JPS5889187A - No14-a substance and its preparation - Google Patents

Abstract

PURPOSE:To prepare a novel substance No14-A substance having antimicrobial actions, its salt and ester, by cultivating a fungus belonging to the genus Aspergillus. CONSTITUTION:A fungus such as Aspergillus terreus Thom No14-strain (FERM- P 6166) belonging to the genus, capable of producing No14-A substance, is subjected to submerged culture at about 30 deg.C for 50-200hr, preferably in a liquid medium, No14-A substance is collected from the culture solution or the culture mold, and, if necessary, it is converted into a salt or ester.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention is a novel substance with antibacterial activity.
- A substance of this invention, its salts and esters, and their production methods, 1
4- The physical and chemical properties of Substance A are as follows.

■Melting point 122°C ■Molecular weight: 248 (Masnuvector method)■Molecular formula: C15H2oO3 ■Specific rotation: [α)D=+29.1° (in ethanol)■Ultraviolet absorption spectrum: λM'-R: 256 nm ax - ■ Infrared absorption spectrum page 5: 2920. λ600-2400 (broad), 1725°1700, 1637.1450
, 1406.1390.1260.

1238.1175.1155.1110.1060.
1040°935.920,890,840,795,
Nuclear magnetic resonance spectrum 1v of hydrogen at 760,640.615o++ 1: As shown in the figure (solvent double chloroform, internal standard: tetramethyl 7 run) ■ Rf value in thin layer chromatography: thin layer Nijiri gel thin layer (thickness 0.25fi) (Merck, pre-coated Kieselgel 60F254) developing solvent
Rf' value benzene:methanol (80:20) 0.3
2 ether: ethyl acetate iv = methanol (45:45:10) 0.62
Rakushu Echi/1/: Methano-μ (93 near)
0.66■ Solubility: Poorly soluble: Water, n-hexane Easily soluble: Methanol, acetone, ethyl acetate, FIV ether, chloroform, benzene [phase] Color reaction: 2.4-dinitrophenylhydrazine reaction Phosphortungstic acid reaction Iron decachloride reaction
-Ninhydrin reaction
-Anthrone reaction -Diphenylamine reaction -Aniline reaction
-〇Crystal color: Colorless The 7a14-A substance of the present invention can be produced, for example, by culturing A14A substance-producing bacteria belonging to the genus Aspergillus in a medium, and separating and collecting the A14-A substance from the resulting culture. Can be done.

Among the A14-A substance producing bacteria used in this invention, a strain newly isolated by the inventors of this invention from the soil of the Osaka Prefectural University farm in Sakai City, Osaka Prefecture (hereinafter referred to as &14 strain)
has the following mycological properties.

■ Morphological properties No formation of sexual reproductive organs is observed.

Numerous asexual reproductive organs are formed and phialoid conidia are produced. Conidiophores are erect, unbranched, and have petropod cells at the base. The tip is swollen and becomes an apical capsule with metre and phialide in the upper half, forming a conidium and forming a cylindrical conidial head.

The conidial head is cylindrical, 50-60μ in diameter and 80-220μ in length, brown to tan, with a white base.

The conidiophore is 50-270μ in length (mostly 100-22μ
0p-), width 4-7μ, smooth surface, colorless, slightly curved.

The apical capsule is 10-15 μm in diameter, hemispherical, and has a metre from the upper 1/2 to 2/3.

Metre is cylindrical, length 4-7μ, width 1.5-2.5μ
,colorless. Followed by one or two Liarrides.

Liaraide has a length of 6-8μ and a width of 1-2μ.

Conidia are spherical or spherical, 2-5 μm in diameter, smooth, and pale colored. No sclerotia are formed.

On malt extract agar medium or MY20 agar medium, spherical cells with a diameter of 5 to 8 μ are formed on vegetative mycelia. .

(4.0 ffi after incubation at 25°C for 110 days). The surface of the colony is woolly and white with shallow grooves, becoming yellowish from the center to lemon yellow. The reverse side develops a dark yellow-orange pigment.

Growth on malt extract agar medium was slightly slower than on Czapek agar medium (colonies with a diameter of 3.0 cll).

The surface has black spots and is powdery to fue/l/)-like. Light brown color with very good conidia formation. The underside is reddish-yellow to yellow-brown;
No pigment is visible.

Growth is fastest on MY2Q agar medium (7.51 diameter).

The colonies are felt-like and light brown, similar to those on malt extraction agar.

As described below, growth is best at 37°C, and 45°C.
It is a thermophilic bacterium that can grow even in the cold.

