KR810001115B1 - Process for the preparation of antibiotics neoviridogriseins - Google Patents

Abstract

Neoviridogriseins having broad spectrum antibiotic effects against bactria, mycoplasmas actincmycetes, and rickettsiae were produced by cultivation of Streptomyces P8648. Thus, St. P8648 was cultured at 28oC for 96 hr with aeration and stirring. The broth was filtered off and extd. twice with 2 EtOAc. The EtOPAc ext. was dried under reduced pressure over Na2SO4. The dried powder was dissolved in MeOH and chromatographed on Sephdex LH-20 and dried. The product was chromatographed on silica gel to yield neovidogrisein.

Description

Method for preparing antibiotic Neovirido Grizeins

Figure 1 shows the UV absorption spectrum of neovirido glycine I (in methanol 0.1N caustic soda in methanol).

Figure 2 shows the ultraviolet absorption spectrum of neovirido glycine II (in methanol 0.1N caustic soda in methanol).

FIG. 3 shows the UV absorption spectrum of neovirido glycine III (in methanol 0.1N caustic soda in methanol).

Figure 4 shows the infrared absorption spectrum of neovirido grizein I (KBr tablets).

Figure 5 shows the infrared absorption spectrum of neovirido grizein II (KBr tablets).

FIG. 6 shows the infrared absorption spectrum of neovirido glycine III (KBr tablet)

Figure 7 shows the UV absorption spectrum of Virido Grizein (in methanol and in 0.1 N methanolic caustic soda).

8 shows the infrared absorption spectrum of Virido Grizein (KBr tablet)

The present invention relates to novel antibiotic neovirido glyzeins and their preparation. More specifically, the present invention relates to peptide-based new antibiotics named neovirido greases I, II and III, their preparation methods and therapeutic agents containing them as active ingredients.

The antibiotics of the present invention have a particularly high activity against Gram-positive bacteria and mycopurazuma.

The so-called antibiotics with mikamycin-vernamycin (or streptogramin) fall into two main groups. -Is a macrocyclic lactone compound of group A (antibiotics III 521-534 (springer verlag Berlin Heidelberg New York 1975)) by D.vazquez and griseoviridin austreo grayin A (OStreo grycin A = Mycamycin A (= mikamycin A) = virginiamycin M (Virginiamycin M) = staphylomycin M (staphylomycin M) = puristinamycin IIA (Vristinamycin IIA) = betunamycin A (Vernamycin A) = streptogramin A (streptogramin A) = synergistin A-1 (synergistin A-1) = compound PA-114A / = compound E-129A factor, etc.], Austraogrycin G (= Vestigermycin MII (Virginiamycin M II = stapylomycin MII) puristamycin IIB (Pristinamycin IIB) = dihydrostereogrecin A = dihydro-streogrycin A = compound E-129-B factor = compound R, P-13920, etc. ) And compounds A-2315 A, B and C.

Antibiotics in this group are bacteriostatic and have activity against Gram-positive bacteria and mycoplasmas.

Grizeoviridin, one of the fermentation products of the present invention, is known to be produced with viridogrisein by streptomycetes.

The production of grzeobiridenes is known from USP 3,023,204 and 3,174,902. The second group is called group B by D, vazqez, and is a macrocyclic depsipeptide which is a subgroup of two subgroups, namely the viridogrize subgroup and the vermycin B subgroup. Classified as Antibiotics in this group are effective in inhibiting the proliferation of Gram-positive bacteria, such as staphylococcus aureus and Bacillus subtilis. Bernamycin B subgroup contains 12 homologues. The synonym relationship of the published name of the antibiotic's name for the bacterium is very complex, and it is impossible to carry all the compound names of this subgroup. Representative scientifically important compounds of this subgroup are as follows.

Verunamycin B _α = Austraograin B = Puristinamycin IA = Micamycin B = Streptogamine B = Syngelgitin B-1 = Compound E-129 = Factor = Compound PA 114 B ₁

2. Verunamycin Bγ = Austraogresin B ₁ = fumastinamycin IC = compound E-129 Z ₁ phosphorus.

3. Verunamycin Bβ = Austraogresin B ₂ = Fumastinamycin IB = Compound E-129Z ₂ = Factor = Compound RP-13919.

4. Factor E-129Z _{3, which is} Osteo Grayin B ₃ = Cargo.

5. Petricin A

6. Petricin B

7. Bernamycin Bδ

8. Bernamycin C = doricin

9. Virgini Mycin (Staphytomycin) S

10.Verginimycin (Staphytomycin) S ₂

11.Veriniomycin (Staphytomycin) S ₃

12.Verginimycin (Staphytomycin) S ₄ (or S ₁ )

Compounds of this subgroup include the same four component 3-hydroxypic Olinic acid, L-threonine, L-Proline and L-phenylglycine (L- Phenylglycin) has a common feature that it is composed of seven components involved.

On the other hand, only one of the subgroups of viridogris is known, the compound viridogrizein, and it is already established that viridogrizein is the same compound as compound K-179 and compound F-1370A of etamycin. Compared to the Bernamycin B subgroup described above, Virido Grizein has 8 component 3-hydroxypicolinic acid, L-steonine, D-leucine, 4-hydroxy-D-proline (4 -hydroxy-D-Proline), Sarcosine, β-N-dimethyl-L-leucine (β, N-dimethyl-L-Leucine), L-alanine and L-phenylzarcosine It consists of (L-phenylsacorsine).

As is clear from the following description of the present invention, the antibiotic neoviiridogrisein of the present invention belongs to this latter bilido-grize subgroup, and the neo-viri is also one of the neo-riridae among the Grizeins. Jane IV was found by the inventors to be identical to Virgi Grizein.

