JP2000189181A - Antibiotic paleic acid a and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new antibiotic, paleic acid A, useful as an antibacterial agent or the like and having excellent antibacterial activity specifically on Pasteurella haemolytica, which is a phlogogenic bacterium against a bovine respiratory disease by culturing a bacterium belonging to the genus Paenibacillus and producing paleic acid A. SOLUTION: A new compound, paleic acid A, expressed by the formula or its pharmaceutically permissible salt, capable of exhibiting excellent antibacterial activity specifically on Pasteurella haemolytica, which is a phlogogenic bacterium against a bovine respiratory disease and useful as an active ingredient of an antibacterial agent, or the like. The new compound is obtained by culturing a bacterium [e.g. Paenibacillus alvei, the strain BKM771-AF3 (FERM P-16999)] belonging to the genus Paenibacillus and producing paleic acid A, and then collecting an antibiotic, paleic acid A, from the cultured product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a new antibiotic, paleic acid A (Palei), which exhibits a novel antibacterial activity specifically against Pasteurella haemolytica, a causative bacterium of bovine respiratory disease.
c) Acid A) or a salt thereof, and also relates to a method for producing paleic acid A. Further, the present invention relates to an antibacterial agent containing a maleic acid A or a salt thereof as an active ingredient. Further, the present invention relates to Paenibacillus albay strain BMK771-AF3, which is a novel microorganism having a property capable of producing maleic acid A.

[0002]

BACKGROUND OF THE INVENTION In chemotherapy of bacterial infections, the emergence of multidrug-resistant bacteria is a serious problem. Known compounds conventionally known or used for respiratory disease in cattle are macrolide antibiotics or β-lactam antibiotics, and these antibiotics are used in human treatment. Has the same molecular structure skeleton. Vancomycin analogs, which were used in animal industry in large quantities in the livestock industry, have produced vancomycin-resistant bacteria, which are at odds with threatening human chemotherapy for bacterial infections. Therefore, it is always desirable to discover or create a new compound having a molecular structure skeleton completely different from that of an antibacterial agent used for treatment of human bacterial infection and exhibiting excellent antibacterial activity. Research has been conducted.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel antibiotic having excellent antibacterial activity which can satisfy the above-mentioned demand.

[0004]

In order to achieve the above object, the present inventors conducted research to find useful antibiotics. As a result, the present inventors succeeded in isolating Paenibacillus albay BMK771-AF3 strain as a novel microorganism. We have also found that this strain produces several antibiotics with new structural backbones. One of those antibiotics was successfully isolated and purified and named as maleic acid A. It has been found that this maleic acid A exhibits an excellent antibacterial activity specifically against Pasteurella haemolytica, a causative bacterium of bovine respiratory disease. Further study continues with maleic acid A
Was analyzed to determine the chemical structure of maleic acid A. And it was confirmed that the maleic acid A was a novel compound. Then, it was found that the maleic acid A can be represented by the following formula (I).

Thus, the present invention provides an antibiotic obtained by culturing Paenibacillus albay BMK771-AF3 strain as a novel microorganism and designated by the present inventors as maleic acid A, and a method for producing the same. To provide. Further, the present invention provides an antibacterial agent comprising a maleic acid A or a salt thereof as an active ingredient.

That is, according to the first aspect of the present invention, the following formula (I) Or a pharmaceutically acceptable salt thereof.

The physicochemical properties of the maleic acid A of the formula (I) according to the first invention are as follows. (1) Appearance Colorless oil (2) Molecular formula _{C 18 H 34 O 3 (3} ) High resolution mass spectrometry (HRFABMS: negative ion mode) Found: m / z 297.2459 (M - H) - Calculated: m / z 297.2430 (4) Specific rotation [α] _D ²⁸ = −5 ゜ (c 0.2, chloroform) (5) Infrared absorption spectrum As shown in FIG. 1 of the accompanying drawings.

