JPH05105657A - Antibiotics wap-5044c and wap-5044a, derivative from wap-5044 c treated with alginase, their production and use - Google Patents

Abstract

(57) [Summary] (Modified) [Objective] The object of the present invention is to provide a novel antibiotic useful for the treatment of fungal infections such as candidiasis and aspergillosis, and a microorganism producing the antibiotic. [Structure] General formula [I] Antibiotics WAP-5044A and WA represented by
A derivative obtained by treating P-5044C and WAP-5044C with arginase, a microorganism belonging to the genus Streptomyces that produces the antibiotic, and an antifungal agent containing the substance as an active ingredient. [Effect] It showed a strong antibacterial activity against Candida albicans and Aspergillus fumigatus. Also,
It showed an excellent therapeutic effect on a model of systemic candidiasis infection by Candida albicans.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel antibiotic WAP-
5044A and WAP-5044C and WAP-50
The present invention relates to a novel derivative obtained by treating 44C with arginase, a method for producing the same, and use thereof. The compounds of the present invention are
It has antifungal activity and is expected as a chemotherapeutic agent against fungal infections.

[0002]

Conventionally, as therapeutic agents for fungal infections, chemotherapeutic agents such as amphotericin B, nystatin, miconazole, fluconazole and pyrrolenitrin have been used.

[0003]

However, these chemotherapeutic agents are not satisfactory in terms of toxicity and efficacy, and the emergence of resistant bacteria due to repeated use is becoming a problem. To solve this problem, novel clinically useful antibiotics with lower toxicity, that is, selective toxicity, are needed.

[0004]

[Means for Solving the Problems] The present inventors mainly isolated a large number of microorganisms from soil for the purpose of searching for new antibiotics, isolated the antibiotics produced by them, and investigated their biological properties. As a result of the investigation, it was found that two types of novel antibiotics described in the literature which have antibacterial activity against other pathogenic microorganisms such as Candida albicans are produced in the culture filtrate of actinomycetes belonging to the genus Streptomyces. .. As a result of isolating and purifying this antibiotic from the culture medium and examining its physicochemical properties, it was confirmed that it was a novel compound having the structure of formula I and formula II described below, and WAP-5044C and WAP-5044A
I named it.

[Chemical 3]

On the other hand, when WAP-5044C having the formula I is treated with arginase derived from bovine liver (manufactured by Sigma), a new derivative in which the guanidino group is decomposed is obtained, and this substance is Candida kefir as a test bacterium. The antifungal activity on the agar plate was about the same as WAP-5044C.

The present invention cultivates a microorganism belonging to the genus Streptomyces which produces the antibiotics WAP-5044C and / or WAP-5044A to obtain the antibiotic WAP-50.
44C and / or WAP-5044A produced and accumulated, and WAP-5044C and WAP-5044A or their salts obtained from this culture, and WAP-5.
Derivatives obtained by treating 044C with arginase, their production methods, and the antibiotic WAP-504.
It is intended to provide microorganisms capable of producing 4C and / or WAP-5044A and their use as pharmaceutically acceptable amounts of antifungal agents.

Antibiotics WAP-5044C and / or WAP-5044A producing bacteria include the antibiotic WAP-5044C.
Any microorganism can be used as long as it produces -5044C and / or WAP-5044A, but is not limited to the antibiotics WAP-5044C and / or WAP-504.
As an example of a 4A-producing bacterium, WAP belonging to the genus Streptomyces separated by the present inventors from soil in Nagano City, Nagano Prefecture.
-5044 strain is mentioned.

(Bacteriological Properties of Producing Bacteria) WAP-5044
The mycological properties of the strain are shown below. Morphological characteristics of WAP-5044 strain and culture findings on the classified medium are as follows. The WAP-5044 strain forms aerial hyphae on various media and they show simple branching. The spore-forming hyphae have a spiral shape and 10 or more spores are linked. The spores are spherical or elliptical, and the size is 0.7 to 1.1 × 0.9 to 1.
At 3 μm, the surface is spiny. No formation of sporangium, flagella spores, sclerotium, etc. was observed on various media.

The growth state of the WAP-5044 strain on the classified medium is shown in Table 1. Observation at 28 ℃, 3 in various media
It was performed after culturing for a week. In Table 1, the numbers in parentheses () indicate the JIS color name book (JIS 28102 compliant, Japanese Standards Association, Showa 62).
First edition, June 1, year, 2nd edition).