The preparation of Virido Grizein is known from USP 3, 023, 204.

Synergism of other compounds called group A and group B is well known in various microorganisms, and it is also known as a drug containing antibiotics of this family as an active ingredient to enhance the therapeutic effect of medicine. It is written, but it is clear that this neobiri also exists in the Gizei.

The present invention relates to antibiotics having novelty and utility.

More specifically, it relates to a group of depsipeptide antibiotics called neovirido glyzenes I, II and III and virido glyzein, which have activity against Gram-positive bacteria and mycopurazma, It can be used with or without glyobiridine as a crude, concentrated or pure form.

The present invention has an excellent effect of inhibiting the proliferation of various Gram-positive bacteria and mycopurazuma. Neoviri also aims to provide a group of new antibiotics called Grizeins I, II and III.

The present invention also relates to streptomyces named Streptomyces SP, P 8648 (microbial fungi: No. 3652) and ATCC 31289 (Strepotmyces sp. P 8648, FERMP-No, 3562), ATCC 31289. It is an object of the present invention to provide a method for recovering vivid neovirido gizein with or without Gizeo viridine by culturing new strains of the genus in a suitable aqueous medium under aerobic conditions.

In addition, an object of the present invention is to disclose a method for selectively increasing the production amount of a kind required among neovirolidogases I, II, and III by adding an appropriate amino acid. In other words, by adding furorine or α-amino-n-butyric acid before or during the culture, the former has I and II of neobiridoglyze and the latter has I and III, respectively. It is possible to selectively accumulate in the land.

Other objects of the present invention will become apparent from the detailed description of the invention.

Neoviridogris of the present invention I, II and III (optionally IV) have a high activity against various pathogens including Gram-positive bacteria and mycopurazuma, and can be effectively used as drugs for humans and livestock. It is.

In more detail, Staphyloocccus aureus, Staphylococcus Pyogenes, Diplococcus Pneumoniae, Mycoplasma garisepticum Gram-positive fungi such as Mycoplasma farmentans and Mycoplasma agalactiae and Mycoplasma farmentans can be effectively used as a therapeutic agent for infectious diseases caused by Mycopurazuma.

As will be described later below, the microorganisms of the method of the present invention are neovirido glyzeins I, II and III and neovirido glycine IV (viridogrizein) and neovirido glyzein A (grizeobiridine). It produces a plurality of antibiotics. Among them, neovirido gizein A was confirmed to be the same as the antibiotic antibiotic Griseoviridin, and neovirido gizein B was neovirido and gizeins I, II and III and neovirido. It is a mixture of a group of peptide antibiotics called Grizein IV, of which neovirido IV is known to be the same antibiotic known as viridogrisein, but the other three are new antibiotics. lost. In addition, in this specification, the name neovirido-grizein is used as the name of not only the mixture of neovirolidogrisin I, II and III, and a biridogrizine, but also the mixture of two or more of these four characters, or these Used as a generic name for the higher-level concept of four characters.

First, the strains producing neovirido glyzenes I, II, III and viridogrine and glyobiridine of the present invention are a type of actinomycetes belonging to the genus Streptomyces Streptomyces SP, P 8648 No. 3562 It can be manufactured by culturing ATCC 31289 (Streptomyces SP 8648, FERMP-No, 3562).

These strains were isolated for the first time by the Fukui-ku Kuzu-Daki River upstream and the Kuz-Daki Dam.

Next, the mycological characteristics of this strain are fascinated.

)에서, 짧은 무색의 기중균사(

)를 신장하고, 그 선단에 느슨한 나선상의 포자연쇄(胞子連鎖)를 형성한다. 그 포자의 표면은 평활한 것이다. 윤생체(輪生體) 및 자낭포자의 형성은 확인되지 않았다. 각종 배지에 있어서의 본 군주의 배양적 특징은 다음과 같은 것이다.The strain of the present application is a well-branched simple branched parasitic mycelia (

), Short colorless aerial hyphae (

) Elongate and form loose spiral spherical chain (胞子 連 鎖) at its tip. The surface of the spores is smooth. The formation of the rotator and follicular spores was not confirmed. The culture characteristics of this monarch in various media are as follows.

(1) Sucrose acetate agar medium

Growth: Slight growth

Airborne hyphae: thin white airborne hyphae

Parasitic hyphae: colorless to off-white

Soluble Color: None

(2) Glucose asparagine agar medium

Growth: Good

Airborne hyphae: almost no formation, white when formed

Parasitic hyphae: pale yellow to light yellow (2db to 2fb)

Soluble Color: None

(3) Glycerin Asparagine Agar Medium

Growth: Medium

Airborne hyphae: almost no formation, white when formed

Parasitic hyphae: pale yellow, gray yellow (db ~ 2dc ~ 3ec)

Soluble Color: None

(4) East malt agar medium

Growth: Good

Airborne hyphae: white to slightly grayish white

Parasitic hyphae: light yellow (2fb) but brownish gray (3li) in the later stages of culture.

Soluble color: absent, sometimes slightly brownish.

(5) Star Arch Agar Badge

Growth: Medium

Aerial hyphae: rarely formed, but when formed, white,

Parasitic hyphae: pale yellow (2db), the center of the corona has a bright gray brown (zge).

Soluble Pigment: None

Fully disassembled: slightly disassembled

(6) tyrosine agar medium

Growth: Medium

Airborne hyphae: Rarely formed, rarely check the white aerial hyphae.

