DD144562A5 - METHOD FOR PRODUCING NEW ANTIBIOTICALLY EFFECTIVE NEOVIRIDOGRISEINS - Google Patents

Abstract

The invention relates to a process for the preparation of novel against gram-positive bacteria and mycoplasma highly effective Depsipeptidantibiotika namely neoviridogriseins I, II and III, by breeding of Streptomyces sp.P. 8648 (FERM-P 3562).

Description

Applicant's Field of the Invention '. ^: Ϊ ν ': The invention relates to novel against gram-positive bacteria and mycoplasmas highly effective Depsipeptidantibiotica, namely Neoviridogriseine I, II and III and their preparation, isolation and use for therapeutic purposes and as an additive to animal feed. '

Characteristic of the known teeh'n. ^Solutions; The so-called micamycins / vernamycins (or streptograms) break down into two groups, of; those the one large group (group A after D. Vazquez: SS. 521-53 ^, ANTIBIOTICS ill, mechanism of action of antimicrobial and antitumor agents, Springer publishing house Berlin Heidelberg New York 1975). a mäkrocyclisches Lac ton represents and griseoviridine, Ostreogrycin A (= mikainycin A = V irginiamycin M = Staphylomyc in M = Pr is inamyc in IIA-Vernainycin A = streptograinine A = synergi st in Al compound PA-ll ^ Al = compound E = I29 Factor A, etc.), Ostreogrycin G 'C = Virginiemyc in MII = Staphylococc in FiIi Pr i st inamyc in I IB ^ Dihydrostreogrycin A = Compound E-I29 Factor B = Compound RP. »13920, etc.) and the connections A-231.5. A, B and C includes. These antibiotics are bacteriostatic and effective against gram-positive bacteria and mycoplasma. One of the breeding products of the invention is griseoviridine, which is known to be co-produced with viridogrisin in the production of streptomycetes and whose production has been disclosed in U.S. Patents 3,023,203 and 3,172,902.

The second large group (group B according to D. Vazquez) is a macrocyclic depslpeptide and breaks down into two subgroups, and zviar into the viridogrisein and vernamycin B group. These antibiotics are mainly active against grain-positive bacteria such as Staphylococcus aureus and Bacillus subtilis. The vernainycin B group comprises 12 homologs. Due to the complicated synonymic conditions of these antibiotics, it is virtually impossible to enumerate all compounds of this subgroup. In the following, only a few representative and scientifically significant compounds

hi 199 3fS

this subgroup called: v

, 1.Vernamycin BoC = Ostreogrycin B = Pristinamycin IA = Mikamycin B = Streptogramin B = Synergistin B-1 = Compound E-129 Factor Z = Compound PA114B1.::

2. Vernamycih Boc = Ostreogrycin BI = Pristinamycin IC = Compound E-129 Factor Z1; ^^^,; :; '.v

3 · Vernamycin Bo <. = Ostreogrycin B2 = Pristinamycin IB = Compound .. '. V .-., BrI29 Factor Z2 = Compound R.P.-13919

4. Ostreogrycin B3 = Compound E-1.29 Factor Z3 ^.

5. Patricin A _; '- ^;; ' -v ·: '- ^ - ^Vr "·"': '·,' /: / '; ^: ':; .. '. ·. ·;', '-' -ϊ '· '.

6. Patricin B : ',:];:; . / ';;\; _;; .- ^. ;; '' .. / ,: " ¹ V ^ o'-: '.' ,. '. ^: .' ^: ·. '. 7 «... Vernamycin'Br / - ^.:.'. ^: . _: ''^:; ^: '" ^ ^: '. ^: ; ';; .. ^; '' ^: '.. / -. , , 8. Vernamycin C = doricin. : ;; ; ;

"9 · virginiamycin (staphylomycin) _S; -

10. Virginiamycin (staphylomycin) S2

11 * Virginiamycin (staphylomycin) S3

12 · Virginiamycin (staphylomycin) S4.;. '.. (or S1).

The common feature of this subgroup is that it is composed of seven components, 4 of which _; namely 3-hydroxypicolinic acid, L-threonine, L-proline and L-phenylglycine are common to all compounds. From the viridogrisein group, in contrast, so far only a single compound has become known, namely the viridogrisin, which is identical to etamycin, compound K-179 and compound F-1370A. It consists of eight components, namely 3-hydroxypicolinic acid, L-threonine, D-leucine, 4-hydroxy-D-proline, sarcosine, β, Ν-dimethyl-L-leucine, L-alanine and L-phenylsarcosine. The neoviridogriseinantibiotica according to the invention now belong to the Viridogr.iseingruppe, wherein the Neoviridogrisein IV corresponds to the Viridogrisein. The preparation of viridogrisein had been disclosed in U.S. Patent 3,023,204.

The synergism between the groups A and B had been examined at various microorganisms "and was successfully used to increase the therapeutic effect of drugs ^that:., Used contained antibiotics of this family as active ingredients is also true for the Neoviridogriseine target Wr invention.: Ν -,; ' , J- '.;,., · Λ .; :: The invention thus relates to novel "valuable depsipeptide antibiotics, in particular to the neoviridogriseins I, II and III, and to viridogrisins which are active against gram-positive bacteria and mycoplasmas and used in dilute form as crude concentrates or in purified form together with or without griseoviridine Explanation of the nature of the invention Thus, according to the invention, they are the depsipeptide antibiotics neovirid agarisin I, II and III, which are highly effective against Gram-positive bacteria and mycoplasmas.

Another object of the present invention is a method for producing these antibiotics by cultivating a new species of Streptomyces, namely Streptomyces sp. P8648 (FERM-P3562) in a suitable aqueous medium under aerobic conditions, whereby .man from the culture solution in addition to griseoviridine or without this the neoviridogriseins

The present invention further provides a process for the selective recovery of the desired neoviridogrisin component (s) by addition of a suitable amino acid (s), in particular proline, to increase the yield of neoviridogrisin I and II, and <2C-amino-n-butyric acid to increase the Yield of neoviridogris in I and III before and during cultivation, respectively. Other subjects of the invention will become apparent from the following description of the invention. The neoviridogriseins I, II and III are highly effective against various pathogenic microorganisms, including

Finally, gram-positive bacteria -and- mycoplasmas, and accordingly may be found in the ¹ human and veterinary application, in particular for the treatment of by gram-positive bacteria and mycoplasma, such as Staphylococcus aureus, Streptococcus pyogenes, Dipiococeus pneumoniae, Mycoplasma gallisepticum, Mycoplasma fermentans, Mycoplasma agalactiae, etc. caused infectious diseases.