(6) Proton nuclear magnetic resonance spectrum The proton NMR spectrum measured at room temperature in deuterated chloroform at 500 MHz is as shown in FIG. 2 of the accompanying drawings. (7) Carbon-13 nuclear magnetic resonance spectrum The carbon-13 NMR spectrum measured at room temperature in deuterated chloroform at 125 MHz is as shown in FIG. 3 of the accompanying drawings. (8) Thin Layer Chromatography The Rf value when developed with chloroform-ethanol (20: 1) as a developing solvent in silica gel 60F ₂₅₄ (manufactured by Merck) is 0.42.

The fact that the novel antibiotic paleic acid A according to the present invention has the chemical structure represented by the above formula (I) was determined as described above by examining the analysis of proton NMR, carbon-13 NMR and the like in detail. .

The pharmaceutically acceptable salts of maleic acid A include, for example, the sodium, potassium and ammonium salts. The maleic acid A of the formula (I) according to the invention has the biological properties mentioned below. Have. That is, maleic acid A
Has a particularly good antibacterial activity against Pasteurella haemolytica and also has antibacterial activity on some strains of Pasteurella maltosida. Maleic acid A has low acute toxicity to mammals.

Test Example 1 The antibacterial spectrum of maleic acid A against various microorganisms was determined on Muller Hinton agar (manufactured by Difco) medium and Brain Heart, based on the standard method of the Japanese Society of Chemotherapy.
The measurement was performed on an infusion agar (manufactured by Difco) medium by a multiple dilution method. The results are shown in Tables 1 and 2, respectively.

[0012]

[0013]

[0014]

Test Example 2 To test the acute toxicity of the maleic acid A of the formula (I) using mice, the maleic acid A was dissolved in a physiological saline solution containing 10% dimethyl sulfoxide and Tween 80, and the resulting solution was used. The mice were injected intraperitoneally and observed for 14 days. As a result, no toxicity was observed for the maleic acid at a dose of 65 mg / kg.

Further, according to a second aspect of the present invention, a bacterium belonging to the genus Paenibacillus which produces the above-mentioned maleic acid A of the formula (I) is cultured, and the maleic acid A is collected from the culture. A method for producing the antibiotic, maleic acid A of formula (I), is provided.

[0017] As the bacteria for producing the maleic acid A used in the method of the present invention, any bacteria capable of producing maleic acid A, which is an antibiotic having the above-mentioned physicochemical and biological properties, may be used. Suitable microorganisms can be selected from genera regardless of their species. Among such microorganisms, a specific and preferred example of a maleic acid-producing bacterium of the present invention includes a microorganism isolated from soil at the Iwatake Ski Resort in Hakuba-mura, Kitaazumi-gun, Nagano Prefecture in May 1997 by the present inventors. Among the bacteria, there is a strain with the strain number of Paenibacillus albay BMK771-AF3.

The following is Paenibacillus Albay BMK
The mycological properties of strain 771-AF3 are described. 1. Morphology (1) The cells of this strain are rods, and the size is 0.7 × 0.8
~ 1.6 x 2.5 microns. (2) Cell polymorphism is not particularly observed. (3) It has peripheral flagella and exhibits motility. (4) It has spores. Its shape is oval, size is 0.45 ~
0.55 x 1.0 micron, central location, swelling of cells is observed.

2. Growth conditions in various media Except for broth gelatin puncture culture, all were cultured at 30 ° C and tested. (1) Gravy agar plate culture The colonies are slightly glossy, opaque circles, and 3
The margins become irregular from around the day. The color is colorless to light brownish gray. No diffusible dye is observed. (2) Liquid broth culture One day after cultivation, the growth of the bacterium was slightly observed, and on the second day, a small amount of bacterium precipitated on the bottom of the tube. One week after the cultivation, it grows slightly white on the surface of the test tube and precipitates on the tube bottom. No significant change in the turbidity of the medium is observed even after 2 weeks from the culture.

