CH623566A5 - Process for producing the new depsipeptide antibiotics neoviridogrisein I, II, III - Google Patents

Description

The present invention relates to a process for obtaining new antibiotics, namely the neoviridogrisein I depsipeptide antibiotic of formula:

CH * / CH3

CH .CH *

OH I X \

atHz CH * CHz

I II

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

N-CH5 I

CH,

I *

CO

i

NH - CO - CH - N ~ CH.

I

CH — CH,

ch3

CH,

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the neoviridogrisin II depsipeptide antibiotic of the formula:

4

CH,

\

CHj

OH

at

N ^ rn-

CH

CH,

CH>

/ \

CHt CH *

CO-NH

CH -

1

-C0-

- NH -

- CH-

- CO-

- N CH-

CO

|

I

CH-

|

i o

V?

N — CH,

1

CHx

1

1

0

1

CO

I

CO 1

N - CH,

CH-

- NOT -

C0-

-CH -

NH-

r o

0

1

.o

X

1

i: h,

CH,

CH —CHà

and the neoviridogrisin III depsipeptide antibiotic of the formula:

CH,

OH

CH CH;

CH,

OC

N CO-NH _ CH-CO

I

CH- CH3

I

0

1

co

NH

X CH

i

CHe

I

CH-

CH,

OH I

CH / \

CHj

I

CO-N

CH2

1

CH-

N -CO —CH— NH - CO— CH

CO

I

N — CH,

I

CHj

I

CO I

NOT

CH,

CH *

CH *

I

CHj

CH

I

ÇH CHS

- CH »

- CH,

Antibiotics of this kind have a strong activity against Gram positive bacteria and mycoplasmas.

Antibiotics of the mikamycinevernamycin family, or streptogramin, are classified into two major groups. A first major group, group A according to D. Vazquez ("Antibiotics", III, pp. 521-534, "Mecanism of action of antimicrobial and antitumor agents", Springer Verlag, Berlin, Heidelberg, New York, 1975), is a macrocyclic lactonic compound which includes griseo-65 viridine, ostreogrycin A (= mikamycin A = virginiamycin M = staphylomycin M = pristinamycin IIA = vernamycin A = streptogramin A = synergistin Al = compound PA-I14A1 = compound E-129 factor A , etc.), ostreogrycin G (= virgini-

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amycin Mil = staphylomycin MII = pristinamycin IIB = dihydrostreogrycin A = compound E-129 factor B = compound RP-'3929, etc.) and compounds A-2315 A, B and C. Although it is bacteriostatic, this group d antibiotic is active against Gram positive bacteria and mycoplasmas. Griseoviridine, which is one of the fermentation products of the process according to the invention, is known to be produced at the same time as viridogrisein by streptomycetes. Processes for the production of griseoviridine are described in US patent specification Nos. 3023204 and 3174902. The other major group, group B according to D. Vazquez, is a group of macrocyclic depsipeptide compounds divided into two subgroups, the viridogrisein subgroup and vernamycin B subgroup. Antibiotics of the latter major group are effective above all in arresting the growth of Gram positive bacteria such as Staphylococcus aureus and Bacillus subtilis. The vernamycin B subgroup has 12 homologs. Because of the many synonyms among the names of antibiotics published in this major group, it is practically impossible to give here a complete list of all the names of compounds in this subgroup. Examples of representative and scientifically important compounds in this group are:

1. Vernamycin Ba = osteogrycin B = pristinamycin IA = mikamycin B = streptogramin B = synergistin B-1 = compound E-129 factor Z = compound PA114B1.

2. Vernamycin By = ostreogrycin B1 = pristinamycin IC = compound E-129 factor ZI.

3. Vernamycin Bß = ostreogrycin B2 = pristinamycin IB = compound E-129 factor Z2 = compound R.P.-13919.

4. Osteogrycin B3 = compound E-129 factor Z3.

5. PatricineA.

6. Patricine B.

7. Vernamycin Bs.

8. Vernamycin C = doricine.

9. Virginiamycin (staphylomycin) S.

10. Virginiamycin (staphylomycin) S2.

11. Virginiamycin (staphylomycin) S3.

12. Virginiamycin (staphylomycin) S4 (or SI).

Each of the compounds in this subgroup has seven constituents. These compounds have the distinction of having in common the four constituents 3-hydroxypicolinic acids, L-threonine, L-proline and L-phenylglycine. The viridogrisein subgroup contains only one known compound, viridogrisein, the identity of which with etamycin, with compound K-179 and with compound F-1370A is well established. In contrast to the vernamycin B subgroup, viridogrisein is composed of the following eight constituents: 3-hydroxypicolinic acid, L-threonine, D-leucine, 4-hydroxy-D-proline, sarcosine, p, N-dimethyl-L-leucine , L-alanine and L-phenylsarcosine. The antibiotics provided by the method described belong to this viridogrisein subgroup and one of them, neoviridogrisein IV, has been identified as viridogrisein. Processes for the preparation of viridogrisein are described in US patent specification No. 3023204.

The synergism of the antibiotics of groups A and B against different microorganisms is well known and used with advantage to reinforce the therapeutic action of drugs containing an antibiotic of this family as active component. This also applies to neoviridogriseins.

The term neoviridogrisein subsequently includes antibiotics called neoviridogriseins in general, that is to say not only neoviridogriseins I, II, III and viridogrisein (= neoviridogrisein IV), but also any mixture of two or more antibiotics from this group.

The present invention relates to a process for the preparation of new depsipeptide antibiotics neoviridogrisein I, II, III, characterized by the steps: cultivating Streptomyces sp. P8648 (FERM-P3562) under anaerobic conditions, at a temperature of around 18 to 37 ° C, in an aqueous nourishing medium containing assimilable sources of carbon, assimilable sources of nitrogen and essential mineral salts, at pH 6 at approximately 9, until appreciable antibiotic activity is imparted to the medium, and collect the fermentation product or else isolate the particular compounds neoviridogrisein I, neoviridogrisein II, neoviridogrisein III.

Streptomyces sp. P8648 was deposited on May 11, 1976 under N ° P3562 at the Fermentation Research Institute, Agency of Industriai Science and Technology, 8-1 Inage-Jgashi 5 chôme, Chiba City, Japan, where it is accessible to the public.

The following description deals, by way of example, with embodiments of the method according to the invention, partially with reference to the drawing. On this drawing:

Fig. 1 shows the ultraviolet spectrum of neoviridogrisein I (NVGI) in methanol.

Fig. 2 shows the ultraviolet spectrum of neoviridogrisein II (NVG II) in methanol.

Fig. 3 shows the ultraviolet spectrum of neoviridogrisein III (NVG III) in methanol.

Fig. 4 shows the ultraviolet spectrum of neoviridogrisein IV (NVG IV, VG) in methanol.