The term "neoviridogrisein" refers not only to a member of the group neoviridogrisin I, II and III and viridogrisin (= neoviridogrisin IV), respectively, but also to a mixture of two or more members of this group. - V

Identification; of the microorganism ; ;

The novel depsipeptide antibiotics according to the invention are, in addition to viridogrisin and griseoviridine, of a new breed of Streptomyces, namely Streptomyces sp. P 86 ^ -8 (FERM-P 35 ^ 2) produced. This microorganism was isolated from a soil sample taken near the Kuzuryu dam in Pukui-ken, Japan. , ::

The microorganism grows long, is colorless, and exhibits an aerial mycelium emanating from a distinctly branched (a single branching) substrate mycelium. The smooth surface spore chains are created in loose loops at the top of the aerial mycelium. 'Neither vortex formation nor ascospores. The behavior in the different agar media is viie

(1) sucrose-nitrate agar ·· '.-

Growth: weak

Aerial mycelium :: thin, occasionally white

Back: colorless to greyish-white

Soluble pigment '·; - - · -

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(2) glucose asparagine agar

Growth ί

Aerial mycelium:

Back:

Soluble s pigment:

(3) G-lycerin-asparagine agar

Growth:

Aerial mycelium:

Back:

Soluble: pigment:

strong, "''';> :: · .; "."^;;;''; .. _ "./ /'', weak or not trained; if present, then white, pale yellowish white to pale yellow

moderate

weak or not trained; if present, then white, pale yellow to greyish-yellow

(4) Yeast Extract-Malt Extract Agar

growth

aerial mycelium

back

Soluble pigment

(5) starch agar

Growth of mycelium

back

Soluble pigment starch hydrolysis

(6) tyrosine agar growth

strongly

white to pale gray to pale yellow, later turning brownish gray

none or rarely slightly brownish

moderate

weak or not trained; if present, then white, pale yellow, slightly greyish brown in the center of the colonies

low

moderate

-6- t $ 9 395

Aerial mycelium. Backside. Soluble pigment

(7) nutrient agar

Growth of aerial mycelium. Backside. Soluble pigment

'(S) oatmeal agar

Growth aerial mycelium backside

Soluble pigment

none or occasionally a few spots-white aerial mycelium greyish-yellow to pale yellowish-brown

first pale purple to light reddish-brown, after days pale brown, somewhat melanoid pigment.

thin, white

pale yellow

white to greyish-white greyish-yellow to light reddish-brown with sting in gray

The optimum growth temperature of the microorganism of the invention is in the range of 25 to 35 ⁰ C. Also, temperatures below or above this range, such as 1O ⁰ C or 45 ⁰ C are still eligible for breeding in question, but then the growth is very low. At a temperature of 52 ⁰ C, however, it comes to no growth.

Gelatin in glucose-peptone gelatin medium is liquefied; Starch in starch-inorganic salts-agar is lightly hydrolysed and skimmed milk is peptonized without coagulation.

Occasional production of melanoid pigment in tyrosine agar, but not in peptone-yeast extract-iron agar and tyrosine

3§S

Yeast extract solution. .

C-assimilation (in Pridham-Gottlieb's medium):

+: D-xylose, D-glucose, D-fructose,

 .; ':.; ...,:,; L-rhamnose, D-mannitol.

weak +: sucrose

'-. \: L-arabinose, i-inositol, raffinose

With respect to the production of the known peptide and nonpeptide macrolide antibiotics, such as Mikamycin A and B, Virginia mycine, Ostreogrycine, Etamycin, Yernamycin, Viridogrisein, Griseoviridin and Pristinamycin, the following microorganisms are treated with Streptomyces sp. Compared to P8648:

Streptomyces griseus NRRL 2426 /

griseoviridus NRRL 2427

sp., etamycin producer

conganensis

ostregriseus' ν

mitakaensis ':; y ^: "'^; · ^;;

''. ^: \ ' '; loidensis .. ^; , , , '''/'^:; ^: - ..: '.. , '·.

The information available to the applicant with regard to the cultural and physiological properties of the mentioned microorganisms show marked differences between the streptomyces claimed according to the invention and the streptomycetes mentioned above. For example, Streptomyces griseus NRRL 2426 belongs to the section Reetiflexibiles with straight or slightly wavy spore chains, while the microorganism according to the invention belongs to the section Spirales; the former produces gray to yellowish-gray aerial mycelia on yeast extract-malt extract agar, the latter white to .gruish-white; the former utilizes L-arabinose, but the latter does not utilize etamycin-producing Streptomyces species _; presented in Antibiotics Annual 1954-1955, SS.728-732,

-a- 199

can be distinguished from the organism according to the invention by the C-assimilation and the culture properties on Czapek agar, glucose-asparagine agar and nutrient agar. Streptomyces conganensis shows marked differences in the morphology of the spores. Among the above-mentioned microorganisms, Streptomyces griseoviridus NRRL 2427 shows the strongest similarity to the streptomycetes of the present invention. The taxonomic comparison of both streptomycetes revealed the following: \ .'-, ·. · ''> ^'·: Λ' .: ^'': '''' - /. - v / A ·, '''' ^{νγ :.} , ' ·. '^;''

Streptorayces gris e o-viridus NRRL 242?

Streptomyces sp. P 8648

Color of the

aerial mycelium ·

Pale orange-yellow to pale-pink with a hint of gray "on yeast extract" malt extract agar, oat agar, starch agar and glycerol-asparagine agar.

White to greyish white aerial mycelium, poorly developed on most ISP media, abundant white aerial mycelium on yeast extract · malt extract agar.

Color of the substrate mycelium

Greyish-yellow to slightly olive-brown or blackish-brown on yeast extract-malt extract agar, oat agar, starch agar and glycerol-asparagine agar.

Pale or light yellow to greyish-brown on most ISP media

Soluble pigment

No melanoid pigment, usually no pigment, rarely weak yellow pigment.

No melanoid pigment, usually no pigment, rarely brown pigment.

Utilization of C sources

L-aräbinose +++ D-fructose + sucrose - '

L-arabinose D-fructose +++ sucrose +

From this table it can be seen that between Streptomyces

- a- 1

griseovxridus NRRL 2427 and the streptomycete according to the invention, there is a clear morphological difference and a clear distinction in terms of cooling properties and C-assimilation. In addition, the microorganism of the present invention produces neo-Yiridpgri.ins I, II and III as well as viridogrisin (neoviridogris in TV) and griseoviridine; Streptomyces griseoviridus KRRL 2427, on the other hand, produces only viridogrisin and griseoviridin under the same conditions.

The microorganism according to the invention is therefore a new species, namely Streptomyces sp. P 8648. A corresponding culture of this microorganism has been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, under the number FERM-P No. 3562. It will be clear to those skilled in the art that the invention is not limited to the specific microorganism defined above and deposited under FERM-P No. 3562 at the Fermentation Research Institute, but also all spontaneously and artificially derived from this microorganism mutants which neo-viridogrisein I , II and III are capable of producing.

Preparation of neoviridogrisins I, II and III.