[0010]

[Table 1]

The physiological properties of WAP-5044 strain are as follows. (1) Growth temperature range: 20 to 40 ° C Optimal growth temperature: 25 to 30 ° C (2) Liquefaction of gelatin: positive (28 ° C), negative (20)
(° C) (3) Starch hydrolysis: positive (4) Coagulation / peptonization of skim milk: positive (peptone) (5) Formation of melanin-like pigment: negative (tyrosine agar medium and peptone yeast iron agar medium) (6) ) Utilization of carbon source (Pridham-Godlieve agar medium) Commonly used carbon source D-glucose, sucrose, raffinose, inositol, D-mannite Medium carbon source D-xylose, D-fructose utilization Sources that are not used L-arabinose, L-rhamnose

On the other hand, LL-diaminopimelic acid was detected in the hydrochloric acid hydrolyzate of all the WAP-5044 strain cells in addition to the above-mentioned morphological characteristics, growth state on the classification medium and physiological properties. From the above properties, the WAP-5044 strain is judged to belong to the genus Streptomyces.

Regarding the taxonomic position of WAP-5044 strain, "International Journal of Systematic Bacteriology" Vol. 18 (1968)
Year), Volume 19 (1969), Volume 22 (1972)
), "Burjay's Manual of Determinative Bacteriology", Volume 8 (1974)
Although the comparison was made with reference to the bacterial strain description of No. 1, no known bacterial strain similar to the WAP-5044 strain was found. Therefore, the present inventors have designated the WAP-5044 strain as Streptomyces sp. WAP-5044 (Streptomyces).
sp, WAP-5044). Streptomyces sp. WAP-5044 is contract number FERM P-125 at Institute of Microbial Science and Technology, Agency of Industrial Science and Technology.
Deposited as 54. As a general property of Streptomyces-containing actinomycetes, the mycological properties are extremely variable, and the WAP-5044 strain is no exception. Therefore, the WAP-5044 strain does not always exhibit the above-mentioned properties. However, the antibiotics WAP-5044C and / or WAP-5044A, even if those mutants are natural or artificial by various mutagens,
Any microorganism that has the ability to produce a can be used in the method of the invention.

(Cultivation and Production) The antibiotic WAP-of the present invention
5044C and WAP-5044A are produced by inoculating a nutrient source-containing medium with the above-mentioned producing bacterium and aerobically culturing. In culturing bacteria producing the antibiotics WAP-5044C and / or WAP-5044A, examples of carbon sources include glucose, fructose, starch, dextrin, glycerin, molasses, starch syrup, oils and fats,
Utilizing organic carbon compounds that can be assimilated, such as organic acids,
On the other hand, as the nitrogen source, for example, soybean flour, cottonseed flour, corn steep liquor, casein, peptone, yeast extract,
Organic nitrogen compounds such as meat extract, germ, urea, amino acids, ammonium salts and inorganic nitrogen compounds can be used. Examples of salts include sodium salt, potassium salt,
Inorganic salts such as calcium salts, magnesium salts and phosphates can be used alone or in appropriate combination. Furthermore, if necessary, heavy metals such as iron salts, copper salts, zinc salts, cobalt salts, biotin necessary for the growth of microorganisms,
It promotes the growth of vitamins such as vitamin B ₁ and other producing bacteria, and WAP-5044C and / or W
It is desirable to appropriately add an organic substance or an inorganic substance capable of producing a large amount of AP-5044A. Further, a defoaming agent such as silicone oil or polyalkylene glycol ether or a surfactant may be added to the medium.

As the culture method, a method generally used for the production of antibiotics is adopted, but in the liquid culture method,
In particular, deep aeration stirring culture is preferable to keep it aerobic,
In the laboratory, ordinary shaking culture using a flask is suitable. The culture temperature is usually 20 to 40 ° C., but preferably 25 to 30 ° C. is desirable. The pH of the culture can be around 6-8, but preferably around 7 is desirable. WAP-5044C and / or in culture
Alternatively, the production amount of WAP-5044A is sufficiently achieved in about 2 to 6 days.

(Isolation / Purification) As described above, WAP-5044C and WAP-5044 accumulated in the culture.
In order to collect A from the culture, it can be advantageously carried out by utilizing the physicochemical properties of the present antibiotic described below. That is, WAP-5044C and WAP-5
Since 044A is contained in the culture filtrate, first, a culture aid is obtained by adding a filter aid such as Celite or Radiolite to the culture and performing vacuum filtration or by centrifuging to remove the cells.