Parasitic hyphae: grayish yellow (3ec) to light yellowish brown (3ge)

Soluble Pigment: At the beginning of the culture, a light purple to light brown pigment is produced, but becomes light brown after about 10 days of culturing, and then thins. (Slightly produces methine pigment.)

(7) nutrient agar medium

Growth: Good

Airborne hyphae: forms lightly white aerial hyphae.

Parasitic hyphae: pale yellow (2db)

Soluble Color: None

(8) Auto Mill Agar Badge

Growth: Good

Airborne hyphae: white to grey-white

Parasitic hyphae: grayish yellow (3ec), light gray-brown (4ge)

Soluble Color: None

This strain grows 25-33 ° C. as the optimum temperature range, but it is poor in growth at 45 ° C. or 10 ° C., but can grow. However, it cannot grow above 52 degreeC.

This strain liquefies gelatin on glucose peptone gelatin medium and hydrolyzes starch, although weak, on inorganic salts and starch agar medium. This strain peptonizes skim milk. Coagulation is not recognized. This strain does not produce metinine pigments in peptone yeast agar media and trypton yeast liquid media, but sometimes in tyrosine agar media. This strain uses D-quisidose, D-gurucose, D-furactose, L-lamnose, D-mannit, on L. arabinose, I Use a little bit of sucrose instead of using inositol and raffinus.

The antibiotic neobiri of the present invention is also known as a strain that produces antibiotics of Grizeins, the following actinomycetes. Namely, Streptomyces griseus NRRL 2426, Streptomyces griseoviridis NRRL 2427, Streptomyces SP.Ethamycin-producing bacteria and Streptomyces conga Encephalitis (Streptomyces Conganensis).

)를 착생하고, L-아라비노오스를 이용하지 않는 등, 양자는 전혀 다른 성질을 나타내고 있다. 다음에 에타마이신 생산균은, 좌벡한천배지, 그루코오스, 아스파라긴한천배지, 영양한천배지 등의 배지상에 있어서의 배양적 특징이나 탄소원 동화패턴 등에 있어서 본원 사용의 균주와 커다란 차이를 나타냄. 스트렙토마이세스·콘가넨시스는, 포자연쇄의 형태적 특징 등 본원 사용의 균주와 명확하게 다르다. 상기 4주(株)의 중에서 본원 사용의 균주와 가장 근연(近緣)이라고 생각되는 스트렙토마이세스·그리제오비티디스 NRRL 2427에 대해서는, 양자를 동시에 나란히 하여 배양해서, 비교시험을 행하였다. 그 결과를 다음 표에 나타냄.Comparing these bacteria with the strains used in the present invention, Streptomyces grizeus NRRL 2426 is a straight or slightly wavy form of a natural chain, and thus has a morphological classification RF (Rectviflexibiles) section. On the yeast malt agar medium, gray to yellowish gray (e to 2ge) airborne hyphae are cultivated, and L-arabinose is assimilated, whereas the strains of the present application use S (Spirales) in morphological classification. ), White to light grayish white aerial mycelium on yeast malt agar medium.

) Are grafted and L-arabinose is not used, and both exhibit completely different properties. Next, etamycin-producing bacteria showed significant differences from the strains used in the present application in terms of culture characteristics and carbon assimilation patterns on media such as left Beck agar, glucose, asparagine agar and nutrient agar. Streptomyces conganenesis is clearly different from the strain of the present application, such as the morphological characteristics of the native chain. In the above 4 weeks, the strain of the present application and Streptomyces grigiovitadis NRRL 2427, which are considered to be the most recent, were cultured in parallel with each other and tested. The results are shown in the following table.

As described above, various obvious differences in morphological characteristics, culture characteristics, carbon mobilization patterns, and the like are shown between Streptomyces Grizeobiridis NRRL 2427 and the strains of the present invention.

In addition, when these bacteria are cultured side by side and produced in the production conditions described below, the strain of the present application produces neoviridagris including the biovirido greases I, II, and III, compared to Streptomyces grezeobiri. This NRRL 2427 is recognized as a physiological difference that it cannot produce these three components.

As judged from these results, there is no known strain corresponding to the strain of the present application, and it is considered appropriate to recognize it as a new species and named Streptomyces SP, P 8648. This strain was usually deposited with the Institute of Microbiological Industry and Technology Research Institute of Industrial Technology and Technology, and received the unconjugated bacteria deposit repair No. 3662 (FERM-P-No, 3562) ATCC 31289.

As is well known, the bacteriostatic properties change due to natural and artificial mutations. The strain used in the present invention is not limited to the strain of the above-mentioned microfluidic accession No. 3652, natural and artificial mutant migration of the present strain having the ability to produce the antibiotic neobirido Grizeins of the present invention It includes (iii).

Next, the method for producing the antibiotic of the present invention using the above-mentioned production bacteria will be described. To produce the antibiotic neovirido glyzeins, the strains of the genus Streptomyces having the ability to produce neoviry glyzeins are inoculated into a nutrient medium suitable for production, and these are treated at 18 to 37 ° C. for 2 to 14 days. Aerobic culture production and extraction and purification from the culture.

As a medium component, the well-known thing used for normal streptomyces culture can be used.

For example, carbon sources include gurucose, glycerin, starch, dextrin, auto wheat, molasses, fats and fats, and most of the nitrogen sources include cottonseed meal, meat extract, peptone, dry yeast and coonstee. Organics such as furaca, yeast extract, casein, casein hydrolyzate and inorganic substances such as ammonium lactate and ammonium acetate may be used.