The antibiotics according to the invention are prepared by inoculation with Streptomyces sp. P 8648 and culturing it in a suitable medium under aerobic conditions at temperatures of 18 to 37 ° C for 2 to 14 days, wherein the enriched antibiotics are separated from the culture fluid and purified by the usual methods. Preferably, work is as follows:

For the breeding of the microorganism. All types of media that would otherwise be considered for Streptomycetes can be used. As carbon sources of the medium are preferably ia question glucose, glycerol, starch, dextrin, oatmeal,

Molasses, fat and oil, etc., as N-sources soybean meal, cottonseed meal, meat extract, peptone, dry yeast, corn syrup liquid, yeast extract, casein and its hydrolyzate and inorganic salts thereof, ammonium sulfate and ammonium nitrate. Optionally, growth-promoting substances may also be added to the medium in small amounts, such as vitamins, amino acids, organic and inorganic salts, e.g. Calcium carbonate, sodium chloride, potassium chloride, sodium phosphate, potassium phosphate and magnesium sulfate. -: ^

The new antibiotics can be produced by cultivating in the usual vessels, such as debris eleven la see, jar fermenter and tank fermenter, however _; From an economic point of view, the Submerskultür with forced ventilation _: most suitable for industrial standards. The cultivation is preferably carried out under aerobic conditions at temperatures of 25 to 35 ° C. When using a shaker bottle or a tank fermenter, the production of neoviridogris usually reaches its maximum after 2 to 10 days. The pH value may exceed that during growth, depending on the type of medium. reach out to the physiological area. However, it is preferable to adjust a pH of 6 to 9 during the culture. Before inoculation, the pH of the medium is usually adjusted to 6.5 to 8.5. · ', / -... λ.'";'V'.,^;; V /. ''' , [' \ -: \ - ': . /'..,;

Control of Neoviridogriseinkomponenten in the culture liquid

As already stated, the microorganism according to the invention produces a mixture of neoviridogrisins I, II, III and IV and griseoviridine. By suitable combination of the C and N sources in the medium, the composition of the neoviridogriseins in the culture fluid can be changed without the addition of a free amino acid or an organic acid. For technical reasons, however, the content of neoviridogrisin I, II and / or III in the culture fluid is more favorable

1 99 3? 5

by adding the appropriate amino acid (η) in free form during the cultivation, depending on the circumstances and the need. It need not be particularly emphasized that the composition of neoviridogriseins in the culture fluid can be varied by appropriate choice of spontaneous or artificial mutants of the culture of the invention, as well as by appropriate adjustment of cultivation conditions such as temperature, pH and aeration and / or by addition of physiologically active substances, such as enzyme inhibitors and promoters. One of the preferred process variants for the selective production of certain neoviridogriseins consists in the supply of the corresponding amino acid (s), namely Oc-amino-η-butyric acid and / or proline during the cultivation. In particular, the administration of proline results in an increase in the proportion of neoviridogris in T and II in the total amount of neoviridogris in I, II, III and IV, the former acting more antimicrobially than the latter. The content of neoviridogrisins I and III can be selectively increased by adding 0l-amino-n-butyric acid to the medium before inoculation or during culture. Since the microorganism of the present invention produces protease during growth, proteinaceous material containing the relevant amino acid (s) may be added in place of the free amino acid (s). For example, proline can be replaced by casein or corresponding hydrolyzates from acid hydrolysis, such as casamino acids.

Insulation and cleaning.

The novel antibiotics neoviridogrisins I, II and III and viridogrisin can be isolated from the culture fluid by conventional methods according to their physico-chemical properties as depsipeptide antibiotics. Optionally, the neoviridogriseins can be obtained from the culture fluid in admixture with the griseoviridine. Become

- AX

199 3

When prepared for feed additives or for veterinary purposes, a crude mixture of neoviridogris and griseoviridine is more advantageous for economic reasons.

The neoviridogriseins and griseoviridine can be extracted from the culture broth with a water-immiscible organic solvent. Sp are e.g. Ethyl acetate, butyl acetate, n-butanol, methylene chloride, chloroform, etc., for the simultaneous extraction of neoviridogris and griseoviridine, but if the neoviridogriseins are to be selectively extracted without griseoviridine, organic isomers methyl isobutylketoh, benzene, toluene, and others are aromatic hydrocarbons Since the mycelium essentially does not contain neoviridogriseins and the extractable lipid in the cells often interferes with the subsequent purification stages, it is better to extract said antibiotics with an organic solvent from the filtered or centrifuged liquid together with the wash water.

The neoviridogrisine and / or griseoviridine extract can then be further isolated and purified in a variety of ways, for example, by combining adsorption and washing with charcoal, Amberlite-XAD- ^ and? (Rohm & Haas Co.), ion exchange resins such as Amberlite IR-120 (Rohin & Haas Co.) and Dowex 5QW-X2 (Dow Chemical Co.), Sephadex LH-20 (Pharmacia Fine Chemicals AB) filtration and equivalent, Adsorption chromatography on alumina and silica gel, etc. In addition, it is additionally possible to work with countercurrent distribution with a suitable solvent system. . "^'...:.'..:."'. \. ·. ' ·. · · '. "·.'.";'''

Physico-chemical properties of neoviridogrids in I ₁ II , '

Neoviridogrisin I, ΙΓ and III are amorphous like viridogris

3Fs

white solids, soluble in methanol, ethanol, n-propanol, iso-propanol, n-butanol, dioxane, ethyl acetate, butyl acetate, acetone, methyl ethyl ketone, methyl isobutyl ketone, benzene, toluene, methylene chloride, chloroform, carbon tetrachloride , N, N-dimethylformamide and dimethyl sulfoxide, sparingly soluble in 1 ^ 0 and ethyl ethers and practically insoluble in petroleum ether and hexane. , ·. ·. _: · '; · -; \ '· V-'. .-Γ : ·. ' , :;;;.: -; / ..

In aqueous solution, they are stable for at least one month at 25 to 37 ⁰ C and 30 minutes at 60 ⁰ C, at a pH of 2 to 9. The melting points of the antibiotics were determined according to Kofier:;

Neoviridogrisin I: not determined

^: - ', -' - ·. ": '': · ·, <'Ii ....:.' 93 ⁰ C "/ - .. /, '']"'/'. [ ^: ;

III: 115 ⁰ C Viridogrisein: 140 ⁰ C.

The UV absorption spectra of neoviridogrisin I, II, III and viridogrisein are shown in Figures 1 to 8, wherein Figures 1 to 4 show the UV spectra of neoviridogrisin I, II, III and viridogrisein in methanol, and Figures 5 to 8 show the corresponding UV Spectra in 0.1 η NaOH of neoviridogrisein:

, , -o o / _a

Show spectra in 0.1 η NaOH-methanol. Maximale.EJ ^, "- values

1 cm

In neutral methanol,

Neoviridogrisein I: 305 nm (-65)

II: 305 nm (88)

III: 305 nm (90) Viridogrisine: 305 nm (90)

In 0.1 η NaOH-methanol,

Neoviridogrisein I: 340 nm (70)

v-II: 340 nm (84)

III: 340 nm (90)

Viridogrisine: 340 nm (96)

The IR spectra of neoviridogrisin I, II, III and viridogrisein in KBr are shown in Figures 9-12. Characteristic maximum and shoulder values:

.Neoviridogrisein I (KBr)

i 3370, 2910, 2850, 1735, 1670 (shoulder), 1635, 1590 i (shoulder) V 1515, 1460 (shoulder), 1445, 1405, 1375, 1290, 1280, 1250 (shoulder), 1200, 1190, 1160, 1125, 1100 '

-.-, · _1 ^: -: '. - '''. ..- ·

and 1080 cm.

Neoviridogrisein II (KBr) V ^

... Γ. , ; .332O, 2950, 2920, 2820, 2800, 1745, 1670 (shoulder),

1630, 1600 (shoulder), 1575, 1515, 1460 (shoulder), -:

1445, 1405, 1390, 1365, 1330 (shoulder), 1295, 1275,

1240, 1200, 1195, 1160, 1130, 1095 and 1065 cm " ¹ .