Fig. 5 shows the ultraviolet spectrum of neoviridogrisein I (NVG I) in methanol / 0.1N NaOH.

Fig. 6 shows the ultraviolet spectrum of neoviridogrisein II (NVG II) in methanol / 0.1N NaOH.

Fig. 7 shows the ultraviolet spectrum of neoviridogrisein III (NVG III) in methanol / 0.1N NaOH.

Fig. 8 shows the ultraviolet spectrum of neoviridogrisein IV (NVG IV, VG) in methanol / 0.1N NaOH.

Fig. 9 shows the infrared spectrum of neoviridogrisein I (NVG I) in KBr disc.

Fig. 10 shows the infrared spectrum of neoviridogrisein II (NVG II) on KBr disc.

Fig. 11 shows the infrared spectrum of neoviridogrisein III (NVG III) in KBr disc.

Fig. 12 shows the infrared spectrum of neoviridogrisein IV (NVG IV, VG) in KBr disc.

Microorganism

According to the described method, the depsipeptide antibiotics neoviridogrisein I, II and III are with viridogrisein and griseoviridine, produced by a new strain of Streptomyces called Streptomyces sp. P8648 (FERM-P 3562). This microorganism was isolated from a soil sample collected near the Kuzuryu dam in Fukui-ken (Japan).

This micro-organism presents short colorless aerial mycelia which extend from mycelium of substrate, or vegetative, in simple fork, well branched. Spore chains, which have a smooth surface, form a loose loop at the top of the aerial mycelia. No vortex or ascospore was observed.

This micro-organism has the following culture characteristics on different agar media:

1) Sucrose / nitrate agar growth: weak aerial mycelium: occasional aerial mycelia thin white reverse: colorless to grayish white soluble pigment: none

2) Glucose / asparagine agar growth: abundant aerial mycelium: little or not; if formed, white reverse: pale yellowish white to light yellow soluble pigment: none

3) Glycerin agar! Asparagine growth: moderate aerial mycelium: little or not; if formed, white reverse: pale yellow to greyish yellow soluble pigment: none

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4) Agar yeast extract / malt growth extract: abundant aerial mycelium: white to white with grayish coloration reverse: light yellow, later turning to brownish gray soluble pigment: none or, rarely, light brown

5) Growth starch agar: moderate aerial mycelium: little or not; if formed, white reverse: pale yellow with a slight greyish brown in the middle of the colonies soluble pigment: none starch hydrolysis: weak

6) Tyrosine agar growth: moderate aerial mycelium: none or a few spots of white aerial mycelia occasionally observed reverse: greyish yellow to light brown yellowish soluble pigment: at first pale purple to light reddish brown turning, ten days later, to pale brown ; formation of a little melanoid pigment

7) Nourishing growth agar: good aerial mycelium: thin, white reverse: pale yellow soluble pigment: none

8) Growing oatmeal agar: good aerial mycelium: white or greyish white reverse: greyish yellow to light reddish brown with greyish coloring soluble pigment: none.

The optimum temperature for the growth of the microorganism described is between 25 and 35 ° C. Although the growth is very weak, this microbe can, however, multiply outside this temperature range, for example at 10 ° C or at 45 ° C, but it can no longer multiply at the temperature of 52 ° C.

This actinomycete liquefies gelatin in a glucose / peptone / gelatin medium, weakly hydrolyzes starch in a starch / inorganic salt agar and peptonizes the skimmed milk without coagulation.

The production of melanoid pigment, occasionally observed in tyrosine agar, occurs neither in peptone agar / yeast extract / iron, nor in tyrosine broth / yeast extract.

The behavior of the microorganism described, with regard to the assimilation of carbon sources in the Pridham-Gottlieb medium, is illustrated by the following diagram:

positive: D-xylose, D-glucose, D-fructose, L-rhamnose, D-mannitol slightly positive: sucrose negative: L-arabinose, i-inositol, raffinose.

As regards the production of known peptide and non-peptide macrolide antibiotics, such as mikamycins A and B, virginiamycins, ostreogrycins, etamycin, verna-mycins, viridogriserin, griseoviridine and pristinamycins, micro- The following organisms can be compared with Streptomyces sp. P8648:

Streptomyces griseus NRRL 2426 Streptomyces griseoviridus NRRL 2427 Streptomyces sp., Producer of etamycin Streptomyces conganensis Streptomyces ostreogriseus Streptomyces mitakaensis Streptomyces loidensis

The information available on the physiological and cultural characteristics of the microorganism described shows a clear difference between this microorganism and comparable microorganisms

listed above. For example, the difference between Streptomyces griseus NRRL 2426 and the microorganism described consists in this: the first belongs to the section of rectiflexibles which has straight or slightly wavy spore chains, while the present microorganism belongs to the section of spirals; the first product on an agar extract of yeast / malt extract from gray to yellowish gray aerial mycelia, while the second produces white or grayish white aerial mycelia; L-arabinose is used by the former but not by the latter. Streptomyces sp., Producer of etamycin, specified in "Antibiotics Annual", 1954-1955, pp. 728-732, can be distinguished from the microorganism described with regard to the behavior of assimilation of carbon sources and the culture characteristics on Czapek agar, glucose / asparagine agar and nourishing agar. Streptomyces conganensis has striking differences in the morphological characteristics of the spores. Among the comparable microorganisms listed above, Streptomyces griseoviridus NRRL 2427 most closely resembles the streptomycetes described. The results of the taxonomic comparison between these two cultures are summarized in the following table:

Streptomyces griseoviridus NRRL 2427

Streptomyces sp. P8648

Color of aerial mycelium

Color of substrate or vegetative mycelium

Soluble pigment

45

So use of carbon sources

Pale yellowish orange to yellowish pink with gray coloring on agar yeast extract / malt extract, oatmeal agar, starch agar and glycerin / asparagine agar

Grayish yellow to oliveish brown or blackish on yeast extract agar / malt extract agar, oatmeal agar,

Starch agar and glycerin / asparagine agar No melanoid pigment formation, usually no other pigment, but rarely little yellow pigment

L-arabinose 4- + + D-fructose +

Sucrose -

White to greyish white and little formed on most ISP media; white and abundantly formed on agar yeast extract / malt extract

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

No melanoid pigment; usually no pigment, but rarely slightly formed brown pigment

L-arabinose

D-fructose

Sucrose

+ + + +

As the preceding table shows, a clear difference exists between Streptomyces griseoviridus NRRL 2427 and the strepto-55 fungus described as regards the morphological and cultural characteristics as well as the use of carbon sources. In addition, when both are fermented under identical conditions, the microorganism described can produce neoviridogriseins I, II and III as well as viridogrisein (neoviridogrisein IV) "o and griseoviridine, while the culture of Streptomyces griseo-viridus NRRL 2427 produces only viridogrisein and griseoviridine, but not neoviridogrisein I, II, and III.