(3) Broth gelatin puncture culture In culture at 20 ° C., growth of bacteria is observed along the surface of the medium and along the puncture line, liquefaction starts about 2 days after cultivation, and progresses slowly thereafter, and the medium grows in 4 weeks. About 2/3 was liquefied.
Liquefaction is moderate. In 30 ° C. culture, good growth of bacteria is observed on the surface of the medium and the whole, and a pellicle is formed two days after the culture. Liquefaction started around the second day after the culture and was completed after 4 weeks of culture. Its effect is moderate. (4) Milk cultivation In the case of culturing with milk, it grows slightly on the tube wall near the surface of the medium from about the second day after culturing. No coagulation is observed 2 weeks after the culture, and coagulation starts 3 weeks later. Peptoneization is
It started immediately after the end of coagulation and was almost completed in 4 weeks after culture. The reaction is slightly acidic.

3. Physiological properties (All culture temperatures are 30 ° C unless otherwise specified) (1) Gram stainability: undefined (positive in immature culture, negative and positive in 48 hours culture, and negative in culture after 72 hours) (2) Reduction of nitrate: negative (3) Denitrification reaction (Komagata et al .: Takeharu Hasegawa, ed .; Classification and identification of microorganisms, 223 pages, University of Tokyo Press, 1975): Negative (4) MR Test: negative (5) VP test: negative (6) Indole formation: negative (7) Hydrogen sulfide formation: negative (8) Starch hydrolysis: positive (9) Utilization of citric acid: Koser's medium, Both negative in Christensen's medium

(10) Use of inorganic nitrogen source: Both sodium nitrate and ammonium sulfate are positive. (11) Pigment formation: No soluble pigment is found in either King A medium or King B medium. (12) Urease (urea medium, Eiken Chemical): Negative (13) Oxidase: Positive (14) Catalase: Positive (15) Growth range: Growth is observed in the pH range of 6.0 to 9.0,
The optimum pH is 7.0. Growth is observed in the range of 20 ° C to 37 ° C, and the optimum temperature is 30 ° C. (16) Attitude to oxygen: facultative anaerobic (17) OF test (by Hugh Leifson method): fermentation type

(18) Production of Acid from Saccharides and Production of Gas (in a Sugar-added Ammonium Salt Medium) The production of acids from 15 kinds of sugars was examined using a BCP indicator and a BTB indicator. The results are shown in Table 3.

[0024]

The generation of gas from the saccharides shown in Table 3 was examined, but no gas was generated in any of the above 15 types of saccharides. (19) Casein hydrolysis: positive (20) Esculin hydrolysis: positive (21) Lysozyme sensitivity (meat broth containing 0.001% lysozyme): resistant (22) Tyrosine degradation: negative (23) Lecithinase: positive (24) Non-acid resistant.

4. Chemical taxonomic properties (1) DNA base composition (G + C content): 44.9%

To summarize the above properties, BMK771-
The AF3 strain is a facultatively anaerobic spore-forming spore-forming bacillus whose gram staining is indefinite. It also has periflagellate and exhibits motility.
The spores are oval, central, swelling of the cells, and are nonacid resistant. The growth on the agar medium is opaque, the edges are irregular, and the colony surface is dull and shiny. The gelatin is liquefied and the milk is coagulated and peptoneized. It does not reduce nitrate and is negative in denitrification, MR and VP tests. Indole is not detected and does not produce hydrogen sulfide. Decomposes starch and does not use citric acid. The urease reaction is negative, and the oxidase and catalase reactions are positive.

The strain grows in the pH range of 6.0 to 9.0, with an optimum pH of 7.0. Growth is observed in the range of 20 ° C to 37 ° C, and the optimum temperature is 30 ° C. It oxidatively degrades glucose and produces acids, but no gases. As described above, acids are generated from saccharides other than glucose, but no gas is generated. It is resistant to lysozyme, degrades casein and esculin, and does not degrade tyrosine.