/ Neoviridogrisin III (KBr); ·

3335, 2960, 2940, 2870, 1750, I67O (shoulder), 1660 (shoulder), 1635, 1590 (shoulder), 1515, 1450, 1405, 1390 (shoulder), 1370, 1340 (shoulder), 1300, 1245, 1200 , 1160 (shoulder), II30, 1100 and 1065 cm " ¹ .

By thin layer chromatography (DSC), neoviridogrisin I, II and III, viridogrisein and griseoviridine show the following

Rf values: ':. λ. " ^: - · 'f,'. '...' ... ';'. '.'. ';''.:' .. '' · ''

(1) DSC plate: Previously coated DSC plate

teSILICA GEL 60 F-254, E. Merck, Darmstadt.

Solvent. : Benzene: methanol = 5: 1

Neoviridogris in I-Rf = 0.66

^; ·· .-. ., '· -. -Ii ". : ';"'' :. ^., - :; : .... Ö, -62 '.:': Ν ^{i; r} "'.'''.;'''·. / "

- 1 99 395

Neoviridogrisin III RF = 0.59 Viridogris in 0.55

Griseoviridine 0.20

(2) DSC plate: as (1)

Solvent: '' chloroform-methanol = 30: 1

Neoviridogrisein H RF = 0.39 III 0.32     , "-. 0.19 Viridogrisein 0.18 griseoviridin 0.02

For analysis of the amino acids, each Neoviridogriseinkomponente in 6 η HCT was hydrolyzed overnight at HO ⁰ C, the hydrolyzate then evaporated to dryness. Upon escape of HGl in traces as a result of repeated induction, the amino acids in the hydrolyzate are detected by DSG (Eastman Ghost Chart sheet 13-254 Cellulose with Fluorescent Indicator, Eastman Kodak Co., solvent system: n-butanol / acetic acid / water = 4/1/1). High Voltage Electrophoresis Paper (Toyo Filter Paper # 5IA, Toyo Soshi Kaisha, Ltd .; buffer system: formic acid / acetic acid / water = 25/75/900, pH 1.8, 60 V / cm at ⁰ ° C, 30 minutes ) and auto-amino acid analysis (Hitachi auto-amino acid analyzer KLA-5, Hitachi, Ltd.). The following amino acids were determined:

Neoviridogrisein I: threonine

; '·.'"'·. ^: . ^: . ^: .'. '/'.; .- -.:. Leucin '. -' / '.:.'^..-...;'' Λ ';.'; ^ '<""Λ' proline '·.'. '.'-."'' / '": "''

'. : - . Oi-amino-n-butyric acid

': -.''"' - -. '. . ' ., '".."'',.'. · Sarcosine - '''^;'";.'".

Phenylsarcosine β, Ν-dimethylleucine

- -4b-

395

Neoviridogrisin II

Neoviridogrisin III

threonine

leucine

proline

alanine

sarcosine

Phenylsarcosine β, Ν-dimethylleucine

Threonine leucine '· hydroxyproline OO -amino-n-butyric acid

sarcosine

Phenylsarcosine β, Ν-dimethylleucine

threonine

leucine

hydroxyproline

alanine

sarcosine

Phenylsarcosine β, N-dimethylleucine ·

The presence of 3-hydroxy-picolic acid was determined by mass spectrometry and by DSC:

Viridogrisein

An original sample of Viridogrisein and Neoviridogrisein I, II, III and IV was hydrolyzed overnight in 6 η HCl at 110 ⁰ C. Each of the hydrolyzates described above exhibited only one UV absorption spot with the same Rf value under the following conditions:

(1) Silica gel DSC ·

DSC plate: Previously plated DSC PIatti

254, E. Merck, Darmstadt.

SILICA GEL.60 F-

- ΊΤ--

Solvent: chloroform: methanol = 2: 1

Rf ·: 0, ^ 6

(2) Cellulose DSC

DSC plate: Eastman Chroinagram sheet

 · Cellulose with fluorescence indica

Tor, Eastman Kodak Co. ' Solvent: n-Butanol! Acetic Acid Water logo CNRS logo INIST

7 · '; ':':.:. .,;:, ·, ... 4: i: i, ;;; .: _. , ; -. ; , ..;

et ; ·. ':';'. ] '<:':;) ^:: /;> - .-. :? o, .62: '·. - ^: -. ; ··. ^{:; .V;} : -,: '":

The MW of this antibiotic was determined directly with the mass spectrometer:

NeoviridGgrisein I - = · ":. : · ·. o7d II : 862 III : 892 Viridogrisein : 878

To elucidate the chemical structure, neoviridogrisins I, II and III and viridogrisin were hydrolyzed overnight in 0.1 N NaOH at room temperature, then inedylated with diazomethane and then mass spectrometrically according to Compernolle et al., (Organic Mass Spectrometry, Vol The structure of neoviridogrisin I, II and III is therefore as follows:

-is 199 3f5

Neoviridogrisin I

CH

CH

CH 'CH,

CH ₂

CH

CO-NH-CH-CO-NH-CH-CO-N

ι * ι

2 CH-CO

CH-CH

: i ':. v ^ co

CH-N-CO-CH-NH-CO-CH

CH.

CH

N-CH,

i .. ·?. CH ₉

io I

N-CH ₃

CH-CH,

CH-CH

CH,

Neoviridogrisin II OH

CH,

CH

CH

? ^H

CO-NH-CH-CO-NH-CH-CO-N

CH-CO

CO-CH-NH-CO-CH-

N-CH, CH ₅

co

N-CH,

CH,

CH-CH

CH-CH,

, :.

CH,

-! 99 395

Neoviridogrisin III

OH

CH ₃

CH,

CH CH,

5 ^0H / CH

CO-NH-CH-CO-NH-CH-CO-N-

CH-CH _x O

CH ₂

CH-CO

N-CH _x ι 3

CH ₉

CH-N-CO-CH-NH-CO-CH - N-CH-

CH-CH ₃

CH-CH, ι 3

CH,

The neoviridogriseins I, II and III according to the invention are thus a group of novel viridogrisin homologous depsipeptide antibiotics. The identity of neoviridogrisin IV with viridogrisein and etamycin was determined by mass spectrometry, by DSC, UY and IR spectrometric and amino acid analysis on original samples of viridogrisine and etamycin,

Antimicrobial activity.

Neoviridogrisin I, II and III have a broad antimicrobial spectrum against bacteria, mycoplasma, actinomycetes and rickettsiae in the laboratory test, especially in vitro against the usual, resistant strains of Staphylococcus aureus, as well as against the strains of Streptococcus pyogenes, Diplococcus pneumoniae, Sarcina lutea, Bacillus subtilis, Mycoplasma gallisepticum, Mycoplasma pulmonis, Mycoplasma fermentans and Mycoplasma agalaetiae. The minimal inhibitory concentrations of the new depsipeptide antibiotics were determined for these alone or together with viridogrisein and griseoviridine on different microorganisms according to the serial dilution test.