As the above shows, the microorganism described is a Streptomyces of a new species called Streptomyces 65 sp. P8648. A typical culture of the microorganism described has been deposited with the Fermentation Research Institute, Agency of Industriai Science and Technology, under the deposit number FERM-P 3562. It should be noted here that the microorganisms

usable in the embodiments of the method according to the invention are not limited to the particular microorganism specified above and classified as FERM-P 3562 with the Fermentation Research Institute, but these microorganisms include all spontaneous and artificial mutants derivatives of the latter microorganism and which are capable of producing the antibiotics neoviridogrisein I, II and III.

Production of neoviridogriseins I, II, III

The process described for producing the depsipeptide antibiotics neoviridogrisein I, II, III, which is an embodiment of the process according to the invention, comprises the following steps: inoculate and allow Streptomyces sp. P8648 in an appropriate medium, under aerobic conditions at a temperature between 18 and 37 ° C, for 2 to 14 days, to collect the antibiotics accumulated from the fermentation broth and to purify them according to usual methods.

All known media which can be used for strepto-fungi can be used for the fermentation of the microorganism of the process described. Examples for preferred medium carbon sources are: glucose, glycerin, starch, dextrin, oatmeal, molasses, fats, oil and the like. Examples of preferred nitrogen sources are soybean meal, cottonseed meal, peptone, dry yeast, soaked corn liquid, yeast extract, casein and its hydrolyzate as well as inorganic salts such as ammonium sulfate and ammonium nitrate. Minor growth factors can be added to the medium. Examples of such factors are vitamins, amino acids, organic and inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, sodium phosphates, potassium phosphates as well as magnesium sulfate.

To obtain antibiotics according to the process described, the fermentation can be carried out in usual containers such as the stirring vessel, the fermentation vessel and the fermentation tank but, from the economic point of view, the culture submerged under forced aeration offers a maximum benefits on an industrial scale. The fermentation is preferably carried out under aerobic conditions at a temperature between 25 and 35 ° C. When a stirring vessel (or a fermentation vessel) is used, the production of neoviridogriseins usually reaches a maximum in 2 to 10 days. The pH value can change during fermentation to beyond the biological range, depending on the type of medium used. It is advantageous that the pH value is maintained, during fermentation, between 6 and 9. Before inoculation, the pH of the medium is preferably between 6.5 and 8.5.

Control of neoviridogriseins in broth

As explained above, the microorganism of the process described produces a mixture of neoviridogriseins I, II, III, IV and griseoviridine. The shares of different neoviridogriseins in the fermentation broth can be changed using an appropriate combination of carbon and nitrogen sources in the medium, without adding a free amino acid or an organic acid. From the point of view of production on an industrial scale, it is however preferable to adjust the shares of neoviridogriseins I, II and / or III in the fermentation broth by adding to the medium, during fermentation, the amino acid constituting relevant free or more of these acids, depending on the circumstances and momentary needs. The shares of the different neoviridogriseins in the fermentation broth can be appropriately varied by selecting spontaneous or artificial mutants derived from the culture of the streptomycet of the process described and / or by adding physiologically active agents such as inhibitory enzymes and promoter enzymes. A preferred process for the selective production of desired particular neoviridogriseins comprises the following step: adding, during fermentation, the amino acid constituting the relevant or more of these acids such as a-amino-n- acid

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butyric and / or proline. The addition of proline, during fermentation, increases the percentage of neoviridogriseins I and II in the total amount of neoviridogriseins I, II, III, IV. Neoviridogriseins I, II have a stronger antimicrobial activity than that of neoviridogriseins II, IV. The addition of α-amino-n-butyric acid to the medium before inoculation or during fermentation makes it possible to selectively increase the shares of neoviridogriseins I and III. As the microorganism of the process described produces protease during its growth, proteinaceous material comprising the amino acid constituting relevant, or several of these acids, can be added instead of free amino acid. Casein or corresponding hydrolysates from acid hydrolysis, for example casamino acids, can thus be substituted for proline.

Isolation and purification

The neoviridogrisein antibiotics I, II, III and viridogrisein can be isolated from the fermentation broth according to a usual process based on the physicochemical properties of pepsipeptide antibiotics. If necessary, neoviridogriseins can be collected from the fermentation broth together with griseoviridine, as a neoviridogrisein / griseoviridine mixture. When the antibiotics described are prepared in the form of an animal feed additive or a veterinary medicinal product, a crude mixture of neoviridogriseins and griseoviridine is economically most advantageous.

The neoviridogriseins and griseoviridine from the fermentation broth can be extracted with an organic solvent immiscible with water. Examples of such solvents for the simultaneous extraction of neoviridogriseins and griseoviridine are ethyl acetate, butyl acetate, n-butanol, methylene chloride, chloroform and the like. Preferred examples of such solvents for the selective extraction of neoviridogriseins are methyl isobutyl ketone, benzene, toluene and other aromatic hydrocarbons. Since the mycelium contains practically no neoviridogrisins and the lipid which can be extracted into the cells may interfere with subsequent purification steps, it is preferable to extract these antibiotics from the broth filtered or centrifuged using a solvent organic, together with the wash water.

The solvent extract of neoviridogriseins and / or griseoviridine can be isolated and purified according to various methods. Examples of such methods are adsorption and elution with active carbon of the brand Amberlite XAD-4 and 7 from Rohm &

Haas Co. with ion exchange resins from the brand Amberlite IR-120 from Rohm & Hass Co. and from the brand Dowex 50W-X2 from The Dow Chemical Co., gel filtration with Sephadex LH-20 from Pharmacia Fine Chemicals AB or its equivalents, chromatographic analysis by adsorption on alumina and silica gel, etc. These methods can be combined for the isolation and purification of these antibiotics. It is advantageous to use a counter-current distribution with an appropriate solvent system.

Physico-chemical properties of neoviridogriseins

Neoviridogriseins I, II, III and viridogrisein are amorphous white solids soluble in methanol, ethanol, n-propanol, isopropanol, n-butanol, dioxane, acetate d ethyl, acetatebutyl, acetone, methyl ethyl ketone, methyl isobutyl ketone, benzene, toluene, methylene chloride, chloroform, carbon tetrachloride, NN-dimethylformamide and dimethyl sulfoxide, barely soluble in water and ethyl ether and practically insoluble in petroleum and hexane.