WAP-5044C and WAP-504
Since substance 4A is a ninhydrin-positive water-soluble amphoteric substance due to the physicochemical properties described below, purification by ion exchange chromatography, which is usually used, is effective in collecting this substance from the culture filtrate. For example, as a cation exchanger, Amberlite IR-12
0B (made by Rohm and Haas), Dowex 50W
(Dow Chemical Co., Ltd.), Diaion SK1B (Mitsubishi Kasei Co.) strong acid cation exchange resin, Amberlite IRC-50 (Rohm and Haas Co.), Diaion WK10 (Mitsubishi Kasei Co.) weak acidity Cation exchange resin, CM-Sephadex, SP-Sephadex (manufactured by Pharmacia) and the like can be used. As the anion exchanger, for example, Amberlite IRA-
401, IRA-68 (made by Rohm and Haas), Dowex 1 (made by Dow Chemical), Diaion SA
10B, PA406, WA30 (manufactured by Mitsubishi Kasei), QA
E-Sephadex, DEAE-Sephadex (made by Pharmacia), etc. can be used. Antibiotics WAP-5044 adsorbed on these ion exchange resins
C and WAP-5044A are various salts, alkalis,
Alternatively, it can be easily eluted with an aqueous solution containing an acid or a buffer solution.

As described below, there are three types of antifungal active substances produced by Streptomyces sp. WAP-5044 strain (WAP-5044C, WAP-5044A, WAP).
AP-5044B) is present, and an adsorption resin such as Diaion HP20, SepaBeads SP207 (manufactured by Mitsubishi Kasei Co., Ltd.), Amberlite XAD-for separating these active substances
2 (manufactured by Rohm and Haas) and the like can be advantageously used, but the separation by activated carbon column chromatography is particularly efficient. That is, when the WAP-5044B component is eluted with water using activated carbon column chromatography, the WAP-5044C component is eluted with the column solution, and then the WAP-5044C component is eluted with water. The WAP-5044A component is adsorbed on an activated carbon column and can be easily recovered with water-containing acetone or water-containing methanol.

As described above, WAP-5044B, WAP-
In order to further remove impurities from the fractions from which 5044C and WAP-5044A components were roughly separated,
Each component can be separated into a single component by using adsorption chromatography using silica gel or the like and developing and eluting with, for example, an acetonitrile-water system. In addition, Sephadex G-10 and other Sephadex G-
It is also possible to make each component single by developing and eluting with water or hydrous methanol using a molecular sieving effect such as 15, LH-20 (manufactured by Pharmacia). Further, separation and purification by high performance liquid chromatography can be advantageously used. Examples of the carrier used include silica gel, chemically bonded silica gel having an octadecyl group, an octyl group, an amino group, or the like bonded thereto, or a polystyrene-based porous polymer gel. As the mobile phase, hydrous acetonitrile, hydrous methanol, a buffer solution or the like can be used.

(Physicochemical properties) WAP-5044 obtained by combining the various chromatographies shown above.
The crystals or powders of B, WAP-5044C, and WAP-5044A have the physicochemical properties shown below. Of these components, WAP-5044B has a physicochemical property very similar to that of β-cyanoalanine. , Directly compared with the standard. As a result, all properties including the instrumental analysis were in agreement, so WAP-5044B was identified as β-cyanoalanine. WAP-5044C and WAP-
The physicochemical properties of 5044A are as follows.