In addition, inorganic salts such as calcium carbonate, salt, potassium chloride, phosphate, magnesium lactic acid, and other trace metal salts are added as necessary to aid the growth of the bacterium, and the neobiori also promotes the production of antibiotics. , Organic substances such as vitamins and inorganics can be added as appropriate. As a culture method, the liquid culture method by shaking culture, tank culture, etc. which are generally used for production of antibiotics is used, but the tank culture method is most suitable for industrial production.

As for this culture, 25-33 degreeC is preferable. In this way, the production of antibiotics reaches the highest concentration in both shake and tank cultures, ranging from 2 to 10 days. Depending on the type of medium, the pH may be greatly inclined to be acidic or alkaline during the cultivation. In this case, it is preferable to maintain the pH of the culture species at 6-9.

The pH at the beginning of the culture is preferably adjusted to 6.5 to 8.5.

The monarch used in the present invention has the ability to produce each component of Grizeobiridine and Neovirido Grizein under culture conditions, and in particular, without adding free amino acids or organic acids, It is possible to control the production rate of the above products by appropriately combining the carbon and nitrogen sources commonly used for actinomycetes fermentation, but free amino acids, in particular, the constituent amino acids of the products are added to the culture, By changing the amino acid composition, it is possible to selectively increase the production of the new antibiotic neobiridoglyzes I, II and / or neobiridogrize III as needed.

In addition, strain Streptomyces SP, P 8648 used in the present invention has the production capacity of neovirolidogases I, II and III invisible to known viridoglyzein producers, but has kazamino acid or putorine. By adding in the appropriate amount of culture, it is possible to selectively increase the production of antibiotic strong neovirido grizein II, and to add n-amino-α-butyric acid during cultivation In this way, the production of neo-olidogrigins I and III can be selectively increased.

The antibiotic neovirido glyzeins accumulated in the culture solution can be recovered and isolated to each component by a known method for isolating depsi peptides and various purification methods based on the physical properties of the substance.

Moreover, neobiri can also be recovered as a mixture of Grizeins or a mixture of these and Grizeobiridine as needed.

When the antibiotics are prepared as feed additives or animal medicines, it is economically advantageous to recover them as a mixture.

The antibiotic neovirido glyzeins present in the culture solution are extracted with a poorly water-soluble organic solvent such as ethyl acetate, butyl acetate, benzene, toluene, n-butanol, methylene chloride, chloroform, and methyl isobutyl ketone. In the case where neovirido is also intended to selectively extract Grizein without extracting Grizeobiridine, the use of aromatic solvents such as benzene and toluene is preferable.

Since neovirido glyzeins are hardly present in the weight of cultures, it is recommended that the cultured cells be removed and washed by filtration or centrifugation before extraction of the antibiotics from the culture filtrate containing the washings. It is preferable because the fat-soluble impurities in the cells can be removed. In addition to the extraction by organic solvents, adsorption and desorption by activated carbon, Ambolite-XAD (Lower, Anda's, Inc.), Ambolite IR-I20 (manufactured by Roum and Huassa), Dow X 50W-X ₂ (manufactured by Dow Chemical) Adsorption, elution with cation exchange resins, gel filtration with Sappadex LH-20 (manufactured by Palomacia, Inc.), adsorption chromatography with alumina, silica gel and the like are suitably combined by column method or thin layer method. In some cases, the antibiotic can be recovered by using a countercurrent distribution method using a suitable solvent in combination.

Next, the neobirido of the present invention describes the physicochemical normal of the Grizeins.

Neovirido glyzeins are either methanol, ethanol, n-propanol, isopropanol, n-butanol, dioxane, ethyl acetate, butyl acetate, acetone, methyl ethyl ketone, methyl isobutyl ketone, benzene, toluene, methylene chloride Soluble in chloroform, carbon tetrachloride, N, N-dimethylformamide, dimethyl sulfoxide, ether in ethyl, poor in water, ether in petroleum, hexane are almost insoluble white amorphous solids. All of them are stable at least one month in the temperature range of 25-27 degreeC in aqueous solution, and are completely stable in the range of pH 2-9 even if it processes 60 degreeC and 30 minutes. .

Melting points measured by the Kopura method are as follows.

Neovirido Grizein II 93 ℃: Neovirido Grizein III 115 ℃: Virido Grizein 140 ℃

Infrared absorption spectrographs of Neovirido Gizein I, Neovirido Gizein II and Neovirido Gizein III and Virido Gizein are shown in FIGS. 1, 2, 3 and 7, respectively. In each of the figures, the solid line ① in the figure indicates ultraviolet absorption in neutral methanol, and the chain line ② indicates ultraviolet absorption in 0.1 N methanolic caustic soda.

값(괄호내)를 다음에 나타냄.UV absorption maximum measured in methanol and its wavelength

The values (in parentheses) are shown below.

값을 다음에 나타냄.Neovirido Grizein I305 nm (65.0): Neovirido Grizein II 305 nm (88.0): Neovirido Grizein III 305 nm (90.0): Virido Grizein 340 nm: 0.1 N methanolic caustic soda solution UV absorption maximum in the inside and the wavelength

The values are shown below.

Neovirido Grizein I 340 nm (70.0): Neovirido Grizein II 340 nm (84.0): Neovirido Grizein III 340 nm (90.0): Virido Grizein 340 nm (96.0).

Infrared absorption spectra of Neovirigo Grizeins I. II and III and Virido Grizein measured as potassium embrittlement purification are shown in FIGS. 4, 5, 6 and 8, respectively. The infrared absorption band is recognized in the wave numbers shown below.