V W -V, Λ. Table 1 '/ Vv-V V, -W.:. ^: ''... MIC values of neoviridogrisin I, II and III (mcg / ml)

, ...,. , , . ·. - 'microorganism (a) medium NV * I NV * II NV * III. VG ** NV * · acc. ; gem. Staphylococcus aureus 209 P V; .-. 'BHIf ₁ 0.2: 0.1 0.2 0.2 0.2 0.1 , ; ν; ; ν / (EM) ^r ... ^: . ·; BHI * 0.4 0.4 0.4 0.4 0.4 Q ^ z V-VV-VV : (STH) ^r BHI ^ 0.8 0.8 1.6 1.6 1.6 0.8 (PCjTC, EM, LM) ^r 1 BHI ^ 6.25 6.25 6.25 6.25 6.25 3.2.7 ^: iV ..; /. , ; .. (PC, TC, EM, LM) ^r 2 BHI * 6.25.  , 6,25V 6.25 6.25 6.25 3.2V-- 7-7 BHI ^ " 6.25 6.25 6.25; V 6.25 6.25 3.2 V ... VVV ^r VV '. (SM, STH) ^r BHI ^ 0.4 0.2 0.2 0,4 : 0.4 0.2 V. :; VV (Pc, KM, NM) ^r V BHI ^ 0.8-V 0.4  0.4 0.8 0.8 0.4 Vi · .. ·; ; ^:: 'V ^; -.j VV-; (EM, CM, SM, PC, TC) ^Γ BHl ^ 0.4 0.2 0.4 0.4 r 0.4 O, 2 .-: V .-- 7 /; V _: - W-: V ν V. , V (EM, QM) ^r V ^: y BHI ^ 0.8V 0.4 0.8 0.8 0.8: o, 47v / .: v;. ;; 7 , '' .. '' '... I J ^ \ ^ ^^ Jn * jj ^^^ J- ''-.' , - '' - ._ ' BHI ^ - ^: V0,2VV _:.   "- 0.2 0,4 ' 0.4 0.4 f \ O - ' , \ .Ο · Λ · I ^ J Jx ~ r - J ν ^^ / ^: '" BHI " ¹ . ; 0.4V 0.2 0.4 0.4 0.47 0, 2 7 '; .- VV- : Russell (PC) ^r .-7, BHI * " '. 0.2 0.2 0.4 0.4 0.4V o, 2 ;;;; V;. V ;; ^V : .V. . V ^: '-'- ··''',) ^: Smith 'V--,'V; .. ' BHI '^ 0.2 0.2 0.2 0.4 0.4; 0.2 Staphylococcus sp. (PC) ¹ V λ ·. , _. · -. ^BHI t 0.8; 0.4 0.8 0.4V 0.8 0.4 -. .. ';: .- V;..' ^S P · V -'-: Ζ; _'-;' / '-_ .....' ·. BHI * · 0.8; 0.8 0.8 0.8 0.8 0.4

(Table 1) Continued

microorganism

medium

NV I

NV II NV III

VG

NV-gem.

NV + GV

Diplococcus pneumoniae type 1 Streptococcus pyogenes Sarcina lutea

Bacillus subtilis ATCG 6653 Salmonella gallinarum Escherichia coli Pseudomonas aeruginosa Proteus vulgaris Candida albicans

BHI + HB 'BHI + BHI ^ BHI ^' BHI BHI

HB

Ψ-

BHI ^ BHI ^ MY ^&

0.2 0.4 0.2 0.4

> 25

> 25

> 25

> 25

> 25

  '- ".Ο, .2

0.4> 25

> ² 5

> 25

> 25

0.2 0.4 0.4 0.8

> 25

0.4 0.4 0.2 0.8

> 25

> 25

> 25 _:;

> 25

> 25

0.4 0.4 0.4 0.8

25 25

25

25

25

0.1 0.1, 0.1 0.4

^ 25

> 25>

NV * = neoviridogrisein; VG ^ * = viridogrisin; GV *** = griseoviridine (a) EM = erythromycin; STH = streptothricin; PC penicillin; TC = tetracycline JLM = leucomycin; CP = chloramphenicol; SM = streptomycin; KM "Kanamycin; NM = neomycin; OM = oleandomycin. ^

() ^r = resistant to the drug (s) indicated in () BHrf = brain-heart extract. ν

BHI + HB ^ = brain heart extract, containing 10 fo Pierdeblood: _

MY ^& = Malt Extract Yeast Extract Medium ν

- ft9 395

The MIC values obtained according to Table 1 at 2-fold dilution show that neoviridogrisine II is more active than neoviridogrise in IV, ie viridogrisine. To differentiate Neoviridogrisein II and Viridogrisein in terms of their anti-. biotic activity, the MIC values were repeated at much lower dilution. Table 2 shows that neoviridogrisin II is 2 to 3 times more active than viridogrisin.

. ": -; '..;' / ^Vj--: Table 2 ^ V - / ^{^:%} >> - ^/;:. '''

Comparison of Neoviridogrisein II with ':: Viridogrisein.

microorganism SM, PC, MIC (Mcg / ml)       , ..-. "Staphylococcus aureus 209 P 0.078 0.133 · :::; : ': V :; (EM, CM, PC) ^r TC) r (PC) ^r 0,125 0.334 (PC) ^r 0.094 0.334 Bx 1633 0,125 0,267 Russell 0,125 0,267 Smith 0,125 0,267

Medium: brain-heart extract · ]

When determining the MIC values of neoviridogrisins I, II and III in the presence of griseoviridine, a synergism is observed among neoviridogris and griseoviridin, as does viridogrisine and griseoviridine. The synergism of the new antibiotics with griseoviridine was investigated in different ratios of neoviridogrisein mixture to griseoviridine (see Table 3):

99

^; -: '? -. ^ Table 3.; "'A ^ VV / Vv V'- -

Neoviridogriseingein synergism with

griseoviridin

Neoviridogris a mixture

100

.:. .- "V ^\:: 90 '80 70 60 V.

 ; · .. 'v-a so;.: "·'"

20 io

eovii in MIC ⁺ (mcg / ml) 0 AAA. vAv. 0.313 10 0.156 20 ; ;;;; ;:; A; .. ;; 0,125 30 0,125 50 0.078 60 70 0,125 80 : VAv AvA. 0 ^ 250 - - 90 0,250 100 0,250

Test Microorganism: Sarcina lutea dilution series test in vitro · with · Brain Heart Extract.

As can be seen from Table 3, the synergism between the neoviridogrins and griseoviridine was greatest at a ratio of 50:50; a 1: 1 mixture of neoviridogris and griseoviridine is 3 to 1 times more active than neoviridogriseins or griseoviridine alone.

Table Λ shows the high activity of neoviridogrisin II in vitro against several species of mycoplasmas and the greater synergism of a neoviridogris II griseoviridine Ge disease with a viridogrisein-griseoviridine mixture. The KIG samples were determined after the dilution series test.

- ζ * - 1

Table 4

Microorganism Medium NV II VG GV NV II VG +

. .;. · Ν -'---; - ';: ": -..-; vv - / ¹ ' - ^; "' ^: ''' - + GV * 'GV *

Mycoplasma galli

septicum KP 13 (1) 0.025 0.10 0.10 0.0063 0.025

Mycoplasma pulmonis ^

PG 22 (2) 0.78 1.56 6.25 0.20 0.39

Mycoplasma fermen->

tans (2) <0.05 <0.05 / 0.05 <0.05 ^ .0.05

Mycoplasma agalactiae.