WAP-5044C (1) Appearance: white needle crystals (strong hygroscopicity) (2) Melting point: 140 to 150 ° C. (decomposition) (3) Molecular weight: 174 [FAB mass m / z: 175
(M + H) ⁺ , m / z: 349 (2M + H) ⁺ ] (4) Molecular formula: C ₅ H ₁₀ N ₄ O ₃ high-resolution FAB mass Measured value: m / z 349.1624 (2M + H) ⁺ theoretical value: m / z 349 1584 (C ₁₀ H ₂₁ N ₈ O ₆ ) (5) Specific optical rotation: [α] _D ²⁰ = + 87.2 ° (c = 0.
50, H ₂ O) (6) Solubility: Soluble in water, sparingly soluble in methanol, ethanol, acetone (7) Ultraviolet absorption spectrum: end absorption (in water) (8) Infrared absorption spectrum: measured by KBr tablet method The infrared absorption spectrum is shown in FIG. The maximum absorption value (wave number cm ^-1 ) is shown below. 3480, 3390, 3170, 1570, 1520,
1460, 1390, 1320, 1300, 1200,
1150, 1060, 970, 890, 840, 79
0,700,590,540,470,420,410 (9) ¹ H NMR spectrum The chemical shift of the ¹ H NMR spectrum of FIG. 2 measured at 270 MHz in heavy water is shown below. δ (ppm): 4.28 (1H, d, J = 10.1Hz),
5.22 (1H, dd, J = 10.1Hz, 12.1H
z), 6.97 (1H, d , J = 12.1Hz) (10) 13 C NMR 13 C NMR of FIG. 3 as measured by the spectral deuterium 67.94MHz
The chemical shifts of the spectra are shown below. δ (ppm): 56.89, 98.97, 155.86, 1
62.59,176.99 (11) Color reaction Positive: Ninhydrin, potassium permanganate, Ehrlich, iodovapor, pentacyanoammonioferate reagent Negative: Dragendorff, phosphomolybdic acid, Sakaguchi reaction (pale yellow) ( 12) Substance classification: Water-soluble amphoteric substances (13) Thin layer chromatography (TLC)

[0022]

[Table 2]

WAP-5044A (1) Appearance: pale yellowish white powder (2) Melting point: No clear melting point (3) Molecular weight: 172 [FAB mass m / z: 173
(M + H) ⁺ , m / z: 345 (2M + H) ⁺ ] (4) Molecular formula: C ₇ H ₁₂ N ₂ O ₃ high-resolution FAB mass Measured value: m / z 345.1760 (2M + H) ⁺ theoretical value: m / z 345 .1774 (C ₁₄ H ₂₅ N ₄ O ₆ ) (5) Specific optical rotation: [α] _D ²⁰ = + 39.18 ° (c =
0.49, H ₂ O) (6) Solubility: Soluble in water and methanol, sparingly soluble in acetone and ethyl acetate (7) Ultraviolet absorption spectrum: end absorption (in water) (8) Infrared absorption spectrum: by KBr tablet method The measured infrared absorption spectrum is shown in FIG. The maximum absorption value (wave number cm ^-1 ) is shown below. 3460, 1624, 1396, 410 (9) ¹ H NMR spectrum The chemical shift of the ¹ H NMR spectrum of FIG. 5 measured at 270 MHz in heavy water is shown below: δ (ppm): 2.03 (3 H, s), 2. 05 (3H,
s), 4.28 (1H, d, J = 10.4Hz), 5.
25 (1H, dd, J = 10.4Hz, 12.4H
z), 7.17 (1H, d , J = 12.4Hz) (10) 13 C NMR spectrum ¹³ C NMR of FIG. 6 as measured in heavy water 67.94MHz
The chemical shifts of the spectra are shown below: δ (ppm): 18.64, 23.47, 56.73, 10
0.40,155.70,167.44,176.55 (11) Color reaction Positive: Ninhydrin, potassium permanganate, Ehrlich, iodovapor Negative: Dragendorff, phosphomolybdic acid (12) Substance classification: water-soluble Amphoteric substances (13) Thin layer chromatography

[0024]

[Table 3]

(Properties of a novel derivative obtained by treating WAP-5044C with arginase) WAP-5044C
Regarding the synthesis of a novel derivative by treating the substance with arginase, it was revealed that the guanidinooxy group existing in the chemical structure of WAP-5044C can be converted into an aminooxy group by the action of arginase from bovine liver (manufactured by Sigma). Obtained 2-amino-4-aminooxy-3-trans-butenoic acid

[Chemical 4] Is unstable and undergoes intramolecular rearrangement and intramolecular dehydration, so that β-cyanoalanine is easily produced.