These antibiotics are stable in an aqueous solution for at least a month at a temperature between 25 and 37 ° C and for about 30 minutes at 60 ° C at pH 2 to 9. The determination

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Mining the melting point of these antibiotics using the Kofler apparatus gave the following results:

Neoviridogrisein I: not determined II: 93 ° C III: 115 ° C Viridogrisein: 140 ° C

Figs. 1-8 show the ultraviolet absorption spectra of neoviridogriseins I, II, III and viridogrisein. Figs. 1-4 show the ultraviolet spectrum of neoviridogriseins I, II, III and viridogrisein recorded in methanol, and Figs. 5-8 show the ultraviolet spectra of these substances recorded in 0.1N methanol / NhOH. The maximum values of E J ° y ° m of neoviridogriseins are as follows:

In neutral methanol,

Neoviridogrisin I: 305 nm (65)

II: 305 nm (88)

III: 305 nm (90)

Viridogrisein: 305 nm (90)

In 0.1N methanol / NaOH Neoviridogrisein I: 340 nm (70)

II: 340 nm (84)

III: 340 nm (90)

Viridogrisin: 340 nm (96)

Figs. 9-13 show the infrared absorption spectra of neoviridogriseins I, II, III and viridogrisein in KBr disc. The characteristic peaks and shoulders are observed in the following regions:

Neoviridogrisein I (KBr disc):

3370,2910,2850,1735,1670 (sh.), 1635,1590 (sh.), 1515,1460 (sh.), 1445,1405,1375,1290,1280,1250 (sh.), 1200,1190, 1160,1125,1100 and 1080 cm-1;

Neoviridogrisein II (KBr disc):

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 and 1065 cm-1;

Neoviridogrisein III (KBr disc):

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 and 1065 cm "K

Neoviridogriseins I, II, III, viridogrisein and griseoviridine have the following Rf values in TLC systems:

0 ° C for 30 min, and by amino acid analysis using the Hitachi KLA-5 type analyzer from Hitachi, Ltd. The presence of the following amino acids has been confirmed:

1) TLC plate:

Solvent:

Neoviridogrisin I: II: III:

Viridogrisein: Griseoviridine:

2) TLC plate: Solvent: Neoviridogrisein I:

II: III:

Viridogrisein:

Griseoviridine:

pre-coated plate of the Silicagel type

60F-254 from E. Merck, Darmstadt.

benzene / methanol 5/1

Rf = 0.66

0.62

0.59

0.55

0.20

identical to that of 1)

chloroform / methanol 30/1

Rf = 0.39

0.32

0.19

0.18

0.02

Neoviridogrisin I

Neoviridogrisin II:

Neoviridogrisin III:

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35

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threonine leucine proline α-amino-n-butyric acid sarcosine phenylsar cosine P, N-dimethylleucine threonine leucine proline alanine sarcosine phenylsarcosine P, N-dimethylleucine threonine leucine hydroxyproline α-amino-n-butyric acid sarcosine phenylcarcinine sarcosine phenylcarcinine hydroxyproline alanine sarcosine phenylsarcosine

P, N-dimethylleucine

The presence of 3-hydroxypycolinic acid was confirmed by mass spectrometry and by TLC as follows:

An authentic sample of viridogrisein and each of neoviridogriseins I, II, III, IV was hydrolyzed overnight in 6N HCl at 110 ° C to produce hydrolysates which had only one UV absorption spot with the same Rf value. , under the conditions indicated.

1) TLC of silica gel

TCL plate: pre-coated plate of the Silicagel 60 F-254 type from E. Merck, Darmstadt chloroform / methanol 2/1 0.46

Neoviridogrisin III:

Viridogrisin

Solvent: 45 Rf;

2) TCL of cellulose TCL plate:

50

Solvent Rf

Eastman Chromagram Sheet 13254 in cellulose with fluorescent indicator, Eastman Kodak Co.

n-buthanol / acetic acid / water 4/1/1 0.62

To analyze the constituent amino acids, each neoviridogrisein was hydrolyzed overnight in 6N HCl at 110 ° C. and the hydrolyzate obtained was concentrated to dryness. After removing all traces of hydrochloric acid by repeated evaporations, the amino acids contained in the hydrolyzate were determined by TLC using an Eastman 13 254 type chromatogram sheet in cellulose with fluorescent indicator, from Eastman Kodak Co. and solvent system n-buthanol / acetic acid / water 4/1/1, by high voltage electrophoresis using paper of the Toyo Filter No. 51A type from Toyo Roshi Kaisha, Ltd., a formic acid / acetic acid / buffer system water 25/25/900, pH 1.8, at 60 V / cm at

The molecular weight of these antibiotics was determined by direct insertion into a mass spectrometer.

55

Neoviridogrisin I: 876 II: 862 III: 892

Viridogrisein: 878

60

To study the chemical structure of neoviridogriseins I, II, III, these antibiotics as well as viridogrisein were hydrolyzed overnight in 0.1N NaOH at room temperature and then methylated before mass spectrometry with diazomethane, 65 according to the method of Compernolle et al. ("Organic Mass Spectro-metry", vol. 6, pp. 151-166,1972). Based on the information set out above, neoviridogriseins I, H, III have the following structure:

Neoviridogrisin I

OH

% ^

\

CH

I

CHj

CO - WH — CH- CO-NH- CH-CO-CK- CH,

4

i co

ÒK-W - CO-CH-NH-

I I

CHi CHt I

CH.

CH2 CH2

-N dH-

CO

h-CHi

I

CK,

it

CQ-CH — N — CHj

Neoviridogrisin II

OH

CHj CHj

X /

CH I

CHj,

CU— H - CH - CO - fJH- CH- C 0

Ctf-CHa

I

0

1

CO

CK-CH,

I

CH-CH,!

VS

/ K,

CH. CH,

-N CH-Cû

Ù-CK * I

CH.

CO

Ctt-N - CO -CH -fc'H -CO-CH— N - CH3

> \ ch3 CH.

Neoviridogrisin III

CH,

at

OH

I

CK

said

CO-KH — ÇH —CO — CVH— Oi — CO-

ch-ch3

i

0

co ch-ch5 I

CH-CHj

I

CH,

OH I

/ ° \ CKf CHj I I -M Ctf-Cö

N-CH,

CHj

I

CO

CH-Kl -CO- Ctf-NH- CO -CH —N— CH.

He J

O ™ '

CH. I

ch3

CH-CHi

CH ~ CHa

I

CH,

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The above structures show that the neoviridogriseins II, III of the method described are depsipeptide antibiotics homologous to viridogrisein. The identity of neoviridogrisein IV with viridogrisein and etamycin was confirmed by mass spectrometry, by TLC, by IV and IR spectrometry and by amino acid analysis, using authentic samples of viridogrisein and etamycin.

Antimicrobial activity

Neoviridogriseins I, II, III have a broad antimicrobial spectrum against bacteria, mycoplasmas, actino-

fungi and rickettsiae, in laboratory tests. They display remarkable activity, in particular against the usual and resistant varieties of Staphylococcus aureus, as well as against those of Straptococcus pyogenes, Diplococcus pneumoniae, 5 Sorcina lutea, Bacillus subtilis, Mycoplasma gallisepticum, Mycoplasma pulmonis, Mycoplasma fermentans and Mycoplasma agalactiae.