Based on the above properties, BMK771-AF
3 strains are Bergey's Manual of Systematic Bacteriology
, Volume 2 (1986), the Bacillus (Bacillu
s), belonging to the genus Bacillus alvei, Bacillus laterosporus and Bacillus brevis.
At present, the genus Bacillus is divided into multiple genera that reflect the lineage, and Paenibacils (Paeniba
cillus) (Literature, Antonivon Leeuwenhock, 64, 2
43-260, 1993), but in 1996 Brevibacillus (Br
evibacillus) and Aneurinibacil
lus) was proposed as a new genus (Literature, International
Journal of Systematic Bacteriology, 46, 937-94
6, p. 1996). That is, each of the three strains proposed in the search was Paenibacillus alvei.
lvei), Brevibacillus laterosporus
s laterosporus) and Brevibacillus brevis
bacillus brevis). BMK771-A
The properties of strain F3 and the above three strains were determined in the literature and Paenibacillus alvei IMC B-0925 (IFO 3343 ^T ), Brev
ibacillus laterosporus IMC B-0514 (purchased from the Ministry of Agriculture, Forestry and Fisheries Livestock Hygiene Laboratory in 1979) and Brevibacill
us brevis IMC B-0115 (IAM1031)
The results are shown in Table 4 below.

[0030]

[0031]

(Note) Each symbol in Table 4 has the following meaning. *: Experimental result ±: Probably determined to be positive. 1), 2), 3): Literature value (+: Positive in 90% or more strains, d: 11)
-: Positive in 89% of strains,-: Negative in more than 90% of strains) ^a : High pressure liquid chromatography (HPLC method)
, ^B : Measured by Thermal Denaturation Method (Tm method), ^c : Measured by Buoyant Density Method (BD method). 1), 2) and 3) shown in Table 4 indicate the following references. 1): Bergey's Manual of Systematic Bacteriology, Vo
lume 2, pp. 1122-1123, Williams & Wilkins, Baltimor
e, 1986 2): The genus Bacillis, Agriculture Handbook No. 42
7, United States Department of Agriculture, Washi
ngton, DC, 1973 3): Topley &Wilson's Principles of Bacteriology,
Virology andImmunity, 6th edition, Volume 1, 1103
Page, Edward Arnold, London, 1975

As is evident from Table 4 above, Brevibacillus laterosporus is BMK in that the position of the spore is canoe-shaped, hydrolysis of starch is negative, and nitrate is reduced.
It is significantly different from the 771-AF3 strain. Brevibacillus brevis is distinguished from the BMK771-AF3 strain in that the VP broth has a pH of 7.0 or more after growth, does not grow under anaerobic conditions, and may grow even at 50 ° C. On the other hand, the BMK771-AF3 strain showed a result different from that of Paenibacillus albay in that the production of indole was negative, but showed extremely closely related properties in the following points. That is, the BMK771-AF3 strain has a
The P-broth has a pH of 6.0 or less, which is positive for starch hydrolysis, does not reduce nitrate, and does not degrade tyrosine. In addition, the VP test was negative as a result of comparative study in the field, and the literature 2) The genus Bacillus,
According to Agriculture Handbook No. 427, it is described as negative with a probability of 1/4. The Egg-yolk lecithinase reaction was reported by Paenibacillus albay 3) Topley.
&Wilson's Principles of Bacteriology, Virology a
nd Immunity is described as positive and BMK771
-Matches the performance of AF3 strain. In addition, about G + C content, both showed very close numerical value. From these results, the BMK771-AF3 strain was identified as Paenibacillus alvei BMK771-AF3.

[0034] The BMK771-AF3 strain was applied to the Institute of Biotechnology and Industrial Technology Research Institute of Industrial Science and Technology, and deposited in September 1998.
Received on 17th as FERM P-16999.