PG 2 ν (2) 0.39 0.78 3.13 <0.05 0.39

Medium (1): PPLO enrichment solution (Eiken, Japan). (2): PPLO solution (Chanock's medium; Difco)

* Mixing ratio 50/50 NV II, VG, GV: s. Table 1

Thus, the novel neoviridogriseins I to III, as such or in combination with viridogrisin (neoviridogrisin I) and griseoviridine, exhibit strong activity against gram-positive bacteria and mycoplasma species, the weight ratios of the individual components of the mixture to each other being unaffected by the surprising antimicrobial and antimicrobial agents antimycoplasmic activity can vary greatly. This is illustrated by a representative but not limiting example in which a mixture of (in% by weight ):

Neoviridogrisein II 27 ^

Neoviridogrisin IV (Viridogrisein) 23 % Griseoviridin V ': 50 %

in vitro to Streptococcus mutans, a caries-causing and peridontal disease-related microorganism

-25- 1

in Todd Hewitt Broth (Difco) with 0.5 % TC lactalbumin hydrolyzate (Difco). The initial number of organisms is cov 3 χ 10 organisms per ml. The cuvatory vials are anaerobically inoculated at 37 ⁰ C for 48 hours. The MIC value and the minimum bactericidal concentration resulted in a concentration of 1.0 ppm for neoviridogrisein. -

In another representative but non-limiting example, the same mixture was tested in vitro on Treponema hyodysenteriae, which causes swine dysentery, in blood agar at concentrations of 100, 50, 10, 5.1, 0.5 and 0.1 ppm. The plates were inoculated with a smear and kept at 42 ^° C. for 4 days. The. MIC · -. was 0.5 ppm. In a further representative but non-limiting example, a blend of (in weight percent) was prepared:

Neoviridogrisein II 25 %

Neoviridogrisin IV (Viridogrisein) 25 % Griseoviridin 50%

tested in vitrq on several mycoplasma species. The following MIC values were found:

Microorganism MIC value

Mycoplasma gallisepticum KP 13 0.07

Mycoplasma pulmonis 0.20

Mycoplasma fermentans 0.05

Mycoplasma agalactiae 0.05.

The said types of neoviridogrisein-griseoviridine mixtures are also suitable for the treatment of bacterial-induced infectious diseases in animals.

Since Mikamycin A and B are known as very effective feed additives "the antibiotics according to the invention were subjected to an animal feed test. The ueoviridogriseins were added to the chicken feed in the form of a mixture in an amount of 2 to 20 ppm and fed to male chicks for 10 weeks. The chicks thus treated showed a greater increase in body weight and feed efficiency over the control group that did not receive neoviridogriseins. The antibiotics according to the invention are thus very suitable as feed supplement. '.:. ; ; ' .:. - _: y ^: ':;' , ·: - ..: ..-;. ^; '.: ..:. . ".-

Compositions with one or more Neoviridogriseinen of group I to III, optionally in admixture with Viridogrisein (Neoviridogrisein IV) and griseoviridine, wherein the weight ratios of the individual components can vary greatly ' have been found to be very suitable feed additives. - .:. , '; - ·; , .. '...' , '.''·':'' / '-'''' · ..

In a representative but nonlimiting experiment, a mixture of (in% by weight ).

Neoviridogrisein II 27%

Neoviridogrisein IY (Viridogrisein) 23% Griseoviridin 50 %

In vivo by addition to chick feed tested. One day old male chicks (15 animals per test) were enriched for 11 days with a feed containing about 55% rye grains. "Vitamins," mineral salts, fat and proteins, fed. The high rye content usually leads to weak: or moderate wax turn. Such compound feed is considered to be the standard diet for the differentiation of growth stimulators and antibiotics as feed additives. Penicillin (100 ppm) was used as a positive control.'The results of the experiment are summarized in tabular form below.

caught. The quotient "feed / gain" ¹¹ means consumed feed in grams per gram increase in body weight, the parameter "body weight" the ratio of the body weight after 11 days to the starting weight The last column indicates the average weight gain per chick (in grams) ,

treatment

Rye Control Penicillin ' Test Mixture Test Mixture Test Mixture

Executives '; .. ''. · ': .- ·;

In the following, the invention is illustrated by further preferred examples, which, however, have no limiting character.

Feed, concentration in ppm Feed / gain Body weight · . , '; Average growth per chick ---. / '.'- 1,334 Yay, 696 .156,47 100 1,197 5,012 163.20 100 1,256 4,789 161.66 "50 1,283 4,892 163.47 : 25 1,204 5,226 174.67

example 1 , '-

A medium consisting of soybean meal, Pharmamedia (Traders Oil Mill Co.), oatmeal, dried yeast and molasses from beet (jewe ils 0.5 '%) _t is adjusted to a pH of 6.5 and in an amount of 50 ml distributed in a 250 μl Srlenmeyer flask. After autoclaving at 120 ° C for 15 min P is a wire loop the culture Streptomyces sp, inoculated on ISP-2 agar slants 8648, after which rährend 48 St is incubated on a rotary shaker at 28 ⁰ C i \. Thereafter, 2 ml of this seed culture are placed in a 500 ml Srlenmeyer flask containing 100 ml of the following fermentation medium:

soy flour 0 , 5 % peanut meal ; ο , 5 % oatmeal 0 , 5 %

(pH 6.5 before autoclaving)

Dry yeast. 0.5% / '^; v..vV '' \ - '... 'λ.''' V ^; . ' Red beet molasses; ^; "0.5;^:; ./ .. ν; _: ;

and at 28 ⁰ C for 96 St on a rotary shaker (200 rev / min, radius 3.5 cm) incubated. The culture liquid from 12 flasks is collected and filtered to give a clear culture filtrate. The filter cake is washed with 100 ml H ₂ O, after which the wash water and the culture filtrate are combined. If the plate test is performed with an 8 mm paper disc, the antibiotic activity of this solution (pH 8.3) is 23.0 mm on a nutrient agar test plate with Sarcina lutea. 800 ml of this aqueous solution are extracted twice with 200 ml of n-butanol. The butanol extracts are combined and evaporated to dryness under reduced pressure to yield 90 mg of a crude powder consisting of neoviridogris and griseoviridine. This is mixed with a small amount of silica gel and applied to a silica column (Wako-Gel C-100, Wako Pure Chemical Industries, Ltd., 1.5 x 25 cm). It is then eluted first with 300 ml of a benzene-acetone mixture (5: 1) and then with a benzene-acetone mixture (2: 1). The 10 g fractions are collected with an automatic fraction collector. The active fractions no. 25 to 35 are combined and evaporated to dryness to give 30 mg of a mixture of neoviridogrisins I, II and III and viridogrisin. In addition, by evaporation of the active fractions Nos. 45 to 54 to dry a crude powder whose antibiotic activity corresponds to the griseoviridine. In both cases DSC is used under the following conditions. The antimicrobial effectiveness is determined on a nutrient agar test plate with Sarcina lutea. " ::::

- as- f

DSC Platter: Previously plated DSC Platter::,:.;. . ' SILICA-GEL 60 F-254, E. Merck, Darm-

:; ';/;:;;;; City. '.'. ... V '/ : \ -; ^: .. /.': ':,.' ^:. ''\\ -

(1) Solvent: chloroform: methanol = 20: 1

Neoviridogrisein RF = 0.45 · 'griseoviridine 0.05

(2) Solvent: Benzene: acetone = 1: 1

Neoviridogriseins Rf. = 0.55 griseoviridine 0.13

Example 2 ''''.'../''.'V;. ^"·';,: ^ :: [ '' ^'; '..' / ':; -.