[Chemical 5] It was confirmed to change to. This novel derivative is silica gel T using the developing solvent acetonitrile: water (3: 1).
LC showed ninhydrin red-purple spot, R
The f value gives 0.19. This ninhydrin-positive spot also has a double bond in the molecule due to color development by iodine vapor and decolorization reaction by potassium permanganate, and is a color reaction of aminooxy group.
fe) because it is positive for the reagent

[Chemical 6] Is determined to have the structure of

On the other hand, although the biological activity of the new derivative is of interest, silica gel TLC-motorography using Candida kefir as a test bacterium shows that the new derivative has almost the same antifungal activity as WAP-5044C. showed that. (Biological properties) WAP-5044C and WAP-5
Table 2 shows the results of the antibacterial activity test of 044A. The minimum inhibitory concentration (MIC) was measured by adding 0.5% glucose to yeast-nitrogen-based without amino acids medium (manufactured by Difco) or pen assay broth (manufactured by Difco) ( This was carried out by the liquid medium dilution method using B). As shown in Table 2, WAP-5
044C and WAP-5044A show antibacterial activity against fungi.

[0027]

[Table 4]

Since WAP-5044C showed a strong antibacterial activity against Candida albicans and Aspergillus fumigatus, the therapeutic effect on the systemic candidiasis infection model obtained by inoculating Candida albicans into the tail vein of mice was shown. Examined. The Candida albicans TIMM0239 strain was cultured overnight at 37 ° C in a Sabro-dextrose liquid medium, and then the cells were collected and suspended in a PBS buffer (pH 7.2) (manufactured by Nissui Pharmaceutical Co., Ltd.). 1 × 10 ^{6 of} this Candida albicans TIMM0239 strain was used as an ICR-MCH mouse, 4 weeks old, male (CLEA Japan, Inc.)
Vaccination from the tail vein, once 2 hours later, then 1
Subcutaneously, once a day, for 4 days, PBS buffer solution of pH 7.2 containing each concentration of the WAP-5044C substance was used for 15 days.
Daily life and death were observed and the therapeutic effect was measured. The results are shown in Table 3.

[0029]

[Table 5]

T / C is a value in% of the average survival days of the administration group with respect to the average survival days of the untreated control group. WA
It was found that P-5044C exhibits excellent therapeutic effect.

(Use) From the above biological properties, W
The novel antibiotics and novel derivatives produced by the AP-5044 strain are useful as therapeutic agents for various fungal infections such as candidiasis and aspergillosis.

When the compound of the present invention is administered as a medicine, the compound of the present invention is used as it is or in a pharmaceutically acceptable non-toxic and inert carrier, for example, 0.1% to 99.
It is administered to animals, including humans, as a pharmaceutical composition containing 5%, preferably 0.5% to 90%. As the carrier, one or more of solid, semi-solid, or liquid diluents, fillers, and other auxiliaries for formulation are used. The pharmaceutical composition is preferably administered in a dosage unit form. The pharmaceutical composition of the present invention can be administered orally, intratically, topically (such as transdermally) or rectally. Therefore, it is preferable to formulate and administer into a dosage form suitable for these administration methods by a known formulation technique.

The dose of the antifungal agent is preferably adjusted in consideration of the patient's condition such as age and body weight, administration route, nature of disease and degree of morbidity. 10 to 2000 mg per day as the active ingredient amount of the invention
The range is generally. In some cases, lower doses may be sufficient, and conversely, higher doses may be required. When administering a large amount (as needed), it is desirable to administer in several divided doses per day.

[0034]

The present invention will now be described in detail with reference to examples, but the present invention is not limited thereto. Example 1 In a 500 ml Erlenmeyer flask, glucose 2.5% defatted soybean flour 2.0%, soybean oil 0.4%, sodium chloride 0.2
Medium consisting of 5% and calcium carbonate 0.5% (pH 7.
2) After injecting 100 ml, prepare 2 sterilized media, inoculate 1 platinum loop of each of them from a slope agar medium of Streptomyces sp. WAP-5044 strain, then 180 strokes / min at a rotary shaker, 30 ° C, 2 Culture was carried out for a day. Eighty 500 ml Erlenmeyer flasks prepared by injecting 100 ml of the above medium and sterilized were inoculated with 2 ml of the culture solution of the Erlenmeyer flasks that had been previously cultured, and 180 revolutions / min at 30 ° C for 4 days on a rotary shaker. Culture was performed. The antifungal activity was measured by the Candida kefir agar plate method.

