JPS6210088A - Novel antimicrobial substance ca-146a and ca-146b - Google Patents

Abstract

NEW MATERIAL:Antibiotic CA-146A and CA-146B shown by the formula (R is methyl or isopropyl) having the following physical and chemical properties (in the case of CA-146A where R is isopropyl). White amorphous powder and ampholytic substance. Molecular weight: MS m/z(M+H)<+>316. Color reaction: positive in Ehrlich reagent positive. Solubility: soluble in water, DMF, and dimethyl sulfoxide, slightly soluble in alkanol, insoluble in chloroform, dichloromethane, benzene, and ether, etc. USE:A mildewproofing agent and an agent for alleviating candidiasis. PREPARATION:A mold [e.g., Streptomyces lavendulae CA-146(FERM P-8314), ETC.,] belonging to the genus Streptomyces, capable of producing CA-146A and/or 146-B, is cultivated preferably by liquid culture at 25-35 deg.C usually for 40-120hrs.

Description

Detailed Description of the Invention A. Field of Industrial Application The present invention relates to novel antibacterial substances, namely, CA-146A represented by the formula (■); CA-146B represented by the formula (■); The present invention relates to pharmaceutically acceptable salts of, methods for producing them, and microorganisms that can produce them.

BACKGROUND OF THE INVENTION Lavam antibiotics, such as the compounds of the present invention, are compounds that have been widely studied, and various lavam derivatives are known (for example, Japanese Patent Application Laid-open No. 53-21194; Japanese Patent Publication No. 57-14).
4290; Tetrahedron Letters (Tatrahad)
Ron Letters), Vol, 22.
No.

27, pp2539-2540. t9st; The Journal of Antibiotics (The J,
Antibiot, 1cs), Vol, 28.
No. 3.208-225.1983). However, the compound of the present invention is a novel compound having a structure different from these.

C.1 Problems to be Solved by the Invention Recently, with the frequent use of various antibiotics, fungal diseases, particularly candidiasis, caused by bacterial alternation have become a problem. Candidiasis is mainly caused by Candida albicans
da albicans).

It is highly pathogenic to humans and animals and can cause fatal infections. Amphotericin B is said to be effective against candidiasis, but it has strong side effects (Dictionary of Medical Sciences,
Although conventional tarabum antibiotics have strong antibacterial activity, their antifungal activity is not very strong.

21. Means for Solving Problems The present inventors conducted intensive research to find flavum antibiotics that have strong antifungal activity, especially anticandida activity, and found that a certain strain belonging to the genus Streptomyces has antifungal activity. It has been found that compounds represented by the following formulas having particularly strong anti-Candida activity are produced and named CA-146A and CA-146B, respectively.The present invention relates not only to these compounds but also to their pharmaceutically acceptable salts. Also includes.

The physicochemical properties of the compound of the present invention are shown below.

a, CA-146A (1) Structural formula (2) Ultraviolet absorption spectrum No specific absorption 3) Infrared absorption spectrum (see Figure 1) IR (KBr
):3380.1775.1670.1605.152
5.1380.1345.1190.1120.104
2.970cm-' (4) C1rcular DichroismCD
λI″10...[θ]: 265(0), 227
゜5(-41600), 20B(0), 200 nm(
+38200) (5) Mass spectrometry spectrum (see Figure 2) MS m/
z: 316 (M+H)'' (6) Nuclear magnetic resonance spectrum (see Figure 3) 'H-NM
R (200MH2, D!O, extTMS) J=Hz
S: 4.79 (4% J-2,6, IH), 3.81
(d, J-3, 1, IH), 3.64 (m, IH), 3
．． 54 (dd, J-7, 4, 3, 1, IH), 3.32
(dd, J ■ 1167.6.4, IH), 3.23 (I
H), 2.71 (dd, J■16.7.2.6, IH)
, 2.41 (dd, J-11, 7, 5, 8, IH), 2
．． 22 (d, J-16,7, IH), 1.66 (m, I
H), o, 59 (4% J-7,1,3H), 0.36
(d, J-7,2,3H)”C-NMR (50MHz,
D*O1e x t CHsCNS CHs” 1,
7 ppm) &: 182.5(s), 176.3
(8), 170.1(a), 85.4(d), a2.5
(d),? 2.6(d), 59.4(d), 57.8(
d), 48.3(t), 45.1(t), 30.8
(d), 1s, 6(q), 17.4(q) (7) Thin layer chromatography developing solvent Chloroform: Ethanol: Water-4Nia:
2 Rf'-〇, 35 (8) High performance liquid chromatography column: Hypersil 5C1g, φ4.6X
150 mm Mobile phase: 0.05M phosphate buffer (pH
6,0) Flow rate: 1ml/min, detection wavelength: 220
nmRt=4.8m1n.