Neovirido Grizein Ⅰ: (KBr Tablet)

3370, 2910, 2850, 1735 1675 (sh) 1635, 1590 (sh), 1515, 1460 (sh), 1445, 1405, 1375 1290, 1280, 1250 (sh), 1200, 1190, 1160, 1125, 1100,

Neovirido Grizein II: (KBr Tablet)

3320, 2950, 2920, 2820, 2800, 1745, 1670 (sh), 1630, 1600 (sh), 1575, 1515, 1460 (sh), 1445, 1405, 1390, 1365, 1330 (sh), 1295, 1275, 1240, 1200, 1195, 1160, 1130, 1095,

Neovirido Grizein III: (KBr Tablet)

3335, 2960, 2940, 2870, 1750, 1670 (sh), 1660 (sh), 1635, 1590 (sh), 1515, 1450, 1405, 1390 (sh), 1370, 1340 (sh), 1300, 1245, 1200 , 1160 (sh), 1130, 1100,

The molecular weight determined by mass spectrometry is shown below.

Neovirido Grizine Ⅰ 876

Neovirido Grizine Ⅱ 862

Neovirido Grize Ⅲ 892

Virido Grizein 878

RF value in the silica gel thin layer chromatography developed as a synergistic method using the silica gel plate 60F Rf by the Malk company is shown next.

(1) For solvent system (benzene: methanol = 5: 1)

Neovirido Grizein Ⅰ Rf 0.66

Neovirido Grizein II Rf 0.62

Neovirido Grizine Ⅲ Rf 0.59

Virido Grizein Rf 0.55

Grigio Birridine Rf 0.20

(2) Solvent system (chloroform: methanol = 30: 1)

Neovirido Grizein Ⅰ Rf 0.39

Neovirido Grizein II Rf 0.32

Neovirido Grizein Ⅲ Rf 0.19

Virido Grizein Rf 0.18

Grigio Birridine Rf 0.02

Hydrolyze each of the Neovirido glyzeins at 110 ° C. in 6N hydrochloric acid overnight, concentrated to dryness to completely remove the hydrochloric acid, and then using thin layer chromatography (Eastman, Klammergram, Sheet 13254, Eastman Kodak), n-butanol: Using acetic acid: water = 4: 1: 1 solvent system or n-propanol: pyridine: acetic acid: water-15: 10: 3: 12 solvent system), high pressure excitation electrophoresis ( Dongyang Filter No. 51 A paper, using Dongyang Filter Co., Ltd., formic acid: acetic acid: water: = 25: 75: 900 using a pH 1.8 buffer solution at 0 ° C. for 30 minutes and energizing at 60 V / cm) and an amino acid analyzer ( When amino acid analysis was carried out on Hidade KLA-5), the amino acids shown below were detected.

Neovirido Grizein I: threonine, leucine, furoline, α-amino-n-butyric acid, salcosine, phenylsalcosine, β, N-dimethylleucine

Neovirido Grizein II: threonine, leucine, purorine, alanine, salcosine, phenylsalcosine, β, N-dimethylleucine.

Neovirido Grizein III: threonine, leucine, oxyfuroline, α-amino-n-butyric acid, salcosine, phenylsalcosine, β, N-dimethylleucine.

Viridogrizein: threonyl, leucine, oxyproline, alanine, salcosine, phenylsalcosine, β, N-dimethylleucine.

The fact that neooxyrido gizein is commonly present in 3-oxypicolinic acid is Rf of 0.46 when developed as a solvent system of chloroform: methanol = 2: 1 using mass spectrometry and thin layer chromatography (melk, silica gel plates). In addition, the Rf value when developed as a solvent system of n-butanol: acetic acid: water = 4: 1 using Eastman Chromagram Sheet 13254 was confirmed by 0.62. It became.

Mass spectrometry was performed in two ways.

Molecular weight determination was carried out by directly introducing each of the neovirido griffins into the mass spectrometer and furthermore, according to the method of F.compernolle et al (organic mass spectrometry vol, 6, 151-166, (1972)) Each of human beings was hydrolyzed with 0.1 N caustic soda at room temperature overnight at room temperature for methylated with diazomethane and introduced into a mass spectrometer to analyze the chemical structure.

In total, the chemical structures of neovirido glyzein I, neovirido glyzein II, and neovirido glyzein III were determined as follows.

Neovirido Grizine Ⅰ

Neobirid Grizein Ⅱ

Neobirid Grizein Ⅲ

Neovirido Gizein II, Neovirido Gizein II and Neovirido Gizein III are novel peptide-based antibiotics similar to viridogrine. Neovirido-Gizein IV equivalents were found to be identical to those of the known samples of viridogrisane and etamycin, as described earlier.

Biological properties

Neovirido glyzeins and mixtures with griseobiridine exhibit a broad spectrum of antimicrobial spectrum, and neovirido glyzenes I, II and III, against bacteria mycopurazuma, actinomyces and rickettase In virto test shows a wide range of antimicrobial spectrum, Streptococcus Pyogenes, including Streptococcus aureus and its resistant bacteria, Streptococcus and Streptococcus Pyogenes, etc. Pneumococcus and Sarcina Lutea, Bacillus Subtilis, Mycoplasma gallisepticum, Mycopurazuma Fullmonis (M, etc.), such as Diplococcus Pneumoniae Pulmonis), mycopurazuma fermentans (M, fermentans), and mycopurazuma agaract (M, agalactiae) and other strains of frostbite and resistant strains It Indicates.

Neovirado Gizein I, Neovirido Gizein II and Neovirido Gizein III and Virido Grizein mixtures, ie Neovirido Grizine mixtures, and Neovirido Grizine and Grizeobiridine About the mixture, the minimum stopping concentration with respect to the various microorganisms performed by the broth dilution method is shown in the following table.

Note: (a) NV is the neovirolidogrizes I, II and III, respectively.

