JPH0479639B2 - - Google Patents

Description

[Detailed description of the invention]

This invention describes the novel pharmacologically active substance FR-
900220 Concerns substance producing bacteria. More specifically, the novel FR-
Amaurocas sp. No. 900220 producing substance.
6237 and its mutant strains. The inventors are in the process of conducting research to screen for substances produced by microorganisms that have a hypotensive effect, and are currently conducting research on No. 1, which belongs to the genus Amauroascus.
They discovered that the 6237 bacterium produced the novel FR-900220 substance, and as a result of further intensive research, they completed this invention. Among the FR-900220 substance-producing bacteria, this bacteria was newly isolated by the inventors from rotten wood in a tree hollow in Mt. Rokko, Hyogo Prefecture (numbered No. 6237).
The mycological properties of the following are shown below. (1) Growth status on each medium Place strain No. 6237 in small dots in the center of a plate made with potato-glucose agar, Tuapetsk agar, malt extract agar, YpSs agar, oatmeal yeast extract agar, and Bennett agar medium. The cells were inoculated and cultured at 30°C for 10 days, and the bacterial colonies during culture were observed. The results are as follows.

[Table] () Fruiting organs: In general, in old cultures with good growth medium, small yellow poppy-like granules appear here and there near the surface of the colony. Observation of this part with an optical microscope reveals that the cleistoma (size:
The presence of ascospores (size: 4 to 600 μm or more, shape: loose net-like oval or spherical, color: yellow) was observed.
Asci (size: 12-14 x 16-20μ, shape: oval, color: colorless and transparent) containing 6μ, shape: spherical with spiny appendages on the surface, color: yellow to orange) They are arranged irregularly. () Asexual spores: All aerial hyphae rupture in old cultures and become asexual spores (arthrospores). () Morphological properties of vegetative hyphae: There are septa and chlamydospores are observed.