In the second method of the present invention, the production of maleic acid A is performed as follows. That is, the production of the maleic acid A is preferably carried out by inoculating a maleic acid-producing bacterium in a nutrient medium and culturing it at 25 ° C. to 30 ° C. while aerobically shaking. A culture containing A is obtained. As a nutrient medium used for such a purpose, a medium that can be used for culturing bacteria is used. As a nutrient source, for example, commercially available nitrogen sources such as peptone, meat extract, yeast extract, corn steep liquor, ammonium sulfate, ammonium chloride, corn gluten meal can be used, and glycerin, starch, glucose, galactose , Dextrins, carbohydrates such as corn starch or carbon sources such as fats. Further, inorganic salts such as salt and calcium carbonate can be added for use. In addition, a trace amount of a metal salt can be added as needed. These can be used as long as they are used by the maleic acid-producing bacteria and are useful for the production of maleic acid A, and any known culture materials of actinomycetes or bacteria can be used.

For the production of maleic acid A, a microorganism which belongs to the genus Paenibacillus and exists widely in the natural world,
Bacteria having the ability to produce E. coli are used. Specifically, our isolated Paenibacillus albay
The present inventors have revealed that the BMK771-AF3 strain produces maleic acid A, but other strains can be isolated from nature by a conventional method for isolating antibiotic-producing bacteria. is there. In addition, there is still room for improving the ability to produce the maleic acid A by subjecting the maleic acid-producing bacteria including Paenibacillus albay BMK771-AF3 to irradiation or other mutation treatment.
Furthermore, it is also possible to modify the maleic acid-producing bacteria by genetic engineering techniques.

The maleic acid A is a bacterium that produces maleic acid A,
Preferably Paenibacillus albay BMK771-
The AF3 strain can be produced by aerobically culturing the strain in an appropriate medium and collecting from the culture solution. The culture temperature is not particularly limited as long as it can produce this substance without substantially inhibiting the growth of the maleic acid-producing bacterium, and can be selected according to the producing bacterium used. May be a temperature in the range of 25-30 ° C, especially 27 ° C.

The inoculum to be inoculated into the medium for the production of maleic acid A includes BMK771-AF3 on agar medium.
Use the growth obtained from the slope culture of the strain.

This Paenibacillus Albay BMK7
Growth of strain 71-AF3 usually peaks in 2-3 days, but generally continues until sufficient antimicrobial activity has been imparted to the medium. The time-dependent change in the titer of the maleic acid A in this culture solution can be measured by a cylindrical plate method using Pasteurella hemoritica BBP0102 N811 as a test bacterium.

In the second method of the present invention, the maleic acid A is collected from the culture obtained as described above. The method for collecting the maleic acid is a method used for collecting metabolites produced by microorganisms. As appropriate. For example, a method of extracting with a solvent that does not mix with water, a method of utilizing a difference in adsorption affinity to various adsorbents, gel filtration, chromatography using countercurrent distribution, or the like is used alone or in combination to collect the maleic acid A. it can. Further, from the separated cells, the maleic acid A can be extracted from the cells by a solvent extraction method using an appropriate organic solvent or an elution method by crushing the cells, and purified and collected in the same manner as described above. Thus, the above-mentioned antibiotic, maleic acid A, is obtained. The maleic acid A obtained from cultures is often obtained in the form of a mixture with several compounds of very similar structure. In order to isolate and purify the maleic acid A, it is convenient to convert it into a maleic acid A derivative that is easily isolated by chemical conversion, separate and purify the derivative, and then make the pure maleic acid A again by chemical conversion. It is.