200 ml of a 48 hour Kulter from Streptomyces sp. P 8648 is in the same nutrient medium as in Example 1 on a 15 1 stainless steel Schüttelferrnenter containing 10 1 of the same inocula vernubule as in Example 1 over vaccines and at 27 to 28 ° C for 96 hours under forced ventilation with 5 incubated in l / min of sterile air. Stirring (200 rpm) is provided by an impeller whose radius is approximately one quarter of the diameter of the chute. After completion of incubation, the mycelia and solids are filtered off. The resulting culture filtrate is adjusted to pH 6.0 and extracted twice with 2: 1 ethyl acetate. The extracts are combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to give 700 mg of a crude neoviridogrisine powder and griseoviridine. Dissolve in a small amount of methanol and place on a Sephadex LH-20 column ( 3 x 50 cm) gives up. The 10 ml fractions are collected using methanol as the eluent. Fraction Nos. 22 to 29 containing the neoviridogriseins are collected, concentrated to dryness, and further passed through a silica gel column (SILICAR CC-7 Special, Mallinckrodt Chemical Works, 1.5 x 20 cm) with chloroform / Methanol (30: 1) as Elutionsmittei

-3O-

cleaned. Eight 5 g fractions from fractions Nos. 5 to 12 are combined and evaporated to dryness under reduced pressure to give a white powder consisting of the Meoviridogrisines.

The percentage of heoviridogrisins I, II and III and viridogrisein is like. follows:

Neoviridogrisein , ι ·. : ·. : 15 II : 20 '' "'." III : 20 Viridogrisein : 45

Griseoviridine is detected by DSC in fractions # 30-34 of the Sephadex LH-20 column. These actives are combined, evaporated to dryness under reduced pressure, and recrystallised from warm methanol to give 30 mg of acicular crystals of griseoviridine. (Identification by DSC or by another physicochemical method). ^^^ ^: - / ^:.

- ^{'^' /:;:} - ..; '. Example 3 ""' .- / ^' "' ·. '· ...'.-' ·. '.'. '. '''"" .-. ·:'';'

The culture is carried out as in Example 1 for 96 hours, except that the culture medium consists of soybean meal, pharmamedia, oatmeal, dry yeast, beet molasses (0.5 % each ) and 0.1 % DL.-06 amino acid. n-butt acid (pH 6.5) exists. The culture solution is collected from 13 flasks and filtered, the filtrate extracted twice with 300 ml of n-butanol. After removal of the n-butanol from the extracts, about 70 mg of a crude powder consisting of the neoviridogris and griseoviridine are obtained. The powder is analyzed with DSG (silica gel) and subsequent bioautography on Sarcina lutea as a test organism. This leads to the following results:

- vif 9 39 $

DSG plate: .Variously coated DSC plate

SILICA GEL 60 P-25 ^, E. Merck, Darmstad. Solvent: Chloroform: methanol.

" ^: ........ ... I Rf = 20: 1 30: 1 40 :1 Neoviridogrisein II 0.60 0.39 °> 20 III 0.56 0.32 0 16 0.50 0.19 0 13 Viridogrisein 0.43 0.18 O" 10 Griseoviridin. 1. . '.'. '. '. ' 0.05 0.02 '' '. '. '' 0 00th •1 . ''': I. ' . Example 4   ·. ,

50 ml seed medium, with 0.3 % Hind extract, 0.5 % .Try.pt on (Difco Laboratories), 0.1 % glucose, 2, k% soluble starch, 0.5 % yeast extract, 0, - % Calcium carbonate and 0.5 % soybean oil (pH 7> 0) are distributed in a 250 ml Erlenmeyer flask and autoclaved at 120 ° C. for 15 minutes. Then the flask is filled with spores of Streptomyces sp. P 8648 on slant agar and shaken at 25 ° C for 3 days to complete the seed culture. The breeding medium consists of 0.5 % soluble starch, 2.0 % glucose, 1.0 % pharinainedia, 0.5 % oatmeal, 0.5 % corn syrup, 0.05 % KpHPO ^ and 0.05 $ MgSO ₂₊ (pH 6.5). 50 ml of this medium are then placed in a conical 250 ml flask and autoclaved at 12O ⁰ C for 15 min. The amount of inoculum is 2 % (v / v). The fermentation flask is inoculated with the seed culture described and incubated at 25 ° C on a rotary shaker. At 24 and 48 hours after incubation respectively, a sterilized solution of casaininic acid (Difco Laboratories) or proline (pH 7.0) is added to a final concentration of 0.4 % , after which the cultivation is continued. Four days after incubation, the culture solution is filtered off and the culture filtrate zv / eimal extracted with the same amount of ethyl acetate. The ethyl acetate extracts are combined and evaporated at reduced pressure to a dry powder. This raw powder will

- "If9 395

taken up in ethyl acetate, after which an appropriate amount of the solution is dropped on a DSC plate (silica gel) on. The plate is developed in chloroform / methanol (50: 1) and the solvent is evaporated in air. Thereafter, the same plate is again developed in the same solvent system. The individual Neoviridogriseine were determined under UV light (3650 2nd) , scraped off the plate and suspended in a known amount of methanol. After removal of the silica gel by decantation, the amount of antibiotic in the extracts is determined by the UV test (£ at 305 nm 8,000: Ov, ..:.;.;.;. ^;; - .. \ _. '.' -. '^;'.'',.ν'.

 '·': Added amino acid

; ; no casamino-proline

Neoviridogrisin I 2% 2% 2%

' ^":;': / ^· · '-;' H -.-"'O-40 60th ' ^: '^;;

Viridogris in 85 55 35

In a HütteIfermenter about 10 1 of a 23-hour old Impfkultur are prepared under the same conditions wi.e in Example 2. The culture medium (600 l), consisting of soybean meal, pharmamedia, oatmeal, dry yeast, beet molasses (each 0.5 %) and 0.1 % DL-öC-amino-n-butyric acid (pH 6.5 before Autoclaving) is steam sterilized at 120 ⁰ C for 15 min in a 1400 1 tank fermenter made of stainless steel and then cooled to 28 ⁰ C. The fermentor the above-mentioned seed culture (10 1) is added, followed at 28 ⁰ C for 75 St with forced ventilation with stirring at 180 U / min (Doppelflügelrad, radius corresponds to 'a quarter _of; fermenter diameter) incubated v / ill, v; obei The sterile air is injected at 300 l / min through the bottom of the tank. After incubation, the culture fluid becomes;

- 33- 1 99 3 * 5

filtered off with a filter press. The culture filtrate is then extracted twice with 150 l each of n-butanol. The n-butanone · nolextrakte are combined together with a small amount of a _saturated} NaCl solution andin concentrated on a rotary evaporator 2. 1 After addition of 20 g of silica gel (Wakogel C-100, Wako Pure Chemical Industries, Ltd.) is further concentrated to complete dryness. The obtained antibiotic-silica gel mixture is suspended in a small amount of chloroform and then applied to a silica gel column (MKOGEL C-100, 6.5 x 75 cm). The antibiotics. are gradually eluated first with 7 liters of chloroform, then with 10 liters of chloroform / methanol (50: 1) and finally with methanol. The fractions # 16 to 29 (300 g fraction) (neoviridogrids, his) found to be Sarcina lutea bioactive are collected and concentrated to dryness under reduced pressure to give a crude powder consisting of neoviridogrisines. This is dissolved in a small amount of methanol and applied to a Sephadex LH-20 column (7.0 x 45 cm), eluting each fraction (100 g) with methanol. Approximately 15 g of crude powder consisting of the Neoviridogriseingemisch are obtained from the fractions Nos. 6 to 15 after abortion of the solvent. The fractions Nos. 50 to 60 of the silica gel column described contain griseoviridine. Purification on a column of Sephadex LH-20 (7, Ox 45 cm), as described above for the neoviridogrisein mixture, gives 4 g of crude griseoviridine.