[Table] (2) Physiological properties () Optimal growth conditions (on YpSs agar medium) pH: 6-8 Temperature: 22-33℃ () Growth range pH: 5-10 Temperature: 14-38℃ (3) Morphological properties based on slide specimens Fruiting organs, asexual spores, vegetative hyphae, shape on the substrate, and color tone are the same as described above. (4) Phenol oxidase reaction negative Regarding the mycological properties of strain No. 6237, F.
The Genera of Huangjiai by FEClemens and CLShear
Fungi (1964), A manual of Soil Fungi (1964), A manual of soil fungi by Joseph C. Gilman.
Soil Fungi (1959), HHKuehn, K.Tubaki
and Mycologia, Vol. 56, pp. 863-872 (1964) by J.F. Or, Persoonis, Vol. 6, Part 3, by JAVon Arx.
Referring to the description on pages 371-380 (1971), No.
From the characteristics of its fruiting organs, it is reasonable to judge that strain 6237 belongs to the genus Amaurcascus of Gymnoascaceae. Therefore, the No. 6237 stock was transferred to Amauroascus.
It was named Amauroascus sp.No.6237. This Amauroascus sp. strain No. 6237 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbial Accession No. 5364, and with the American Type Culture Collection as ATCC No. 20595. The FR-900220-producing bacteria belonging to the genus Ayauroascus used in this invention can be treated with irradiation such as X-rays or ultraviolet rays, such as nitrogen mustard, azaserine, nitrous acid, 2-aminopurine, N
- Production of FR-900220 substance by commonly used bacterial strain mutation treatment methods such as treatment with mutagenic agents such as methyl N'-nitro-N-nitrosoguanidine (NTG), phage contact, transformation, transduction, and conjugation. ability can be improved. The production of the FR-900220 substance by culturing the FR-900220 substance producing bacteria in a medium is in principle based on the culture method of general microorganisms, but the deep culture method using a normal liquid medium is advantageous. The medium used for culture may be any medium containing a nutrient source utilized by the FR-900220 substance-producing bacteria belonging to the genus Amauroascus. That is, a synthetic medium, a semi-synthetic medium or a natural medium is used,
The medium composition includes carbon sources such as glucose,
Sucrose, maltose, glycerin, starch, liquefied starch, etc. are used, and as nitrogen sources, for example, meat extract, casein hydrolyzate, peptone,
Gluten meal, corn meal, cottonseed meal, soybean flour, corn steep liquor, dried yeast, yeast extract, urea, ammonium phosphate, etc. are used.
In addition, inorganic salts such as disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium chloride, magnesium sulfate, and calcium carbonate are also added to the medium as necessary. Further, when foaming is significant during culturing, antifoaming agents such as vegetable oils such as soybean oil and linseed oil, higher alcohols such as octadecanol, tetradecanol, and heptanol, and silicon compounds may be appropriately added. A suitable culture temperature is around 30°C, and good results are often obtained by carrying out seed culture as appropriate as the culture volume increases. The appropriate culture time for the main culture is about 50 to 100 hours, and the culture time may be further extended as the medium becomes more concentrated. The culture conditions described above are applied by selecting the optimum conditions for each depending on the characteristics of the production strain used. Next, since the FR-900220 substance produced by culture usually accumulates both inside and outside of the bacterial cells in the culture, it is generally necessary to remove the bacterial cells and the filter by means such as centrifugation or filtration. After separating into a liquid (supernatant), both are separated, purified, and collected by means used in the production of general antibiotics. That is, the target substance within the microbial cells is usually separated and extracted from the microbial cells by oil extraction means such as solvent extraction. This extract and the filtrate (supernatant) are subjected to vacuum concentration, solvent extraction, liquid conversion, treatment with resins such as anion exchange resins, cation exchange resins, and nonionic adsorption resins, such as activated carbon, silica, etc. Separate and purify the target substance, FR-900220 substance, by combining or repeatedly applying methods such as treatment with adsorbents such as acid, silica gel, alumina, and cellulose, crystallization, and recrystallization in any order. be able to. By treating the FR-900220 substance obtained in this way with an acid such as hydrochloric acid, sulfuric acid, maleic acid, or fumaric acid in accordance with a conventional method, an acid addition salt thereof can be obtained. FR−900220 obtained by the above manufacturing method
Substances have the following physical and chemical properties. (a) Elemental analysis value (%) C74.00; H7.18; N10.64 (b) Molecular weight: 508.2825 (high resolution mass spectrometry) (c) Molecular formula: C ₃₂ H ₃₆ N ₄ O ₂ (d) Melting point : 156-158℃ (e) Specific optical rotation: [α] ²⁸ _D = -583° (C = 1, in chloroform) (f) Ultraviolet absorption spectrum (Figure 1): λethanol max: 245nm (ε = 11000 ), 300nm (ε=4200) (g) Infrared absorption spectrum (Figure 2): ν ^KBr _nax : 3350, 2940, 2850, 1650, 1600, 1480,
1464, 1415, 1380, 1362, 1310, 1300,
1287, 1270, 1247, 1208, 1180, 1145,
1115, 1075, 1055, 1032, 1015, 1005, 980,
967, 912, 890, 830, 817, 795, 780, 752,
738, 690, 655, 595, 565, 512, 480, 453cm
^-1 (chi) ¹ H-NMR spectrum: δppm (CDCI ₃ ): 1.01 (3H, s), 1.10 (3H, s),
2.45 (2H, d, J = 9Hz), 3.75 (1H, t, J
=9Hz), 4.9-5.2 (2H, m), 5.10 (1H, s,
Disappeared with D ₂ O), 5.45 (1H, s), 5.8-6.1 (1H,
m), 6.4-7.2 (4H, m) (li) ¹³ C-NMR spectrum: δppm (CDCI ₃ ): 22.5 (q), 22.81 (q), 35.19
(t), 40.71(s), 60.25(d), 61.76(s),
77.11(d), 109.15(d), 114.25(t), 118.61(d),
124.74(d), 128.69(s or d), 128.81(d or s), 143.49(d), 149.92(s), 166.06(s) (nu) Crystal form and color: Colorless columnar crystals Above physical and chemical properties and From the results of separate research
The chemical formula of the FR-900220 substance was determined as follows. Next, the pharmacological effects of the FR-900220 substance will be explained using the following test examples. Test example 1 [Spontaneous hypertensive rats]
Blood pressure lowering effect on Hypertensive Rat (hereinafter abbreviated as SHR) Male SHRs, 6 months old and weighing 300 to 380 g, were used as test animals. FR-900220 substance was suspended in physiological saline containing gum arabic, and this was administered intravenously, intraperitoneally and orally to rats.
Blood pressure was measured invasively through a cannula inserted into the abdominal aorta of the rat. The results are shown in Tables 1, 2 and 3 below.

【table】

【table】

[Table] Test Example 2 [Hypertensive effect on Donryu rats] Using 12-week-old male Donryu rats weighing 350 to 400 g as test animals, FR-900220 was administered in the same manner as Test Example 1. The substance was tested for its hypotensive effect. The results are shown in Table 4 below.

[Table] Test Example 3 [Relaxation effect on isolated blood vessels] Aortas isolated from rats and guinea pigs were treated by the conventional method using a tank of Magnus.
The relaxing effect of FR-900220 was measured. Table 5 shows the results.
Shown below.