The minimum inhibitory concentrations, hereafter designated by the symbol MIC, of the depsipeptide antibiotics described were determined, both separately and in combination with viridogrisein and griseoviridine, against different microorganisms, by means of test tests. dilution, the results of which are shown in the following tables.

Table 1: MCI values of neoviridogriseins I, II, III (mcg / ml)

Microorganism

Middle

NV * I

NV * II

NV * III

VG **

NV * e-mail

NV

Staphilococcus aureus

209P

BHI (1)

0.2

0.1

0.2

0.2

0.2

0.1

(EM) r

BHI (1)

0.4

0.4

0.4

0.4

0.4

0.2

(STH) r

BHI (1)

0.8

0.8

1.6

1.6

1.6

0.8

(PC, TC, EM, LM) r 1

BHI (1)

6.25

6.25

6.25

6.25

6.25

3.2

(PC, TC, EM, LM) r 2

BHI (1)

6.25

6.25

6.25

6.25

6.25

3.2

(TC, EM, LM, CP) r

BHI (1)

6.25

6.25

6.25

6.25

6.25

3.2

(SM, STH) r

BHIa)

0.4

0.2

0.2

0.4

0.4

0.2

(PC, KM, EM) r

BHI (1)

0.8

0.4

0.4

0.8

0.8

0.4

(EM, CM, SM, PC, TC) r

BHI (1)

0.4

0.2

0.4

0.4

0.4

0.2

(EM, OM) r

BHI (1)

0.8

0.4

0.8

0.8

0.8

0.4

(TC, CP, PC) r

BHI (1)

0.2

0.2

0.4

0.4

0.4

0.2

(BX-1633) (PC) r

BHI (1)

0.4

0.2

0.4

0.4

0.4

0.2

Russell (PC) r

BHI (1)

0.2

0.2

0.4

0.4

0.4

0.2

Smith

BHI (1)

0.2

0.2

0.2

0.4

0.4

0.2

Staphylococcus sp.

(PC) r

Staphylococcus sp.

Diplococcus pneumoniae type 1 Streptococcus pyogenes Sorcina lutea

Bacillus subtilis ATCC 6633 Salmonella gallinarum Escherichia coli Pseudomonas aeruginosa Proteus vulgaris Candida albicans

BHI (1)

0.8

0.1

0.8

0.4

0.8

0.4

BHI (1)

0.8

0.8

0.8

0.8

0.8

0.4

BHI + HB'2 "

0.2

0.4

0.2

0.4

0.4

• 0.1

BHI + HB (2)

0.4

0.2

0.4

0.4

0.4

0.1

BHIU)

0.2

0.4

0.4

0.2

0.4

0.1

BHI (1)

0.4

0.4

0.8

0.8

0.8

04

BHI (1)

> 25

> 25

> 25

> 25

> 25

> 25

BHI (1)

> 25

> 25

> 25

> 25

> 25

> 25

BHI (1)

> 25

> 25

> 25

> 25

> 25

> 25

BHI (1)

> 25

> 25

> 25

> 25

> 25

> 25

my (3)

> 25

> 25

> 25

> 25

> 25

> 25

* N V = neoviridogrisin la) EM = erithromycin LM = leucomycin NM = neomycin

** VG = viridogrisein STH = streptothricin

*** GV = griseoviridine PC = penicillin TC = tetracycline SM = streptomycin KM = kanamycin

CP = chloramphenicol OM = oleandomycin

() r = Resistant to the drug (s) in the parenthesis.

(1) BHI = Broth and heart infusion broth.

<2) BHI + HB = Broth and heart infusion broth comprising 10% horse blood.

, 3) MY = Malt extract / yeast extract medium.

As the previous table of ICD values shows, neoviridogrisein II is more active than neoviridogrisein IV, i.e. viridogrisein. This result was obtained on the basis of two dilution tests. To differentiate neoviridogrisein II and viridogrisein with regard to their antibiotic activities, the test to determine the ICD was repeated with a significantly lower dilution. Table 2 indicates that neoviridogrisein II is 2 to 3 times more active than viridogrisein.

(Table at the top of the next page)

When testing the ICDs of neoviridogriseins I, II, III in the presence of griseoviridine, synergism was observed between neoviridogrisein and griseoviridine as in the case of viridogrisein 60 and griseoviridine. This is why the synergism of the antibiotics described with griseoviridine has been studied in more detail in various proportions of the mixture of neoviridogriseins compared to griseoviridine. Table 3 shows the results obtained:

65 (Table on next page j

As this table shows, the maximum activity of the neoviridogrisein group in synergism with griseoviridine is located at

Table 2:

Comparison of neoviridpgrisein II with viridogrisein

11

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Table 3:

Synergism of the mixture of neoviridogriseins with griseoviridine

Microorganism

ICD (mcg / ml) NV * II VG **

Neoviridogrisein mixture: griseoviridine

CIM * (mcg / kl)

Staphylococcus aureus

100: 0

0.313

209 P

0.078

0.133

90: 10

0.156

(EM, CM, SM, PC, TC) r

0.125

0.334

, n 80: 20

0.125

(TC, CP, PC) r

0.094

0.334

70:30

0.125

Bx-1633 (PC) r

0.125

0.267

60:40

0.094

Russel (pc) r

0.125

0.267

50:50

0.078

Smith

0.125

0.267

40:60

0.094

15 30: 70

0.125

Medium: Broth with infusion of brain and heart.

20: 80 10: 90 0: 100

0.250 0.250 0.250

Abbreviations: according to table 1.

the proportion of 50:50, that is to say a mixture of neoviridogriseins / 20 griseoviridine 1/1 and 3 to 4 times more active than the mixture of neoviridogriseins or griseoviridine alone.

Table 4 illustrates the strong in vitro activity of neoviridogrisein II against different varieties of mycoplasmas as well as the superiority of the synergism displayed by a neovirido- mixture.

* Test microorganism: Sartina lutea.

Dilution test with broth with brain and heart infusion.

grisein II / griseoviridine in comparison with that of a viridogrisein / griseoviridine mixture. The MICs were determined by means of dilution tests.

Table 4

Microorganism

Middle

NV II

VG

GV

NV 11 + GV *

VG + GV

Mycoplasma gallisepticum 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 ferment

(2)

<0.05

<0.05

<0.05

<0.05

<0.05

Mycoplasma agalactiae PG 2

(2)

0.39

0.78

3.13

<0.05

0.39

Medium (1): PPLO enrichment broth (Eiken, Japan).

Middle (2): PPLO broth (middle of Chanock, Difco).