Example 2 The culture broth obtained in Example 1 was centrifuged at 5,000 rpm for 15 minutes to obtain about 6 liters of culture supernatant. The supernatant was passed through Diaion HP-20 (1.5 liter) and washed with water (4 liter). The passing solution and the washing solution were combined, and subjected to column chromatography with Amberlite IR-120B (H ⁺ type, 1 liter), and eluted with 8 liters of 0.5N ammonia water. The eluate was concentrated, subjected to activated carbon column chromatography (300 ml), developed with 4.5 liters of water, and then eluted with 30 ml of 80% acetone water. Although the active substance was present in all of the passing solution, the washing solution, and the 80% acetone eluate, different Rf values (WAP-5044A: Rf0.40, Wf-5044A: Rf0.40, in the silica gel TLC developed with acetonitrile = water (3: 1). WAP-504
4B: Rf0.27, WAP-5044C: Rf0.1
Since each of them has activity in 2), it was revealed that they are different active substances. Since there are three types of active substances obtained in the above crude purification process,
044% acetone elution area is 5044A, passing fraction is 5044
B, the water development fraction was tentatively named 5044C.

Example 3 The crude products 5044A, B and C obtained in Example 2 were isolated and purified by the method described below. 5044A WAP-5044A-containing activated carbon column chromatography containing 80% acetone eluate was concentrated, and the concentrate (1.3 g) was purified by silica gel (Amerck, 50 g).
Subjected to column chromatography with acetonitrile:
It was eluted and fractionated with a solvent system of water (8: 1). Active fraction (1
000 ml) was collected and concentrated, and then the concentrate (38.64 mg) was subjected to Sephadex G-10 column chromatography (Pharmacia), eluted with water and fractionated. Fractions showing a single spot by ninhydrin color development (yellow) on silica gel TLC (acetonitrile: water = 3: 1) were collected and concentrated to obtain 9.5 mg of WAP-5044A pale yellowish white powder.

5044B WAP-5044B-containing activated carbon column fraction passed through the column was concentrated, and the concentrate (3.3 g) was subjected to column chromatography on silica gel (120 g, manufactured by Amerk) to give acetonitrile: water (8: 1). The solvent system was used for elution and fractionation.
The active fraction (200 ml) was collected, concentrated, and then concentrated (20
2.6 mg) of SepaBeads SP-207 (manufactured by Mitsubishi Kasei)
The product was eluted and fractionated with water. Fraction containing active substance (30
ml) is collected and concentrated, and further, Dowex 50W × 8H ⁺
A mold (manufactured by Dow Chemical Co.) was washed with water and then eluted with 0.5N ammonia water. After the eluate was concentrated, it was crystallized from water-methanol to give WAP-5044B white needle crystals 7
Obtained 3.9 mg. This substance was in agreement with β-cyanoalanine from various instrumental analyzes and comparison with a standard product.

5044C WAP-5044C-containing activated carbon column chromatography water-developed elution fraction was concentrated, and then the concentrate (1.56 g)
Was subjected to column chromatography on silica gel (Amerck, 60 g), and acetonitrile: water (5: 1) was used.
The solvent system was used for elution and fractionation. The active fraction (3 liters) was collected and concentrated, and the concentrate was subjected to Sephadex G-10 column chromatography (Pharmacia) and eluted with water to fractionate. Fractions showing a single spot (yellow) on ninhydrin on silica gel TLC (acetonitrile: water = 3: 1) were collected and concentrated to give WAP-5044C white powder 25.
0 mg was obtained. Further, this was crystallized from water-methanol to obtain 85 mg of WAP-5044C white needle crystals.

Example 4 Reduction product of WAP-5044C 5.2 mg of WAP-5044C was dissolved in 50% methanol water, 1 drop of acetic acid was added, and 10% palladium-carbon 5 was added.
After adding mg, catalytic reduction was carried out with hydrogen under normal pressure overnight. After the reaction was completed, the catalyst was recovered and the filtrate was concentrated to give 5.3 mg of white crystals.
Got To confirm this reduction product, developing solvent n-butanol: pyridine: acetic acid: water (15: 10: 3: 12)
Cellulose TLC (Avicel) using
Ninhydrin spots (magenta) with the same Rf (0.15) value as DL -homoserine were given. In order to determine the absolute configuration of this reduced product, Crown Pack CR
(+) (Φ4 × 150mm, Daicel Chemical Industries)
The analysis was carried out by a high-performance liquid chromatography using a perchloric acid aqueous solution (pH 1.5), a flow rate of 0.4 ml / min, and a detection wavelength of 200 nm for elution. As a result,
Consistent with L -homoserine having a retention time of 4.4 minutes, D
-It was found to be different from the retention time of homoserine of 3.2 minutes. Therefore, this reduction product is L -homoserine, and the absolute configuration of the α-carbon of WAP-5044C is L.