For example, in collecting the maleic acid A from the culture of the maleic acid-producing bacteria, it is convenient to use the following collection and purification methods. That is, the obtained culture solution was centrifuged to separate cells into culture cells and a culture filtrate, and the culture filtrate was extracted with butyl acetate. The cells were extracted with methanol, concentrated, added with a small amount of water, and extracted with butyl acetate. The obtained butyl acetate extracts were combined and concentrated to dryness. The obtained oily residue was partitioned with hexane-methanol-acetonitrile (70:20:30 volume ratio). A crude oily substance containing acid A was obtained, and then this oily substance was subjected to silica gel column chromatography (developing solvent: chloroform) to obtain a primary purified product of maleic acid A, which was further subjected to centrifugal liquid-liquid distribution chromatography ( Solvent system: hexane-ethyl acetate-acetonitrile, 7: 2: 3 by volume) to obtain a secondary purified oil of the maleic acid A. Further, the maleic acid A in the oil is reacted with diazodiphenylmethane to produce the secondary oil. Diphenylmethyl ester is produced, the reaction solution containing it is concentrated to dryness, and the resulting residue is purified by silica gel column chromatography. Chromatography (eluted with toluene-ethyl acetate), and then the resulting oily substance consisting of maleic acid diphenylmethyl ester was treated with p-nitrobenzoyl chloride to obtain Op-nitrobenzoyl-maleic acid diphenylmethyl ester. The crude product was collected and subjected to silica gel column chromatography and silica gel thin layer chromatography to obtain a purified product of Op-nitrobenzoyl-maleic acid A diphenylmethyl ester. Next, the compound is subjected to a hydrolysis reaction with an aqueous NaOH solution, and then the free acid, maleic acid A, is extracted with ethyl acetate under acidic conditions.The obtained ethyl acetate extract is washed with water, dried over anhydrous sodium sulfate, and further concentrated. By drying the oily residue and subjecting the resulting oily residue to purification by centrifugation liquid-liquid partition chromatography (solvent system: hexane-ethyl acetate-acetonitrile, 5: 1: 4 by volume). Can be obtained as a pure product.

Further, according to a third aspect of the present invention, there is provided an antibacterial agent comprising, as an active ingredient, the maleic acid A of the formula (I) or a pharmaceutically acceptable salt thereof.

In this antibacterial agent, the composition is prepared by mixing the maleic acid A or a salt thereof as an active ingredient with a pharmaceutically acceptable conventional solid or liquid carrier such as ethanol, water, starch and the like. There can be.

In the fourth aspect of the present invention, the above formula (I)
Paenibacillus, which has the property of producing
Albay BMK771-AF3 strain is provided.

[0045]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in more detail with reference to the following examples. Example 1 Production of Antibiotic Paleic Acid A Paenibacillus albay B cultured on an agar slant
MK771-AF3 strain (FERM P-16999) was prepared using corn starch 2%, corn gluten meal 2%, glucose 1%, corn steep liquor 1%, ammonium chloride 0.25%, sodium chloride 0.3%, calcium carbonate 1000 ml of a liquid medium containing 0.6% (adjusted to pH 6.4) was dispensed in an Erlenmeyer flask (500 ml volume) in an amount of 110 ml and sterilized at 120 ° C. for 20 minutes by a conventional method and inoculated.
Thereafter, the cells were cultured at 27 ° C. for 3 days with rotary shaking.

The culture thus obtained was centrifuged to separate the cells. The obtained culture filtrate (800 ml) was extracted with butyl acetate (800 ml), and the extract was dehydrated with anhydrous sodium sulfate and concentrated to dryness. The residue was partitioned with hexane-methanol-acetonitrile (70 ml: 20 ml: 30 ml), and the lower layer was concentrated to dryness to obtain 175 mg of an oily substance.

Further, methanol (200 ml) was added to the cells, and the mixture was filtered by stirring, concentrated, and then water (200 ml) was added.
Extraction was performed with butyl acetate (200 ml), and the extract was treated in the same manner as the culture filtrate to obtain 64 mg of a crude oily substance containing maleic acid A. These oily substances obtained above are combined and subjected to silica gel column chromatography (Wa
kogel C200, 18 g), and purified using chloroform as a developing solvent to obtain a primary purified product of maleic acid A (yield;
46mg). Hexane-ethyl acetate-acetonitrile (7: 2: 3) by centrifugation liquid-liquid distribution chromatography.
The primary purified product is purified using a solvent system of
The product contained 34.7 mg of a secondary refined oil. This maleic acid A
The secondary refined oil contained was dissolved in 5 ml of methanol, and 50 mg of diazodiphenylmethane was added to the solution.
Allowed to react for hours. In order to complete the reaction, 9 mg of diazodiphenylmethane was added, and the mixture was further reacted for 9 hours. The resulting reaction solution containing the diphenylmethyl ester of maleic acid A was concentrated to dryness, and the residue was subjected to silica gel (Wakogel C2
00, 10 g) to purify by chromatography using a column. That is, the silica gel column was washed with 10 ml of toluene and then eluted with toluene-ethyl acetate (30: 1) to obtain 44 mg of an oily substance composed of diphenylmethyl ester of maleic acid A.