(9) Color reaction di-Rietsch reagent Positive (10) Solubility Soluble in water, dimethylformamide, dimethyl sulfoxide, alkanol! ! 11 soluble, insoluble in chloroform, dichloromethane, benzene, and ether (11) Properties amphoteric substance, white amorphous powder b, CA-146B (1) Structural formula (2) Nuclear magnetic resonance spectrum (see Figure 4) 'H- N.M.
R (200MHz, D*O, extTMS)
J=Hz &:4.79 (d, J-2,6, IH),
3.81 (4% J■2.6, IH), 3.62 (m,
IH), 3.57 (m, IH), 3.46 (IH), 3
．． 31 (dd% J■11°5.6.0, IH), 2.
72 (dd, J-16, 7, 2, 7, IH), 2.42
(dd, J ■ 11.5.5.5, IH), 2.23 (4
% J-16.7, IH), 0.89 (d, J-7,
0°3H) (3) Thin layer chromatography development ig Chloroform: Ethanol: Water■4Nia:
2 Rf'-0,25 (4) High performance liquid chromatography column: Hypersil 5C18, φ4.6 x
150 mm mobile phase, 0.05 M phosphate buffer (pH
7,0) Flow rate: 1ml/min, detection wavelength: 220
nmRt=3.1m1n.

(5) Color reaction technique - Riech's reagent Positive (6) Soluble Soluble in water, dimethylformamide, dimethyl sulfoxide, sparingly soluble in alkanol, Insoluble in chloroform, dichloromethane, benzene, ether (7) Amphoteric substance, white non-soluble Crystalline powder As mentioned above, CA-146A and CA-146B have properties different from known flavum antibiotics,
It is judged to be a new flavum derivative.

The bacteria producing CA-146A and/or CA-146B include: - actinomycetes CA-14 isolated from a soil sample;
Six stocks are listed.

Based on the results of taxonomic examination, this strain is classified as Streptomyces lavendule
Streptomyces labengulee CA-146 was identified as being the same species as Streptomyces labengulee
, has been deposited as Microtechnical Research Institute No. 8314.

The mycological properties of this strain are as shown below.

(1) Morphological properties (Pennet agar medium, 28°C1
14-day culture) On this medium, this strain exhibits good growth, formation of aerial hyphae, and spore settlement. The morphology of the spore chain is straight. The surface structure of the spores was observed by electron microscopy and was smooth, with no sporangia, flagellated spores, or sclerotia observed, and no fractures due to fragmentation in the basal hyphae.

(Margin below) Growth temperature (Pennet agar medium, cultured for 14 days at each temperature) 10°C: No growth.

28°C: Growth, aerial mycelium formation, and spore settlement were all good.

37°C: Growth is quite good, but no aerial mycelium is formed.

45°C: No growth.

(3) Physiological properties (cultured at 28°C for 14 days) Melanoid pigment production ability Positive tyrosinase reaction Coagulation of weakly positive milk Peptonization of negative milk Positive gelatin liquefaction ability Negative starch ice-melting ability Positive (margin below) (4) Availability of carbon source (5) Composition of cell wall The type of diaminopimelic acid is LL-type.

From the above properties, it is clear that this strain belongs to the genus Streptomyces. Waxman, The Actinomycetes
es) Volume 2 (1961), Scherling and Gottlieb's I, S, P, (Intern
a-tional Steptomyces Proj
ect) report [International Journal of Systematic Bacteriology (Interna
tional Journal of Systemat
ic Bacteriology) Volume 18 (1968
), Volume 19 (1969), Volume 22 (197
2 years)] and Bargey's Manual of Determinative Bacteriology (Bargey's Manual of Determinative Bacteriology)
Manual of Dataminative B
When we searched for related species of this strain from the 8th edition (1974) and other literature announcing new species of actinomycetes, we found that Streptomyces vendulaa
) [International Journal o
f Systematic Bacteriology Vol. 18, pp. 137-138 (1968), Berge
y's Manual of Determinati
veBacteriology, 8th edition, pages 808 and 810 (1974)] are listed as the most closely related species. Streptomyces labengelae and CA-14
As a result of comparing the properties of the six strains, the main properties of both strains were in good agreement. Therefore, strain CA-146 was identified as being homologous to Streptomyces lavengulee, and Streptomyces lavengulee CA-146
mycas 1avendulae CA-146).