(b) EM: erythromycin, STH: streptolysine, PC: penicillin, TC: tetracycline

LM: leucomycin, CP: chloramphenicol, SM: streptomycin, KM: kanamycin,

㎚: Neomycin, OM: Oreandomycin, () r: () Resistant to drug resistance

(c) BHI: Plaine, Fine Art, Infected Zone, Buros, MY Maltoisex East Badge

Neovirido glyzenes I, II and III coexist with glyobiridine to increase antimicrobial activity. When the minimum ratio is measured by changing the mixing ratio of neovirido glyzein mixtures and grzeobiridine, the result is shown in the following table. When added to each other, the antimicrobial activity shows the antimicrobial activity of individual multiples of each.

* Broth Dilution Method (BHI Medium) Using Salusina Lutea

Therefore, of course, the new antibiotic neovirido glyzes I, II and III of the present invention, respectively, are effective antibiotics alone, and by combining them with neoviride glyzevirides, It is an antibiotic.

Neovirido glyzein and the mixture of Grizeobiridine can be said to be an effective antibiotic for the disease, such as streptococcal infection, live purulent disease staphylococcal infection, pneumococcal infection, especially for livestock mastitis.

The synergistcaction is most pronounced when the mixing ratio of neoviridoglyzein and griseobiridine in the table is 50:50, i.e., compared with only neovirido grizinori or griseobiridine, It was confirmed that mixing them simultaneously showed 3-4 times the activity.

As shown in the table, Neovirido grizein II has stronger antibacterial activity than virido grizein. In this experiment, the minimum blocking concentration was measured by a 2-fold dilution method, but each pixel blocking concentration was determined by lowering the dilution ratio. The results were as shown in the following table, and it was clear that neovirido-grizein had two to three times stronger antimicrobial activity than that of virido-grizein.

Note: Ellipses, same as documents

In addition, the neoviridae mixture was added to 2-20ppm feed in a 10-week office building test by Osvina, and the added portion was larger than the non-added control in terms of weight gain and feed efficiency. Was outstanding.

Thus, it was found that the neovirido gizein mixture was also used as a feed additive.

Next, examples of the present patent manufacturing method will be given, but the scope of the claims is not limited to these examples.

The following table shows the high activity in vitro of neovirido glyzein II against various strains of Mycoplasma, as well as neoviry compared to the mixture of virido glycine III-grizeovidin. It also shows that the mixture of Grizein II-grizeobiridine has an excellent synergistic effect.

Note: Badge (1) PPLO reinforced broth (Japan: Sanitation Research Institute)

(2) PPLO broth (Difco), chanocks

50/50 medium mix

NV: Neovirido Grizane

VG: Birido Grizein

GV: Griseo Viridil

M. Mycoplasma

As shown in the table above, the novel compound neovirido glycine I-III of the present invention may be used alone or separately for the gram-positive bacteria and mycopurazuma strains, or viridogrizein (neovirigo glyzein). IV) and Grizeobiridine also show significant activity when used in various mixtures.

The mixed weight ratios in the mixture of neovirido glyzein I-III, viridoglyzein (neovirido glyzein IV) and glyobiridine are remarkable antimicrobial activity and antimycopurazma even if they are changed within a very wide range. It is recognized that activity remains. A non-limiting example is a representative example of a mixture of neovirido grizein II 27%, neovirido grizein IV (virido grizein) 23%, and gizeobiridine 50% (all weight ratios). Microorganisms of Streptococcus mutans related to dental, caries) and alveolar thick were tested in vitro. The test was conducted as a medium of hydrolyzate of Difco (Difco) with 0.5% TC lactoalbumin (Difco).

The microbial count at the start of the test was 3 × 10 ⁴ / ml. The test tube was incubated anaerobicly at 37 degreeC for 48 hours. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration were 1.0 ppm of neovirido-grizein.

In addition, although this is not a limiting example, as another example, the same mixture was tested in vitro against Treponema hyodysenteriae, a microbial microorganism of swine. The mixture was done at a concentration of 100, 50, 10, 5, 1, 0,5 and 0.1 ppm by dilution in blood agar. The bacterial solution attached to the cotton swab was inoculated under the dead, and incubated at 42 ° C for 4 days. MIC was determined to be 0.5 ppm. In another example, but not by way of limitation, mixtures of the weight percent composition of the heart were tested in vitro against several species of Mycopuraprise strains.

The MIC in this case is as follows.

Neovirido Grizein II 25%; Neovirido Grizein Ⅳ (virolidogrizane) 25%; Grizeobiridine 50% (all by weight)

The above-mentioned neobirido-grizein-grizeobiridine mixture is also effective for the sagging of animals of the above-mentioned pathogens.

In addition, since it is known that mikamycin A and B are very effective as feed additives, the present invention was conducted on animal feed tests for the compounds. Neovirido was also added to the chick's feed as a mixture in the ratio of 2 to 20 ppm, and male chicks were bred for 10 weeks. Compared to the chicks of the control group fed with the same feed without adding neovirine glyzene, the chicks reared as food supplemented with neovirido glycein are superior in weight gain and feed efficiency. . Therefore, the neobiridogizein of the present invention has proved to be very useful as a feed additive.