[Table] Test Example 4 [Infection Prevention Effect] 5-week-old DDY male mice (5 mice per group) were given the following predetermined concentration of FR-900220 substance suspended in physiological saline containing gum arabic once a day. Administer intraperitoneally and orally for 4 days. The day after the final dose,
Candida albicans
Mice are infected by intravenously injecting 0.25 ml of a diluted overnight culture (4×10 ⁶ viable cells/ml) of C. albicans. The results of determining whether the mice were alive or dead on the 5th day after infection are shown in Table 6 below. Note that control mice were administered only physiological saline containing gum arabic before infection.

[Table] In the same manner as above, the infection prevention effect of substance FR-900220 against Escherichia coli was investigated. The results are shown in Table 7 below.
(The amount of Escherichia coli inoculated was 10 ⁵ viable bacteria/mouse.)

[Table] Test Example 5 [Acute Toxicity] Substance FR-900220 was suspended in an aqueous solution containing 0.5% methylcellulose, and this suspension was intraperitoneally administered to eight 7-week-old DDY male mice (dose
200mg/Kg) and observed for one week after administration. As a result, there were no deaths. As mentioned above, the FR-900220 substance of this invention is
In addition to having a blood pressure lowering effect and being useful as a blood pressure lowering agent, it also has an infection prevention effect against pathogenic bacteria and is useful as an agent for preventing and treating infectious diseases. FR−900220
Although the substance and its pharmaceutically acceptable acid addition salt can be administered to mammals including humans as such, they are generally administered as a preparation in combination with various pharmaceutically acceptable carriers. Examples of such formulations include capsules, tablets, granules, powders, solutions, and the like. Further, examples of pharmaceutically acceptable acid addition salts include hydrochloride, sulfate, and the like. Further, pharmaceutically acceptable carriers include, for example, sucrose, starch, mannitrate, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, methyl cellulose,
In addition to hydroxypropyl cellulose, gelatin, gum arabic, carboxymethyl cellulose, magnesium stearate, surfactants, water and the like, carriers commonly used in the above formulations may be used. The dosage of FR-900220 substance and its pharmaceutically acceptable salts depends on the type of disease, patient weight,
It varies depending on age and administration method, but usually 10Kg or more
The optimal dosage is often selected within the range of 100 mg/Kg/day. Next, using the FR-900220 substance-producing bacteria of this invention.
An example of manufacturing the FR-900220 substance will be explained. Production example: 2.0% cornstarch, 1.0% soybean flour, dry yeast
100 ml of medium (PH 7.0) containing 1.0% corn steep liquor and 0.5% glucose
Dispense each into 25 500ml shake flasks and heat at 120°C.
After sterilization for 20 minutes, one platinum loopful of Amauroascus sp. No. 6237 was inoculated and cultured at 30°C for 72 hours. Separately, a medium containing 2.0% glucose, 0.25% corn steep liquor, 0.25% dry yeast, 0.5% cottonseed meal, and 0.5% wheat germ (PH 7.0) was divided into 5 x 30-volume jar fermenters. Note,
Sterilize at 120 °C for 20 minutes. Inoculate each jar fermentor with 400 ml of the preculture obtained above.
Incubate at 30°C for 72 hours. An equal volume of acetone is added to the obtained culture 75, stirred and filtered. Concentrate the filtrate under reduced pressure to a volume of 3. Add this concentrate to 6N hydrochloric acid.
After adjusting the pH to 2.0, extract twice with 3 portions of ethyl acetate.
The extracts are combined, neutralized with 6N aqueous sodium hydroxide solution, and then concentrated to dryness under reduced pressure. This concentrate is subjected to column chromatography using silica gel (1.2). The column was washed with 3 portions of benzene, and the target substance was eluted with 800 ml of a mixture of benzene and ethyl acetate (10:1). The eluate was concentrated to dryness under reduced pressure to obtain 2.08 g of an oil. This oil was dissolved in 30 ml of a mixture of chloroform and methanol (98:2) and subjected to column chromatography using silica gel (1.0). When the column is developed with the same solvent, the target substance is eluted in fractions 1.2 to 1.4 from the start of elution. This fraction is concentrated under reduced pressure to obtain 1.5 g of crude crystals of the target substance. The crude crystals were recrystallized from hot methanol to obtain 1.35 g of colorless prismatic crystals of FR-900220 substance.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of the FR-900220 material, and FIG. 2 shows the infrared absorption spectrum of the FR-900220 material.

Claims (1)

[Claims] 1 Amauroascus sp No. 6237, which is characterized by producing FR-900220 substance.