Example 5 Treatment of WAP-5044C with Arginase 50 mg of WAP-5044C obtained in Example 3 was dissolved in 2 ml of distilled water, 0.2 ml of 10 mM MnCl ₂ was added, and 1N was added.
The pH was adjusted to 9.4 with NaOH. To this was added 0.16 ml of a solution (3125 units / ml) in which beef liver arginase (manufactured by Sigma) was dissolved in 10 mM MnCl ₂ and the mixture was added at 37 ° C. for 3.
The reaction was carried out for 5 hours. Part of the reaction solution is silica gel TLC
(Acetonitrile: H ₂ O = 3: 1). As a result of ninhydrin color development, as shown in FIG. 7, in addition to the unreacted raw material WAP-5044C (Rf value 0.12, yellow), 2
Various kinds of ninhydrin positive substances (Rf value 0.19, red purple, Rf value 0.27, green) were observed. Rf value 0.
Twenty-seven substances were in agreement with β-cyanoalanine by comparison with the standard. The substance having an Rf value of 0.19 was positive for ninhydrin, iodine vapor, potassium permanganate, and the Jafe reagent reaction. These color reaction results and WAP-5044C
In consideration of the structure of and the reaction specificity of arginase, this substance is considered to have the structure of the following formula in which the guanidino group of WAP-5044C is hydrolyzed.

[0041]

[Chemical 7]

Since the novel derivative obtained by the treatment with arginase is unstable, it is considered that β-cyanoalanine was produced by intramolecular rearrangement and intramolecular dehydration. Due to such instability, a part of the reaction solution was subjected to the above-mentioned silica gel TLC and then subjected to motorcycle ographography using Candida kefir as a test bacterium. As a result, the novel derivative was almost the same as WAP-5044C. It was found to have antibacterial activity.

[0043]

As described above, according to the present invention, the novel antibiotics WAP-5044C and WAP-5044A and WAP are used.
Provided are novel derivatives obtained by treating -5044C with arginase, and methods for producing them. The present antibiotics WAP-5044C and WAP-5044A and WAP
The novel derivative obtained by treating -5044C with arginase exhibits antibacterial activity against fungi, and thus can be used for the treatment of fungal infections.

[Brief description of drawings]

FIG. 1 is an infrared absorption spectrum (KBr method) of an antibiotic WAP-5044C.

FIG. 2 is a ¹ H NMR spectrum (270 MHz, heavy water) of antibiotic WAP-5044C.

FIG. 3 is a ¹³ C NMR spectrum (67.9 MHz, in heavy water) of the antibiotic WAP-5044C.

FIG. 4 is an infrared absorption spectrum (KBr method) of antibiotic WAP-5044A.

FIG. 5 is a ¹ H NMR spectrum (270 MHz, in heavy water) of the antibiotic WAP-5044A.

FIG. 6 is a ¹³ C NMR spectrum (67.9 MHz, in heavy water) of the antibiotic WAP-5044A.

FIG. 7 is a silica gel thin layer chromatogram of a reaction solution obtained by treating the antibiotic WAP-5044C with arginase. Developing solvent acetonitrile: water = 3: 1. Coloring agent ninhydrin.

Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12P 13/04 6977-4B // A61K 39/00 8413-4C (C12N 1/20 C12R 1: 465) (C12P 13/04 C12R 1: 465) (72) Inventor Masayoshi Goto 4-2-5-408 Higashi Naruse, Isehara City, Kanagawa Prefecture

Claims (5)

[Claims] 1. The following formula: Antibiotics WAP-5044A and WAP-5
044C or a salt thereof. 2. A derivative represented by the following formula or a salt thereof obtained by treating the antibiotic WAP-5044C with arginase. [Chemical 2] 3. An antibiotic W belonging to the genus Streptomyces
A microorganism having the ability to produce AP-5044A and / or WAP-5044C is cultivated in a medium, and the antibiotics WAP-5044A and / or WAP are contained in the culture.
Antibiotics WAP-5044A and / or characterized in that -5044C is produced and accumulated, and then collected.
Alternatively, a method for producing WAP-5044C. 4. An antibiotic W belonging to the genus Streptomyces
A bacterium producing AP-5044A and / or WAP-5044C. 5. An antifungal agent comprising as an active ingredient at least one compound selected from the antibiotics or derivatives according to claims 1 and 2.