0 Growth temperature range and optimal growth temperature 14-45°C: Optimal 35-39°C O Growth pH range and optimal growth pH pH 2-9: Optimal pH 3 K, B, Raper), 7' Eli Fuenne IV (D, Eng, Fennell) co-authored 1
'rhe genusas
pergj-11us), page 568, Willi, ams &
WilkinS) Co., Ltd. (1965), Uda Yotoshi - 1. Mycology Illustrated by Keisuke Tsubaki et al., 1000 pages, Kodansha (197
As a result of comparing this A14 strain with the description of Aspergillus terreus (L.
lus terreus Thom). In addition, this Aspergillus 4us tom & 14 strain was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute No. 6166.
It has been deposited as No.

The &14 substance-producing bacteria belonging to the genus Aspergillus used in this invention can be treated with irradiation such as X-rays and ultraviolet rays, such as lytlodiene, mustatoceazaserin, nitrous acid, etc.
2-7 Minopurine, N-Mef-yv-N'-Nitro-N
- Black 14-A
The production capacity of substances can be increased.

A9 is carried out by culturing A14-A substance-producing bacteria belonging to the genus Aspergillus in a medium. Production of 14-A substances is in principle based on the culture method of general microorganisms, but is usually deep cultured in a liquid medium. The law is favorable. The medium used is A14, which belongs to the genus Aspergillus.
-A medium may be used as long as it contains a nutrient source used by substance-producing bacteria. That is, a synthetic medium, a semi-synthetic medium, or a natural medium is used, and the medium composition includes carbon sources such as glucone, shakerose, maltose, glycerin, starch, and liquefied starch, and nitrogen sources such as meat. Extract, casein hydrolyzate, peptone, gluten meal, corn meal, cottonseed meal, soybean flour, corn steep liquor, dried yeast, yeast extract, urea, ammonium sulfate, etc. are used. In addition, inorganic salts such as disodium hydrogen phosphate, potassium dihydrogen sinate, magnesium chloride, magnesium sulfate, and calcium carbonate are also added to the medium as necessary.

In addition, when foaming is significant during culturing, for example, vegetable oils such as soybean oil and linseed oil, octadecanol, tetrazomenol,
Antifoaming agents such as higher alcohols such as heptatool and silicon compounds may be added as appropriate.

A suitable culture temperature is around 50°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 200 hours, and as the medium becomes more concentrated, the culture time may be further lengthened.

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 &14-A substance produced during culture usually accumulates both inside and outside the bacterial cells in the culture, it is generally After separating into a filtrate (supernatant) and a filtrate (supernatant), both are separated, purified, and collected by means used in the production of general antibiotics. That is, the target substance within the bacterial cells is usually separated and extracted from the bacterial cells by 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, A14-A substance, by combining or repeatedly applying methods such as treatment with an adsorbent such as acid, silica gel, alumina, or cellulose, crystallization, or recrystallization in any order. be able to.

First, by treating the A14-A substance thus obtained by a conventional method, a salt thereof can be obtained.

Examples of such salts include pharmaceutically acceptable salts such as alkali metal salts such as sodium salts and potassium salts.

The presence of a carboxy group in the 414-A substance is recognized from the above-mentioned physical and chemical properties.

Therefore, when the Mura 4-A substance is subjected to an esterification reaction by a conventional method, an ester form of the A14A substance can be obtained.

Preferred examples of the ester form of the A14-A substance obtained in this way include ethyl ester, ethyl ester,
Propyl ester, isobutyl ester, t-butyl ester, lower alkyl ester such as hexyl ester, benzyl ester, phenethyl ester, etc.
Examples include L/(lower) alkyl ester.

Next, the biological properties of the A, 14-A substance of this invention will be shown.

■ Measurement of antibacterial activity in vitro: Test Example 1 The minimum inhibitory concentration (MIC) of the A14A substance against each microorganism was determined by the agar dilution method using bouillon agar for bacteria and Sabouraud agar for mold and yeast.
was measured.

(ES(3heri, Qhla stop stop 1'1.-12
) ■ Toxicity test: Intraperitoneal administration of A14-A substance to mice (2004119
/kq) showed no toxicity. .

Next, the biological properties of the ester of substance 71114-A of this invention will be shown.

■ Measurement of in vitro antibacterial activity: Test Example 2 The in vitro antibacterial activity of the methyl ester of substance 14-A obtained in Example 2 was measured in the same manner as in Test Example 1.

