl acetate. On the other hand, the culture filtrate was extracted with ethyl acetate, anhydrous sodium sulfate was added to both extracts for dehydration, and the filtrate was concentrated to dryness. A column of silica gel (Kiesel gel 60 manufactured by Merck & Co., Inc.) (3 cmφ x 3
0 cm) and eluted with chloroform. The active fraction was concentrated to dryness to obtain 1.5 g of a crude sample of k-18. Add this to a small amount of chloroform: methanol (1:
It was dissolved in 1), applied to a "Sephadex LH-20" column, and the active fraction was eluted with chloroform: methanol (1: 1) and concentrated to dryness. Furthermore, this was dissolved in ethyl acetate and EDTA diammonium salt aqueous solution (0.1%) was added.
And 0.01 N hydrochloric acid, then water, and the ethyl acetate solution was concentrated. 40 in this concentrate
A double amount of n-hexane was added to cause precipitation, which was collected and dried to obtain 700 mg of a pure k-18 product (yellow powder).

Experimental Example 1: Calcium over k-18
Load Inhibitory Effect Experiment Calcium ionophore A23187 and the compound of the present invention were allowed to act on mouse 3T3 cells cultured in a 96-well microplate for 90 minutes to examine the inhibitory effect of the compound on the toxicity of A23187. Cell survival was measured by the MTT method (J. Immuno. Methods, 65 , 55 (1983)). Table 1 shows the drug concentration range that recovers survival by 50% or more. The compound of the present invention has a calcium ionophore A2 similar to that of tocopherol and flunarizine hydrochloride.
It antagonized the toxicity of 3187.

Experimental Example 2: Inhibition of k-18 production of lipid peroxide
Action experiment The forebrain of Wistar male rat was excised and homogenized with 67 mM phosphate buffer (pH 7.4) to control brain homogenate. Incubation with shaking for 1 hour at 37 ° C in the presence of ascorbic acid generated reactive oxygen species in the brain homogenate, resulting in the production of lipid peroxide. In this reaction, a test drug was added, incubation was carried out in the same manner, and malondialdehyde (MDA) produced in the incubation mixture was measured by the thiobarbituric acid method. Ferrous sulfate was added depending on the conditions as a factor for amplifying lipid peroxidation.
The inhibitory effect on lipid peroxide production of a drug is obtained by determining the inhibition rate of MDA production with respect to the MDA value of a control without addition of a drug.
The concentration at which 50% of the control value was suppressed was shown as an IC ₅₀ value. As shown in Table 1, the compound of the present invention was stronger than tocopherol and exhibited a lipid peroxide production-inhibitory effect similar to that of flunarizine hydrochloride. Table 1 _{Tocofe Furnari Hydrochloride k-18 roll gin} Ca overload inhibitory action 10-80 10-80 10-80 μg / ml Lipid peroxide production inhibitory action IC ₅₀ 10.0 67.1 11.5 μg / ml

[0013]

The compound k-18 of the present invention has a calcium overload inhibitory action and an excellent lipid peroxide production inhibitory action by eliminating reactive oxygen species. Inhibitor,
Specifically, for example, prevention / prevention of various diseases such as inflammation, edema, etc. based on circulatory disorders in the brain, heart, and periphery
It can be expected as a therapeutic agent.

[Brief description of drawings]

FIG. 1 is a copy of an ultraviolet absorption spectrum of k-18 in methanol.

FIG. 2 is a copy of an infrared absorption spectrum of a k-18 KBr disk method.

FIG. 3 is a copy of a 500 MHz proton nuclear magnetic resonance spectrum of k-18 in deuterated chloroform.

FIG. 4 is a copy of a 125 MHz carbon-13 nuclear magnetic resonance spectrum in k-18 deuterated chloroform.

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Claims (1)

[Claims] 1. A compound k-18 represented by the following formula (I) or a base addition salt thereof. [Chemical 1]