Example 6 . ,; .- .. ·. '. : ..: . ''":.

For final cleaning, a DSC silica gel plate is used. Ig of the crude Neoviridogriseingemisches of Example 5 is dissolved in 2 ml of ethyl acetate and layered on 10 SiIicagel plates (previously coated DSC plate SILICA GEL 60 F-254) applied. These plates are first developed with a solvent system of chloroform and methanol (50: 1). After stripping off the solvent in the air

- ·: 1f9 3

The plates were once again developed with chloroform / methanol (25: 1). Neoviridogrids I, II and III and viridoglysin are labeled and scraped on the BSC plates under UV light (3650 'S;-; BLAKRAY UVL-22, Ultra-Violet Products, Inc.). Each Neoviridogriseinkoinponente is eluted with a small amount of methanol and evaporated to dryness. The following quantity distribution of pure products is obtained:. ν , ^; - ¹ ./;/ '. '. - ^ - 'V ^ v ^ \'^;'/ ^: ··: / '. ^ {. _: \ '' / ', ''''..".'

/ Neoviridogrisein I: 16.7 mg (not completely pure, oily)

: '·;: II'. ;;,. ': 11.1 mg (white powder)

^::: III: 15.2 mg (white powder)

Viridogrisein: 30.0 mg (white powder)

In each case about 5 mg of all Keoviridogriseine be hydrolyzed. subjected in 6N HCl at 110 ⁰ C for 36 hours in a sealed tube and then the DSC, paper chromatography, PapiereIektrophorese at high voltages and Autoaminpessigsäure analysis. The following components are found: ''^;' ^: ';, ..;. '/.''^;' ^:. '· ^: -: ·'; .λ ... ···

Neoviridogrisin I: 3-hydroxy-picolinic acid

:: - ":. ^: · ':". ^: '·' Threonine · ...; . . ' , ':. ''

\:. , "..;·;^·" - '. , · V. ';;. ·. /. ·.:. ";.,. '.''.''-Proline';'';' - '-J. - :'/;:.'..' -. ·; ^: ; · ^; ". ' ^: - '-;"..": · ;; "". ^:: "'· ·"",.'" ^: -. Sarcosine ':'"·.; ·. ^{; :} " -. '. '"

ß, N-Dimethylleucin

, ' ^: ' .. ''':; ö <i - amino-n-butyric acid '' ..

Phenylsarcosin

Neoviridogrisin II: 3-hydroxypicolinic acid

, -. - . ':.' - k ' - ' - . . ' , ": \ :.";;. - Thre onxn '. .. · ^: '"'". , ,. '

_: .; , ;.; , :: '...>;^:;''_; -. ''."':" , "· Prolin;:" -

^; '' ,, '·.'.;. ·. '..-; .. "· - ...".;: /.'''.' - -. · ^: Sarcosine: · ..- ';. .-.'. '.:'':-.'.'""; · · ;. " · ''. ' ^:: ,' . ' : .'.-;" ,. '.. · "Alanin'',''"; -. ^::

Neoviridogrisin II Neoviridogrisin III

Viridogrisein

Phenylsarcosin

3-hydroxy-picolic acid threonine leucine hydroxyproline sarcosine β, Ν-dimethylleucine δ6-amino-n-butyric acid phenylsarcosine

3-hydroxy ω-γ-ureine threonine leucine hydroxyproline sarcosine ß, N-whine thyleucine alanine phenylarsarcosine

In addition, the identity of neoviridogrisin IV with yiridogrisine was still detected by IR, UV, NMR and mass spectrometry, by DSC, hydrolyzate analysis and antimicrobial spectrometry. -

The griseoviridine prepared according to Example 5 was recrystallized from warm methanol to give acicular crystals. Part of this was compared with an original sample of griseoviridine IR, UV, NM and mass spectrometry, DSC, elemental analysis and other physico-chemical properties, which could reveal complete identity.

Claims (4)

Erfindungsanspruoh. 1. A process for the preparation of the depsipeptide antibiotic neoviridogrisein I of the formula CH, CE CH CH ₂ CH, ZT ¹ Z CO-NH-CH-CO-NH-CH-CO-N-CH-CO CH-CH CO Ή-Κ-CO-CH-NH-CO-CH-N-CH Γ I ί CH * CH, CH CH-CH, : -CH-CH, the depsipeptide antibiotic Neoviridogrisein II of the Formula -γ'- .-.) /: ' Υ- _Λττ ''; _CH :.: / Γ γ; "" - " : ^; , CH, CO-NH-CH-CO-NH-CH-CO-N CH-CH, -. ι : ' j :. '- ..- : ^r Ο ": · ·· ·., .. Ζ"'·. : CH ₂ CH-CO N-CH ₃       >.: CH-ψ-CO-CH-NH-CO - CH-N-CH ₃ OH j - CH-CH ₃ CH-CH ₃ CH ₃ - I $ 9 395 and the depsipeptide antibiotic neoviridogrisin III of the formula "-. - '^;-:.': \ ^:. "'-' V. ' , \ "., ''"> \. .. '' CH, CH y 3 ^CH 2; CO-NH-CH-CO-NH-CH-CO-N : CH-CH ₃ · · "O": ·: '- /; ^.-: - T.---.:-. : · Co OH I.: V; CH CHo ^X CH. CH-CO N-CH, CH ₉ ι ' :: co CH-N-CO-CH-NH-CO-CH-N-CH "CH- CH 'CH-CH, I
CH-CH, That is, that under aerobic conditions at temperatures of 18 to 37 ° C in an aqueous nutrient medium containing assimilable C and N sources as well as essential mineral salts at a pH of approx. 6 to 9 the microorganism Streptomyces sp. P 8648 (FERM-P3562) until the medium has significant antibiotic activity and separates the growth product from the medium and optionally separates into the individual components neoviridogrisin I, neoviridogrisin II and neoviridogrisin III. 2. Method according to. Point 1, since you are crude, that in addition to neoviridogrisin I, neo- viridogrisein II and neoviridogrisin III nor viridogrisein and griseoviridine. 3. The method according to item 1, characterized in that the cultivation is carried out in the presence of 0-> amino-η-butyric acid and / or natural α-amino-n-butyric acid sources. ^; · / ^:: 4. Procedure after point ;; 1, thus "ge k e η η .. · I ze η e t that the cultivation in the presence of proline and / or a natural proline source is performed. For this 12 pages tables