Furthermore, in some cases, compositions containing one or more of neovirido glyzein I-II, mixtures of virido glyzein (neovirido glycine IV) and gliobiovirine are very wide in the weight ratio of the respective components. It was very useful as a feed additive within the range of mixing ratio. As a representative example, but not limited to, breeding of chicks with a composition consisting of neovirido grizein 27%: neovirido grizein IV (viridogrizein 23%: grizobiridine 50% by weight). It was added to the feed of the biotest. One day after birth, 15 male chickens were used for 1 day, and about 55% of the rye was bred for 11 days as a food supplemented with vitamins, fats, and proteins. Many feeds in rye grains are commonly used as standard screening feeds for antibiotics for growth accelerators and feed additives because only poor to moderate growth is obtained. Penicillin (100 ppm) was used as a positive control. The result is shown next. The feed / weight ratio is the ratio of the weight of the feed consumed to the weighted weight, the weight ratio is the weight ratio of the chicks after 11 days of testing to the weight at the start of the trial, and the average weight of the last egg. (g) represents the mass (g) of the microbial mass per chick

Next, an embodiment of the present invention will be shown again, but the present invention is not limited only to this embodiment.

Example 1

50 ml of medium consisting of 0.5%, pearler media (made by traders oil, wheat) 0.5%, 0.5% of auto wheat, 0.5% of dry yeast, 0.5% of molasses (adjusted to pH 6.5) After sterilization into the nose, Streptomyces SP, P 8648 was inoculated, and rotary shaking culture was carried out at 28 ° C. for 48 hours to make it a seedling.

100 ml of medium containing 0.5% soy flour, 0.5% Venus-Miles, 0.5% Automiyl, 0.5% dry yeast, 0.5% molasses (pH 6.5) was placed in a triangle fresco, and 2 ml of seedlings were inoculated after sterilization. Then, the agitated shake culture (200 rpm x 7 cm) was performed at 28 ° C for 96 hours.

The culture solution for 12 scores was collected and the culture filtrate was obtained by filtration. The cake was washed with 100 ml of water and the washing solution was added to the culture filtrate.

The pH of this solution was 8.3, and on the NUTRIENT agar plate in which Sarusina lutea was buried, a bioassay was performed using a pulp disk of 8 mm in diameter. Gave.

This solution was extracted twice with 200 ml of n-butanol, and the n-butanol layer was concentrated and solid to obtain 90 mg of solid. This was filled in a silica gel column (1.5 ø × 25 cm) of Wago Gel C-100 (manufactured by Hwagwang Pure Chemical Co., Ltd.), first as 300 ml of a mixed solvent of benzene-acetone = (5: 1), followed by benzene-acetone (2: The elution was carried out as a mixed solvent of 1), and 10 g of the eluate was collected.

Fraction No. The active fractions of 25 to 35 were collected and concentrated to dryness to obtain a mixture of neovirido glyzeins (i.e., neovirido glyzenes I, II, III and viridogris 30 mg. This was benzene: acetone = 1: 1 A mixed solvent was used to give a spot of Rf = 0.05 on the thin layer chromatography on silica gel plate F254 manufactured by Melk Corporation.

Example 2

10 l of medium having the same composition as that of the seedling medium of Example 1 was placed in a 15 L stainless steel spermator, and sterilized, and then inoculated with 200 ml of the fresco culture medium incubated at 28 ° C. for 48 hours in a medium of the same composition, and 5 l of If swelled through air, stirring was incubated at 27-28 ° C for 96 hours while stirring 200 revolutions per minute of putraroshi per minute. After completion of the culture, the mixture was filtered and the filtrate was adjusted to pH 6.0, and then extracted four times with 2 L of ethyl acetate. The ethyl acetate layer was taken out, the dehydrated forget-me-not was added, concentrated and solid, and 700 mg of crude powders were obtained.

This was dissolved in a small amount of methanol, charged to a Sephadex LH-20 column (3 x 50 cm), developed as methanol, and the eluate was collected in 10 ml. Flexion No. The active fractions of 22 to 29 were collected, and the resultant was suspended in a silica gel column (1.5 χ x 20 cm) manufactured by Marine Crot Co., Ltd. and developed and eluted as a mixed solvent of chloroform: methanol = 30: 1, and 5 g of the eluate was collected. The active fractions of flexion numbers Nos. 5 to 12 were concentrated to dryness to obtain 25 mg of white powder. This white powder was dissolved in ethyl acetate and subjected to silica gel thin layer clototo confirm that this was a neovirido glyzein mixture.

The composition ratios of I, II, III, and Virido Grizein, which are neobiride Grize in the powder, are as follows.

Neovirido Gizein Ⅰ: 15% Neovirado Gizein III: 20%

Neovirado Grizein II: 20% Virido Grizein: 45%

Flexion No. of the LH-20 column. 30 to 34 active portions were collected, concentrated under reduced pressure, and recrystallized from warm methanol to obtain 30 mg of needle crystals. The crystals were characterized by the behavior of thin-layered chromatographs, and their chemical properties were consistent with those of grizeobiridine.

Example 3

The composition of this culture medium was mostly 0.5, permeer media (manufactured by traders oil, wheat, wheat) 0.5%, oatmeal 0.5%, dry yeast 0.5%, molasses 0.5%, DL-α-amino-n-butyric acid 0.1% ( pH 6.5) was incubated for 96 hours in the same manner as in Example 1. The 13 fiscal filtrates were extracted with 30 ml of n-butanol, and the butanol layer was concentrated to dryness to obtain 70 mg of crude powder. This crude powder was subjected to thin layer chromatography on a silica gel plate (F254) manufactured by Melk Corporation and subjected to a biotograph using Sarusina lutea as a test bacterium. Neovirido was also obtained from the higher antimicrobial activity spot Rf value as shown below. Glyzenes I, II and III and virido). The relationship between the developing solvent and the Rf value of each component was as follows.