In the present invention, not only the above-mentioned CA-146 strain and its natural and artificial mutant strains, but also all strains that belong to the genus Streptomyces and produce CA-146 can be used, and are within the scope of the present invention. shall be.

Production of CA-146A and/or B is based on CA-14
CA-146A and/or B producing strains are cultured in a nutrient medium under aerobic conditions, and CA-146A and/or B are separated and collected from the culture after completion of the culture.
A general method for manufacturing A-146A and/or B is described.

As for the culture medium composition, culture conditions, etc., those generally used in the production of antibiotics may be used. In principle, the medium contains carbon sources, nitrogen sources, inorganic salts, etc., and vitamins, precursors, etc. may be added as necessary. As the carbon source, for example, glutenose, starch, dextrin, glycerin, molasses, organic acids, etc. can be used alone or as a mixture. As the nitrogen source, for example, soybean flour, cornsteep liquor, meat extract, yeast extract, cottonseed flour, peptone, wheat germ, ammonium sulfate, ammonium nitrate, etc. are used singly or as a mixture. Examples of inorganic salts include calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, copper sulfate, manganese chloride, zinc sulfate, cobalt chloride, and various phosphates.
Add to the medium as needed.

Cultivation may be carried out according to methods generally used for the production of antibiotics, preferably liquid culture, and deep aeration culture for mass production. If the pn of the medium is variable, a buffer such as calcium carbonate may be added to the medium, and the culture is preferably carried out at about 20 to 45°C.
Particularly preferred is 25 to 35°C. Although the culture time largely depends on the scale of fermentation, the culture time required for mass production is about 40 to 120 hours. If severe foaming occurs during culturing, for example, before or during culturing,
Antifoaming agents such as vegetable oil, lard, and polypropylene glycol may be added as appropriate.

After culturing, CA-146A and/or B are extracted from the culture.
In order to separate and collect the fermentation products, a conventional method for separating and collecting fermentation products is used as appropriate. For example, filtration, centrifugation, adsorption/desorption using various ion exchange resins or other active adsorbents, chromatography, extraction using various organic solvents, etc. may be appropriately combined.

CA-146A and B are used for separation operations and also for pharmaceuticals,
For convenience of use as a veterinary drug, it may be desirable to form it into a salt. Bases that can form salts with CA-146A and B include alkali metals such as potassium and sodium;
alkaline earth metals such as aluminum and magnesium;
Examples of acids include inorganic acids such as hydrochloric acid and sulfuric acid, and organic acids such as acetic acid and fumaric acid.

CA-146A, B and their pharmaceutically acceptable salts can be administered orally or parenterally to humans or animals as active ingredients of antifungal agents. Using general-purpose excipients, stabilizers, preservatives, wetting agents, surfactants, etc., it can be administered orally as a tablet, capsule, or powder, or parenterally as an injection or liniment. You can also do that. The dosage varies depending on the therapeutic purpose, age of the patient, symptoms, etc., but when administered orally, it is approximately 50 to 500 mg per day for adults.

E. Examples Production examples of CA-146A and CA-146B are shown below, but these Examples are not intended to limit the present invention in any way.

Example 1 Fermentation process Soluble starch 0.5%, glucose 0.5%, polypeptone 0.5%, beef extract 0.5%, yeast extract 0.25
%, 0.25% salt, and 800 ml of medium consisting of deionized water (pH 7.0, before sterilization) 2f! Inoculate a seed culture slant of Streptomyces labengelae CA-146 (Feikoken Bacteria No. 8314) into an Erlenmeyer flask, and perform shaking culture at 28°C for 148 hours at 180 revolutions per minute.
Add 800ml each of this culture solution to 2% glucose and raw! Flour 2%, defatted soy flour 2%, yeast extract 0.5%, salt 0.
25%, sulfur #copper heptahydrate 0. QOO5%, manganese chloride tetrahydrate o, ooos%, zinc sulfate heptahydrate o, ooo
s%, precipitated calcium carbonate 0.35%, water (pH 7,
0.0, before sterilization) was inoculated into a 3ON jar fermenter containing 2ON of a medium, and the aeration volume was 20! /min, internal pressure 0.
2 kg/cm"G, stirring number 150-350 rpm, 2
Incubate at 8°C for 65 hours.

Example 2 Separation Step A supernatant liquid 130P is obtained from the culture solution obtained in the above step by centrifugation. After adjusting this supernatant to pH 6.5,
Pour it into an ON activated carbon column for chromatography, wash it with 4ON water, and then wash it with 30j of 60% acetone water! Elute with 400ml
The active fractions were collected by pulp disk diffusion method using yeast, adjusted to pH 7.0, concentrated under reduced pressure, and lyophilized to yield 36 g of coarse powder containing CA-146A and coarse powder containing CA-146B. 632
get g.