* Proportion of the mixture: 50/50.

NV II, VG, GV: see table 1.

In summary, the described neoviridogriseins I-III display remarkable activity against Gram positive bacteria and mycoplasmas, both alone and in various mixtures with viridogrisein (neoviridogrisein IV) and griseoviridine. It has been found that the mutual proportions by weight of neoviridogriseins I-III, viridogrisein (neoviridogrisein IV) and griseoviridine, in a mixture, can vary within wide limits while retaining the antimicrobial and antimycoplasma activity of these mixtures. By way of nonlimiting example, a mixture comprising, by weight: Neoviridogrisein II 27%

Neoviridogrisein IV (viridogrisein) 23% Griseoviridine 50%

has been tested in vitro against Streptococcus mutans, a microorganism that causes dental caries and periodental diseases. The test was carried out in a Todd Hewitt broth (Difco) with 0.5% of TC lactalbumin hydrolyzate (Difco). The initial count of microorganisms gave approximately 3 x 104 organisms per milliliter. The culture tubes were incubated under anaerobic conditions at a temperature of 37 ° C for 48 h. Both the ICD and the minimum bactericidal concentration were found at a neoviridogrisein concentration of 1.0 ppm (parts per million).

Still by way of nonlimiting example, the same mixture was tested in vitro against Treponema hyodysenteriae, a microorganism which causes swine dysentery. The mixture was tested, diluted in blood agar at concentrations of 100, 50.10,

5,1,0,5 and 0,1 ppm. Once inoculated, the plates incubated for 4 days at a temperature of 42 ° C. The ICD was 0.5 ppm. By way of nonlimiting representative example, a mixture comprising, by weight:

Neoviridovisein II 25%

Neoviridogrisein IV (viridogrisein) 25% Griseoviridine 50%

50 has been tested in vitro against several varieties of mycoplasma. The following CIMs were found:

CIM species

Mycoplasma gallisepticum KP 13 0.07

Mycoplasma pulmonis 0.20

Mycoplasma ferment 0.05

Mycoplasma agalactiae 0.05

These typical neoviridogrisein / griseoviridine mixtures are also useful in the treatment of animals suffering from infectious diseases caused by the above pathogenic bacteria.

Since mikamycins A and B are known to be effective as a food additive, the antibiotics described have been tested in combination with animal food. A mixture of neoviridogriseins was added to a chicken feed in a proportion of 2 to 20 ppm and the resulting mixture was administered to male chickens for ten weeks. Compared to a reference group of chickens fed the same food devoid of neoviridogriseins, chickens fed neoviridogro-

623,566

12

grisein were superior in terms of weight growth rate and food yield, proving that the neoviridogriseins described are very useful as a food additive.

A composition comprising one or more of the neoviridogriseins I-III mixed or not mixed with viridogrisein (neoviridogrisein IV) and griseoviridine, and in which the mutual proportions by weight of the components can vary to a very large extent, has been shown to be very useful as a food additive. By way of nonlimiting representative example, a mixture comprising, by weight:

Neoviridogrisin II 27%

Neoviridogrisein IV (viridogrisein) 23% Griseoviridine 50%

has been tested in vivo by adding it to chicken feed during the growing season. Fifteen 1-day-old chicks were used for a test in which they were subjected to a diet comprising, during

11 days, 55% of rye grains supplemented with vitamins, minerals, fat and protein sources. The high grain content of rye usually causes little or moderate growth. This standard diet is commonly used to test growth promoters and antibiotic food additives. Penicillin was used as the reference additive, in a proportion of 100 ppm.

The method described makes it possible, in summary, to obtain the depsipeptide antibiotics neovirideogriseins I, II, III and virideogrisein which are active against Gram positive bacteria and against mycoplasmas and which can be used diluted, as crude concentrates or in pure form, with or without griseoviridine. The method also makes it possible to obtain these depsipeptide antibiotics by simple fermentation of an aqueous medium under aerobic conditions using Streptomyces sp. P8648 (FERM-P 3562) and to collect the neoviridogriseins formed from the fermentation broth with or without griseoviridine. This process has, moreover, the advantage of allowing the selective production of the desired neoviridogriseins by using, during or before fermentation, relevant amino acids, for example proline to increase the production of neoviridogriseins I and II. α-amino-n-butyric acid to increase the production of neoviridogriseins I and III. The antibiotics obtained according to the process described can be used in human and veterinary medicine, in particular for the treatment of infectious diseases caused by Staphylococcus aureus, Streptococcus pyogenes, Diplicoccus pneumoniae, Mycoplasma gallisepticum, Mycoplasma fermentans, Mycoplasma agalactiae, etc.

Example 1

50 ml of a sprout culture medium comprising 0.5% soy flour, 0.5% Pharmamedia from Traders Oil Mill Co., 0.5% oat flour, 0.5% dry yeast and 0.5% beet molasses were adjusted to pH 6.5 and distributed in a 250 ml Erlenmeyer flask. After treatment in an autoclave at 120 ° C for 15 min, a small amount of Streptomyces sp. P8648, cultured on an agar plate, was inoculated in the Erlenmeyer flask which incubated on a rotary shaker for 48 h at 28 ° C. 2 ml of the culture of sprouts thus obtained were transferred to an Erlenmeyer flask containing 100 ml of '' a fermentation medium which included 0.5% soy flour, 0.5% peanut flour, 0.5% oat flour, 0.5% dry yeast and 0.5% molasses beets, and cultivated at 28 ° C for 96 h on a rotary shaker at 200 rpm on a circle with a radius of 3.5 cm. Filtering the culture broth collected from

12 Erlenmeyers gave a clear broth filtrate. The residue, or cake, obtained on the filter was washed with 100 ml of water. The wash water and the broth filtrate were combined. The antibiotic activity of the aqueous solution at pH 8.3 thus obtained was found to be 23.0 mm on a nourishing agar test plate,

by running the disc test with an 8 mm paper disc. 800 ml of this aqueous solution were extracted twice using each time 200 ml of n-butanol, the butanol extracts were combined and concentrated to dryness under reduced pressure to produce 90 g of crude powder of neoviridogriseins and griseoviridine . This crude powder was mixed with a small amount of silica gel and applied to a 1.5 x 23 cm column of silica gel of the registered trademark Vako-Gel C-100 from Vako Pure Chemical Industries, Ltd. The silica gel column was eluted first with 300 ml of a 5/1 benzene / acetone mixture and then with a 2/1 benzene / acetone mixture. 10 g fractions were collected on an automatic fraction collector. The active fractions numbered 25 to 35 were combined and concentrated to dryness to provide, or produce, 30 g of a mixture of neoviridogriseins I, II, m and viridogrisein. The dry concentration of these active fractions numbered 45 to 55 produced a crude powder whose antibiotic activity corresponded to that of griseoviridine, determined by thin layer chromatography, hereinafter designated by the symbol TLC. These two preparations were analyzed by TLC. Antimicrobial activity was determined using a nourishing Sorcina lutea agar plate.