This diphenylmethyl ester was dissolved in 4 ml of pyridine, 30 mg of p-nitrobenzoyl chloride was added, and the mixture was reacted at room temperature for 29 hours to protect the hydroxyl group of diphenylmethyl ester of maleic acid A with p-nitrobenzoyl group. To complete the reaction, p-nitrobenzoyl chloride
After adding 20 mg and reacting for 19 hours, 10 mg of p-nitrobenzoyl chloride was further added and reacted for 8 hours. O generated
10 μl of water was added to the reaction solution containing -p-nitrobenzoyl-maleic acid A diphenylmethyl ester, left for 1 hour, and concentrated to dryness. Silica gel (Wakogel C200,
2g) Chromatography using a column, eluting with hexane-ethyl acetate (20: 1) gave 54
mg was obtained. The obtained oily substance is put on a silica gel plate (M
erck, Art 11798) and chromatographed with hexane-diisopropyl ether (10: 1).
It was spread once and separated by scraping to obtain 11.7 mg of a single maleic acid A derivative, Op-nitrobenzoyl-maleic acid A diphenylmethyl ester.

11.7 mg of this derivative was dissolved in 5 ml of ethanol, 1 ml of 0.5N NaOH was added, and the mixture was reacted at room temperature for 14 hours. This hydrolysis eliminated the p-nitrobenzoyl group and the diphenylmethyl ester group. Further, 4 ml of water and 0.1 ml of 1N HCl were added to the concentrate, and the resulting aqueous layer was extracted three times with 4 ml of ethyl acetate. The ethyl acetate layer (extract) was washed with water, dried over anhydrous sodium sulfate, and concentrated to dryness. The residue was subjected to centrifugal liquid-liquid partition chromatography to obtain hexane-ethyl acetate-acetonitrile (5: 1:
Purified using the solvent system of 4), and purified pure maleic acid A 5.5
mg was obtained as a colorless oil.

[Brief description of the drawings]

FIG. 1 is an infrared absorption spectrum of a maleic acid A measured by a KBr tablet method.

FIG. 2 is a proton nuclear magnetic resonance spectrum at 500 MHz measured in a deuterated chloroform solution of maleic acid A at room temperature.

FIG. 3 is a carbon-13 nuclear magnetic resonance spectrum at 125 MHz measured in a deuterated chloroform solution of maleic acid A at room temperature.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) // (C12P 7/42 C12R 1:01) (72) Inventor Umekita Maya Mitsuzawahigashi-cho, Kanagawa-ku, Yokohama-shi, Kanagawa-ken 5-46 (72) Inventor Hiroshi Naganawa 3-17, Denon-chofu-honmachi, Ota-ku, Tokyo (72) Inventor Riki Sawa 4-6-1, Ayanishi, Ayase-shi, Kanagawa Prefecture F-term (reference) BH01 BH02 BH04 BH05 BH07 BH08 CA02 DA04 4B065 AA01X AC14 BA22 CA10 CA43 4C206 AA01 AA02 AA03 AA04 DA07 MA01 MA04 NA14 ZB35 ZC61 4H006 AA01 AA03 AB03 AB20

Claims (4)

[Claims] 1. The following formula (I) Or a pharmaceutically acceptable salt thereof. 2. An antibiotic, paralein, which comprises culturing a bacterium belonging to the genus Paenibacillus which produces the maleic acid A of the formula (I) according to claim 1, and collecting the maleic acid A from the culture. Method for producing acid A. 3. An antibacterial agent comprising, as an active ingredient, the maleic acid A of the formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof. 4. Paenibacillus albay B having the property of producing the maleic acid A of formula (I) according to claim 1.
MK771-AF3 strain.