Example 3 Purification of CA-146A 36g of crude powder of CA-146 was made into a 120ml aqueous solution and Q
Apply to a column of AE-Sephadex A-25 (Pharmacia Fine Chemicals) 1 OOml. Elute with water, collect active fractions, make 20% saline solution, MCI
Pass through a 100ml column of GEL CHP-20P (Mitsubishi Kasei), elute with 15% saline to water, collect active fractions, concentrate under reduced pressure below 30°C, and re-inject into MCI GEL C
Pour into HP-20P, elute with 5% saline, collect active fractions, adjust to pH 6.5, concentrate, and flow into 600 ml of Biogel P-2 (Bio-Rad Lab). Collect pure fractions while tracking with HPLC. , after concentration SP-Sephadex C-25 (Pharmacia Fine Chemicals) 10
0 ml, eluted with water, and lyophilized the active fraction.
182 mg of -14aA amorphous powder is obtained.

Example 4 Purification of CA-146B 60 g of crude powder of CA-146 was dissolved in 240 ml of Q
It was applied to a 200 ml column of AE-Sephadex A-25 (Pharmacia Fine Chemicals), eluted with water, the active fraction was collected, made into a 15% saline solution, and added to MCI.
Pass it through a 200 ml column of GEL CHP-20P (Mitsubishi Kasei), elute with 15% saline, collect the active fraction, and collect the active fraction.
Adjust to H7, o, wet activated carbon (Wako Tsumugi) 200
ml to desalt. Then, it was eluted with 60% acetone-water, the active fraction was adjusted to pH 7.0, frozen, and
Obtaining 3.05 g of residue, then biogel P-2
(Bioland Lab) Pour into 600 ml and elute with water, adjust the active fraction to pH 6.5, concentrate, and then Sephadex G-
10 (Pharmacia Fine Chemicals). Elute with water, concentrate the active fraction, Dowex IX2 (CI
, Bio-Rad Lab) and elute with water.
The active fraction was concentrated and frozen at pH 6.5, and CA-146B
37 mg of containing residue are obtained. Make this a 15% salt solution and M
(r Pour into 100 ml of GEL CHP-20P and elute with 15% saline, adjust the active fraction to pH 6.5 and concentrate, then pass through 600 ml of Biogel P-2 and elute with water.
After concentrating the active fraction, it was poured into SP-Sephadex C-25 (Pharmacia Fine Chemicals) and eluted with water, and the active fraction was concentrated, frozen, and dried to obtain 2 mg of CA-146B.

CA-146A(7)i was determined by the minimum inhibitory concentration (MIC) by broth dilution method and agar plate dilution method.
n shows antibacterial activity in vitro.

liquid dilution method (Margin below) Agar plate dilution method

[Brief explanation of the drawings] Figures 1, 2, and 3 are of CA-146A.
0 infrared absorption spectrum, mass spectrometry spectrum in Br tablet,
Figure 4 shows 'H-NMR of CA-146B.
Shows NMR. Patent applicant: Shionogi & Co., Ltd. Figure 1 Figure 2

Claims (1)

[Scope of Claims] 1) A compound represented by the following formula; ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (wherein R represents methyl or isopropyl) and a pharmaceutically acceptable salt thereof. 2) Compound CA-146A according to claim 1, wherein R is isopropyl. 3) Compound CA-146B according to claim 1, wherein R is methyl. 4) CA-146A and/or CA-146B, which is characterized by culturing CA-146A and/or CA-146B-producing bacteria belonging to the genus Streptomyces in a medium, and separating and collecting CA-146A and/or CA-146B from the culture. /
Or a method for producing CA-146B. 5) The production method according to claim 4, wherein the producing bacterium is Streptomyces labengelae. 6) The producing bacterium is Streptomyces labengulee CA
-146, the manufacturing method according to claim 4. 7) A bacterium belonging to the genus Streptomyces that produces CA-146A and/or CA-146B. 8) The bacterium according to claim 7, wherein the bacterium is Streptomyces labengulee. 9) The bacterium is Streptomyces labengulee CA-1
46. The bacterium according to claim 7, which is No. 46. 10) An antifungal agent containing CA-146A and/or CA-146B as an active ingredient. 11) The antifungal agent according to claim 10, wherein the antifungal agent is an anticandidiasis agent.