TLC plate: pre-coated plate of the Silicagel 60 F-254 type, from E. Merck, Darmstadt.

1) Solvent: chloroform / methanol 1/1

Neoviridogriseins Rf: 0.45

Griseoviridine: 0.05

2) Solvent: benzene / acetone 1/1

Neoviridogriseins Rf: 0.55

Griseoviridine: 0.13

Example 2

200 ml of the culture of Streptomyces sp. P8648 in a culture medium identical to that of Example 1 were inoculated in a stainless steel fermentation vessel containing 101 of the culture medium of Examples 1 and cultured at 27-28 ° C with stirring for 96 h, under forced ventilation of 5 1 / min of sterile air. The stirring took place at a rotation speed of 200 rpm by means of a propeller whose radius was equal to a quarter of that of the fermentation vessel. After this, the mycelia and solids were removed by filtration. The filtrate obtained was adjusted to pH 6.0 and extracted four times, each using 21 ethyl acetate. The ethyl acetate extracts were combined, dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure to produce 700 mg of crude powder of neoviridogriseins and griseoviridine. This powder was dissolved in a small amount of methanol and loaded onto a Sephadex LH-20 column of 3 x 50 cm. 10 ml fractions were collected with methanol as the eluent. Neoviridogriseins were found in fractions numbered 22-29 which were collected, concentrated to dryness and purified by 1.5 x 20 cm silica gel column chromatography of the Silicar CC-7 type from Mallinckrodt Chemical Works. Elution was carried out using a 30/1 chloroform / methanol mixture. The eight 5 g fractions numbered 5 to 12 were combined and concentrated to dryness under reduced pressure, to produce 25 mg of white neoviridogrisein powder which included 15% neoviridogrisein 1.20% neoviridogrisein II, 20% neoviridogrisein III and 45% viridogrisein. Griseoviridine was found by TLC in fractions numbered 30 to 34 from the Sephadex LH-20 column. These active fractions were combined, concentrated to dryness under reduced pressure and crystallized from hot methanol to produce 30 g of acid crystals, the identity of which has been proven by TLC as well as by other physicochemical determination methods.

Example 3

The same fermentation as that of Example 1 was carried out for 96 h, except that the fermentation medium comprised 0.5% of soy flour, 0.5% of pharmamedia, 0.5% of oat flour, 0.5% dry yeast, 0.5% beet molasses and 0.1% DL-a-amino-n-butyric acid at pH 6.5. The fermentation broth was collected from 13 Erlenmeyer flasks and filtered. The filtered liquid was extracted two

5

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20

25

30

35

40

45

50

55

60

65

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623,566

times using 300 ml of n-butanol each time. Removal of n-butanol from the extracts provided approximately 70 mg of crude powder of neoviridogriseins and griseoviridine. This crude powder was analyzed by TLC of silica gel followed by writing with Sorcina lutea as the test organism. The TLC plate used in this test was a pre-coated plate of the Silicagel 60 F-254 type, from E. Merck, Darmstadt. The Rf values and the solvent systems were as follows:

Chloroform / methanol

1/20

1/30

40/1

Neoviridogrisin I

Rf = 0.60

0.39

0.20

II

0.56

0.32

0.16

III

0.50

0.19

0.13

Viridogrisin

0.43

0.18

0.10

Griseoviridine

0.05

0.02

0.00

Example 4

50 ml of a germ culture medium comprising 0.3% of beef extract, 0.5% of tryptone from Difco Laboratories, 0.1% of glucose, 2.4% of soluble starch, 0.5 % yeast extract, 0.4% calcium carbonate and 0.5% soybean meal at pH 7.0 were distributed in a 250 ml Erlenmeyer flask and autoclaved at 120 ° C for 15 min . Spores of Streptomyces sp. P8648 grown on an agar plate were introduced into the Erlenmeyer flask and cultured at 25 ° C for 3 days, to produce the culture of sprouts. The fermentation medium included 0.5% soluble starch, 2.0% glucose, 1.0% pharmamedia, 0.5% oatmeal, 0.5% soaked corn liquid, 0, 05% bipotassium phosphate, 0.05% magnesium sulfate (pH 6.5). 50 ml of this fermentation medium were placed in a 250 ml conical vase and treated in an autoclave at 120 ° C for 15 min. The inoculum share was 2% of the volume. Germs from the above culture were inoculated into the fermentation vessel which incubated in a rotary shaker at 25 ° C. 24 or 48 h after inoculation, a sterilized casamino acid solution from Difco Laboratories or proline (pH 7.0) was added to a final concentration of 0.4% and fermentation was continued. Four days after inoculation, the fermentation broth was filtered and the broth filtrate was extracted twice using each time an equal volume of ethyl acetate. The ethyl acetate extracts were combined and concentrated to dryness under reduced pressure to produce a dry powder. This crude powder was dissolved in ethyl acetate and an aliquot of the solution was distributed on a TLC plate of silica gel, in the form of spots of determined size. The TLC plate was developed in a solvent system of chloroform and methanol (50/1) and the solvent was evaporated into the atmosphere. The TLC plate was then developed again in the same system of chloroform and methanol solvents (50/1). Each component of neoviridogrisein was located under ultraviolet light (3650 Å), scraped off the surface of the TLC plate and suspended in a known volume of methanol. After removing the silica gel by decantation, the amount of antibiotics contained in the extracts was determined during tests by irradiation with UV rays, knowing that at 305 nm the value of s was 8000. The ray tests UV gave the following results:

Amino acid added

no proline casamino acid

Neoviridogrisin I

2%

2%

2%

Neoviridogrisin II

9%

40%

60%

Neoviridogrisin III

4%

3%

3%

Viridogrisin

85%

55%

35%

Example 5

About 101 of the 23 h old sprout culture were prepared in a fermentation vessel, under conditions identical to those of Example 2. The fermentation medium (6001), which included 0.5% soy flour , 0.5% pharmamedia, 0.5% oatmeal, 0.5% dry yeast, 0.5% beet molasses and 0.1% DL-a-amino-n- acid butyric, was steam sterilized at 120 ° C for 15 min in a 14001 steel fermentation tank, and cooled to 28 ° C. The 101 seed cultures were added to the middle of this tank and the whole was cultivated at a temperature of 28 ° C. for 75 h with forced aeration with stirring with a rotational speed of 180 rpm using a double helix whose radius corresponded to a quarter of the diameter of the fermentation tank. A flow rate of 3001 / min of sterile air circulated from a disperser from the bottom of the tank. After fermentation, the broth was filtered by a filter press. The broth filtrate was extracted twice using 1501 n-butanol each time. The n-butanol extracts were combined, washed with a small volume of saturated NaCl solution, and concentrated to 21 in a rotary evaporator. At this stage, 20 g of silica gel of the brand Wakogel G-100 from Wako Pure Chemical Industries, Ltd., were added and the concentration in the rotary evaporator was continued until complete dryness. The antibiotic / silica gel mixture thus obtained was suspended in a small amount of chloroform and loaded at the top of a column of silica gel of the Wakogel C-100 type of 6.5 × 75 cm. The elution to determine the antibiotic activities was carried out in stages, first using 7 l of chloroform, then with 101 of a chloroform / methanol mixture (50/1) and finally with methanol. The 300 g fractions numbered 16-29 which exhibited bioactivity against Sorcina lutea (neoviridogrisein) were collected and concentrated to dryness under reduced pressure to produce a white powder of neoviridogriseins. This powder was dissolved in a small volume of methanol and passed through a 7.0 x 45 cm Sephadex LH-20 column, each of the 100 g fractions being eluted with methanol. After removal of the solvent by evaporation, approximately 15 g of crude powder of a mixture of neoviridogriseins were collected from fractions numbered from 6 to 15. Fractions numbered from 50 to 60 from this column of silica gel contained griseoviridine . Purification with the 7.0 x 45 cm Sephadex LH-20 column, which was used for the neoviridogrisein mixture, produced 4 g of crude griseoviridine.

Example 6

For final purification, TLC was used, with a TLC plate of silica gel. 1 g of the crude mixture of neoviridogriseins of Example 5 was dissolved in 2 ml of ethyl acetate and loaded in the form of strips on TLC plates of silica gel of the Silicagel 60 F-254 type. These TLC plates were first developed with a chloroform / methanol (50/1) solvent system. Then the solvent was evaporated into the atmosphere and the TLC plates were further developed with a chloroform / methanol solvent system (25/1). Neoviridogriseins I, II, III and viridogrisein were found on TLC plates under ultraviolet light at 3650 Å, using the Blak-Ray UVL-22 type instrument from Ultra-Violet Products, Inc ., and scrapings of the elution. Each component of neoviridogrisein was eluted with a small amount of methanol and concentrated to dryness, to produce the following amounts of components of pure neoviridogrisein:

Neoviridogrisein I: 16.7 mg, less pure, oily Neoviridogrisein II: 11.1 mg, in the form of a white powder Neoviridogrisein III: 15.2 mg, in the form of a white powder Viridogrisein 30.0 mg in the shape of a white powder.

About 5 mg of each of these neoviridogriseins were hydrolyzed in 6N HCl at a temperature of 110 ° C. for 36 h in a sealed tube and analyzed by TLC, by paper chromatography, by high voltage electrophoresis as well as by a

5

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25

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35

40

45

50

55

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amino acid analysis. These neoviridogriseins included the following compounds:

Neoviridogrisein I: 3-hydroxypicolinic acid, threonine, leucine, proline, sarcosine, P-N-dimethylleucine, α-amino-n-butyric acid, phenylsarcosine.

Neoviridogrisein II: 3-hydroxypicolinic acid, threonine, leucine, proline, sarcosine, p, N-dimethylleucine, alanine, phenylsarcosine.

Neoviridogrisein III: 3-hydroxypicolinic acid, threonine, leucine, hydroxyproline, sarcosine, p, N-dimethylleucine,

α-amino-n-butyric acid, phenylsarcosine.

Viridogrisein: 3-hydroxypicolinic acid, threonine, leucine,

hydroxyproline, sarcosine, P, N-dimethylleucine, alanine, phenylsarcosine.

In addition, the identity of neoviridogrisein IV with viridogrisein was confirmed by IR, UV, paramagnetic resonance, mass spectrometry, TLC, hydrolysate analysis and antimicrobial spectrometry. The preparation of griseoviridine obtained in Example 5a, moreover, was crystallized from hot methanol, to provide acid crystals. A part of these crystals was then compared with an authentic preparation of griseoviridine and identified with it by irradiation with IR, UV, by RNM, by mass spectrometry, by TLC, by elemental analysis and by other physico-chemical processes.

R

12 sheets of drawings

Claims (5)

623,566 1 1 CHt j (ni) CO) N -CO - CH— NH— CO— CH N - CH, CH * CH * CH— CH * I I CHj ÇH— CR, CH3 1. Process for obtaining the depsipeptide antibiotic neoviridogrisein I of formula: GH * ^ CH3 X CH / CH * OH î X \ aCHe CHZ CHz I II CO- NH- CH - CO - NH - CH— CO - N CH - CO cIh n-ch5 I CH, I (i) CO n - co - ch — nh - co - ch— n - ch. i i i CHj CH, CH — CH, CH, V "CH3 the neoviridogrisin II depsipeptide antibiotic of the formula: CHj CHi .CH, X chi chi OC v i "- i" - n co — nh - ch - co - nh - ch - co— n ch-co I n-ch3 I chx I (ii) CO I co - ch - nh - co - ch - n - ch. I I ch, ch — ch " i ch ch » ch; and the neoviridogrisin III depsipeptide antibiotic of the formula: (Formula at the top of the next page) comprising the following stages: cultivating Streptomyces sp. P8648 (FERM-P3562) under aerobic conditions, at a temperature of 18 to 37 ° C, in an aqueous nourishing medium containing assimilable sources of carbon, assimilable sources of nitrogen and essential mineral salts, at pH 6 to 9 until appreciable antibiotic activity is imparted to the medium, and collect the fermentation product, or else isolate the particular compounds neoviridogrisein I, neoviridogrisein II, neoviridogrisein III. 2. Method according to claim 1, comprising the following steps: producing, in addition to neoviridogrisein I, neoviridogrisein II and neoviridogrisein III, viridogrisein and griseoviridine. 2 3. Method according to claim 1, in which the fermentation of the seine I, of the neoviridogrisein II, of the neoviridogrisein III or one is carried out in the presence of α-amino-n-butyric acid mixture of these substances. and / or from natural sources comprising α-amino-n-6 acid. Use according to claim 5, wherein the butyric ingredient. active is neoviridogrisein II. 3 623,566 OH CH5 y "- CH, I CH 1 CH ^ \ CH, CH * CH2 i '' NH-CH - CO— NH - CH —CO - N CH - CO CH-CH. N —CHj 4. Method according to claim 1, in which the fermentation 7. Use according to claim 5, in which the neo-is carried out in the presence of proline and / or a viridogrisein source is mixed with viridogrisein and grise-natural containing proline. viridine. 5. Use, as a fodder supplement, of a composition. 8. Use according to claim 7, in which the neo-sition comprising as active ingredient neoviridogri-viridogrisein is neoviridogrisein II.