Example 4

50 ml of medium of beef extract 0.3%, tryptone 0.5%, glucose 0.1%, soluble starch 2.4%, yeast extract 0.5%, calcium carbonate 0.4%, soy flour 0.5% (pH 7.0) After the sterilization of the spores of Streptomyces SP.P 8648 by incubation with incubation at 120 ° C. for 15 minutes, the cells were inoculated by triangular fresco, followed by shaking culture at 25 ° C. for 3 days. 250 ml of 50 ml of medium containing 0.5% soluble starch, 20% glucose, 1.0% permeermedium, 0.5% oatmeal, 0.5% cornstalker, 0.05% potassium diphosphate, 0.05% magnesium lactate (pH 6.5) After the sterilization, sterilization was carried out and the seedling culture was inoculated every 2%, followed by shaking culture at 25 ° C. After incubation for 24 to 48 hours, sterilized kazamino acid (manufactured by Diff Co.) or furoline solution (pH 7.0) was added to 0.4%, followed by shaking culture. After incubating for 4 days at inoculation, the culture solution was extracted twice using an equivalent amount of ethyl acetate, the extract was concentrated to dryness, dissolved in a certain amount of ethyl acetate, and a predetermined amount was spotted on a silica gel thin plate, and chloroform: methanol = 100: Multiple development was carried out twice using a mixed solvent of 2. The spot part detected by the ultraviolet-ray of 3650 kV after development was scraped, and it extracted and eluted as a fixed amount of methanol, and the amount of each component was bioassayed as a test bacterium using satusina lutea. The production rate of each Neovirido Grizeins was as follows.

Example 5

Medium containing 0.5% soy flour, 0.5% permeer media (traders oil, wheat mill), 0.5% oatmeal, 0.5% dry yeast, 0.5% molasses, and 0.1% DL-α-amino-n-butyric acid 600 liters (pH 6.5 L) was put into a 1.4 KL stainless steel culture tank, heat sterilized, and incubated under the same conditions as in Jasper Aman culture of Example 2 after cooling. About 10 liters of 23-hour seed cultures were inoculated.

Through 300 liters of sterile air was passed through a downward spar, and aeration stir culture was carried out at 28 ° C. for 75 hours while stirring 180 revolutions per minute in a two-point furorera (two stages). After incubation, the solids were filtered out, and the culture filtrate was extracted twice with 150 L of n-butanol.

The butanol layer was collected, washed with a small amount of saturated brine, and concentrated as a rotary evaporator to make the liquid amount 2 liters. To this, 20 g of silica gel (WaKO C-100, manufactured by Chemical Engineering Co., Ltd.) was concentrated and dried. A small amount of chloroform was suspended, which was then charged into a silica gel column (6.5 cm x 75 cm, 24 l, silicagen-WaKO C-100). I did not know. This column was eluted as a mixed solvent of chloroform-methanol (firstly, chloroform alone, inoculated with methanol), and 500 g of the eluate was collected. Flexion No. 16 to 29 synthetic fractions (neovirido grizeins) were collected, concentrated, and introduced into a Celadex LH-2-column (7 x 45 cm), eluted with methanol, and 100 g of the eluate was collected. Flexion No. 6-15 active fractions were collected and concentrated to dryness to obtain 15 g of crude powder.

Again, the flexion number No. 50 to 60 active fractions (grizeobiridine fractions) were collected, concentrated, and introduced into a Sephadex LH-20 column (7 x 45 cm), eluted with methanol, and 100 g of the eluate was collected. The active fractions of flexion number No-18-23 were collected and concentrated to dryness to obtain 1 g of crude powder.

Example 6

1 g of the crude powder of the neo-virido-grizein fraction obtained in Example 5 was dissolved in 2 ml of ethyl acetate, and it was wired to 10 silica gel plates made by Delx Corporation. This was first developed with a mixed solvent of chloroform: methanol = 50: 1, then developed as a mixed solvent of chloroform: methanol = 25: 1 to carry out a preparative thin layered chromatography.

After the development, the portion of neovirido glyzein detected by 3650 Å ultraviolet rays was scraped off, extracted as a small amount of methanol, and concentrated to dryness to obtain 16.7 mg of oily neovirido glycine I1.

In this way, Neoviri was also scraped off the part of Grizein, thereby obtaining 11.1 mg of white powder of the same substance. In this manner, Neoviri was also scraped off the part III of Grizene to obtain 15.2 mg of white powder of the same substance. Thus, the part of the fish oil was scraped off, and 30.0 mg of white powder of the same substance was obtained from it.

Each component was hydrolyzed at 110 ° C. for 36 hours using 6N hydrochloric acid in a sealed tube, and the amino acid and organic acid were analyzed by thin layer chromatography, fair perma chromatography, and an amino acid analyzer for the hydrolyzate.

The amino acids and organic acids shown below were detected in each component of neovirido glyzeins.

Nearly behavior in ultraviolet, infrared absorption characteristics, mass spectrometry and NMR analysis, etc. The majority of neovirido-Gizein IV is determined as the Virido-Gizein, and the chemical structure of other Neovir-Gizeins is determined.

Claims (1)

General formula (Ⅰ) belonging to the Streptomyces genus in nutrient media containing carbon, nitrogen, inorganic salts and trace components

Wherein R ₁ is -CH ₃ or -CH ₂ CH ₃ and R ₂ is H or -OH Microbial Streptomyces S.PP 8648 microreactor No. 3562, which has the ability to produce antibiotics, is cultured aerobicly at a temperature of 18 to 37 ° C. and a pH of 6 to 9, and in the medium, a depsipeptide of formula (I). A method for producing a tepsipeptide antibiotic neovirido glyzenes, characterized by accumulating and separating an antibiotic or a mixture thereof.

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