Strain MIC (mQE!, /yxl) Staphylococcus aureusae'F0 3060
12.5 Norirarasu) - Masu A chair IFO122
1025 Microconocanus roseus EFO3768
25 Eshilichia coli K-12>50 tγ″f7 $Kinshishi=≠=・Malto1z→?ance IFO1264
8>50Nyudo Seven eggplant aeruginosa IFO3923
>50 Satsukaromyces cerevisiae DingFO13461
2,5 Cancita albicans IFO1594>5
0 Penicillium chrysogenum F'0 4897
12.5 Fusarium oxpo! Rem EFO5
880>50 As mentioned above, /15.14- of this invention
Substance A, its salts, and its esters are each new substances, have antibacterial effects, and are useful as pharmaceuticals or veterinary drugs.

The A14-A substance, its salts, and its esters are combined with various carriers and administered as preparations commonly used as agents for preventing and treating bacterial infections. Examples of such formulations include capsules, granules, powders, tablets, troches, pills, syrups, ointments, suppositories, injections, and injections.

Next, the present invention will be explained with reference to examples.

Example 1 // Production of a14-A substance Glucose 5.0y, defatted soybean flour 2.5y, yeast extract 0.5F, monopotassium phosphate 1.0y% magnesium sulfate heptahydrate 1. OjE, sodium chloride 05y, calcium chloride dihydrate o, 5y.

Ferric chloride dihydrate 2.0■, zinc sulfate heptahydrate 2
．． A medium consisting of 0.0 ■ and water 11 was adjusted to pH 5.5,
Put the 100+ w/ into a 500+/ flat bottom flask,
After sterilization at 120°C for 15 minutes, Aspergillus terreus tom A1 strain was inoculated and cultured at 30°C for 5 days. The culture solution 1e obtained as described above was filtered, the pH of the furnace solution was adjusted to pH 3.0 with hydrochloric acid, extracted by shaking with ethyl acetate, dehydrated with anhydrous sodium sulfate,
It was concentrated under reduced pressure at °C to obtain a crude extract.

Silica gel containing 1y of this extract dissolved in benzene (
benzene,
Elution was performed with a mixed solvent of ethyl acetate. The fractions eluted with a mixed solvent of benzene: ethyl acetate/l' (93 nia) were collected and concentrated to obtain an oily /Fl114-A substance (70
0~) was obtained.

Example 2A, Preparation of methyl ester of substance 14-A - Calbitort P in the presence of an aqueous solution of potassium hydroxide
-)/reninsulfonyl-N-methyl-N-nitrosamide in ether solution was added to generate diazomethane gas to obtain 500η of A14-A substance as an oily substance in a 10% methanol-containing ether content of approximately 90%. .

[Brief explanation of drawings]

The drawing shows the nuclear magnetic resonance spectra of hydrogen of the A14A material obtained according to the present invention. Applicant: Hira Sakai -

Claims (1)

[Claims] 1. Substances 14-A, salts thereof, and esters thereof, having the following physical and chemical properties (1) to (2). ■Melting point: 122°C ■Molecular M: 248 (mass spectrometry) ■Molecular formula: C
15H2oO5 ■ Specific rotation: [α)2D0= + 2'9.1°C (
(in ethanol) ■ Ultraviolet absorption spectrum: λ1 tanol: 236 nm a
1'), 1725, 1700.1637, 1450
．． 1406.1390. 1260.1258.1175, 1155, 1110,
1060°1040.935,920,890,840
,795,760°640.6151' ■ Nuclear magnetic resonance spectrum of hydrogen: As shown in the figure (molten g
: deuterated chloroform, internal standard: tetramethylsilane) ■ Rf value in thin layer chromatography: thin layer of Nijiri gel (thickness: 0.251111) (manufactured by Merck & Co., Ltd.)
Pre-coated Kieserge/L/60F254) Benzene: Methanol) lz (80:20) 0.3
2 ether per ethyl acetate) v: methanol (45:45:10) 0.62 ethyl acetate lv: methanol (93 nia) 0.66■ Solubility: poorly soluble: water, n-hexane easily soluble: methanol, Acetone, ethyl acetate, ethyl ether, chloroform, benzene [phase] Color reaction: 2.4-dinitrophenylhydrazine reaction Tenphosphotungstic acid reaction Decachloride reaction
-Ninhydrin reaction Anthrone reaction -Diphenylamine reaction -Aniline reaction
-〇 Crystal color: colorless 2.7 Production of &14 substance, characterized by culturing A14-A substance producing bacteria belonging to the genus Svergillus in a medium, and separating and collecting black 14-A substance from the resulting culture Law. 5. A method for producing an ester of &14, which comprises esterifying the &14 substance to obtain an ester of &14.