CA1088441A - Antibiotics, neoviridogriseins, and their method of production - Google Patents

Abstract

ABSTRACT
A group of novel depsipeptide antibiotics, neo-viridogriseins I, II and III are produced by fermentation of Streptomyces sp. P8648 (FERM-P3562; ATCC 31289). The antibiotics are highly active against Gram-positive bac-teria and mycoplasmas.

Description

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DEPSIPEPTIDE ANTIRIOTICS NEO'VIRIDOGRISEINS
FROM STREPTOMYCES
" '.

This invention relates to new depsipeptide anti-blotics, called as neoviridogriseins I, II and III; to their production and isolation; and to their utilization as therapeutic drugs and animal feed additive~

The so-called mikamycin-vernamycin (or ~trepto-; gramin) family antibiokics are classified into two major groups. One major group (Group A according to D. Va2quez;
pages 521-534, ANTIBIOTICS III, Mechanism of action ~f anti-microbial and antitumor agents, Springer-Verlag Berlin Heidelberg New York 1975) is a macrocyclic lactone compound, and includes griseoviridin, ostreogrycin A(=mikamycin A=virginiamycin M=staphylomycin M=pristinamycin IIA=verna-mycin A=streptogramin A=synergistin A-1-Compound PA-114A1-=Compound E-129 Factor A, etc.), ostreogrycin G (-virginia-mycin MII=staphylomycin ~II=pristinamycin IIB=dihydrostreo-grycin A=Compound E-129 Factor B=Compound R.P.-13920, etc.), and Compounds A-2315 A, B and C. Though bacteriostatic, this group of antibiotics are active against Gram-positive ~ -, 1: , ~ .

' 18,374-F -l--~1i8~4g~

bacteria and mycoplasmas. Griseoviridin, which is one of the fermentation products in this invention, is known to be produced together with viridogrisein by streptomycetes, and the production of griseoviridin is disclosed in U.S.
Patents 3,023,204 and 3,174,902.

The other major group (Group B according to - D. Vazquez) is a macrocylic depsipeptide compound and divided into two subgroups, that is, the viridogrisein subgroup and the vernamycin B subgroup. The anti-10 biotics in this major group are mainly effective in `
suppressing the growth of Gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis. The ~-vernamycin B subgroup contains 12 homologues. Because the synonym relationships among the published names o~
antibiotics in this group are so complicated, it is practically impossible to list up all the compound names o this subgroup. Some representative, and scientifically important, compounds of this subgroup are as follows:

1. Vernamycin B =ostreogrycin B=pristinamycin IA=mikamycin B=streptogramin ~=synergistin B-1=compound E-129 Factor Z=compound PA114B1

2. Vernamycin B =ostreogrycin B1=pristinamycin IC=compound E-129 Factor Zl ,

3. Vernamycin B =ostreogrycin B2=pristinamycin 25 IB-compound E-129 Factor Z2=compound R.P.-13919

4. Ostreogrycin B3 =compound E-129 Factor Z3 ', ' ':

5~ Patricin A

.
, . - .
~, 18,374-F -2 , 1~8~}4~

6. Patricin B

7. Vernamycin B~ ~-

8. Vernamycin C=doricin

9. Virginiamycin (Staphylomycin)S

10. Virginiamycin (Staphylornycin)S2

11. Virginiamycin (Staphylo~ycin)S3

12. Virginiamycin (Staphylomycin)S4 (or ';l) The compounds of this subgroup hav~ the common feature that they are composed of seven constituents sharing in the same four constituents of 3-hydroxypico-linic acid, L-threonine, L-proline and L-phenylglycine.

On the other hand, in the viridogrisein sub-group there has been known only one compound, virido-grisein, and the identity with viridogrisein of etamycin, compound K-179 and compound F-1370A is well established.
In contrast to the above vernamycin B subgroup, virido-grisein is composed of 8 constituents, that is, 3-hydroxy-picolinic acid, L-threonine, D-leucine, 4-hydroxy-D-~proline, sarcosine, ~,N-dimethyl-L-leucine, L-alanine and L-phenylsarcosine. As apparent in the followincJ
detailed description of the invention, the neovirido-grisein antibiotics of this inven~ion belong to this viridogrisein subgroup, and one of the compounds, neo viridogrisein IV, has been identified as viridogrisein.
Preparation of viridogrisein is disclosed in U.S.
Patent 3,023,204. -. .

18,374-F -3-L `

~L13~

The synergism between Group A and Group B
is well known on various microorganisms, and profitably utilized to potentiate the therapeutic efficiency of -pharmaceutical drugs containing the a~tibiotic of this 5 family as active components. This is true of neovirido- ~1 griseins, too.

This invention provides a process for preparing depsipeptide antibiotic neoviridogrisein I of the formula :' . ' ' CH~ /CH3 :~
~ OH lH2 1H2 1H2 CO-NH-ICH-CO-NH- H-CO- CH-fO
1H-CH3 ~ 3 1 fH2 co fo CH- ~ _CO_CH_NH_CO_fH - N CH3 CH3 CIH2 fH-CH3 ~ ~ H3 CH-CH3 depsipeptide antibiotic neoviridogrisein II of the formula lg,374-F -4-B

. ` . ` . ~ . .. ` . ` .. ~ . .. ... .. . . ... .. ., `.. ; . ~. .. ` .. . .. . ` ` .. . . " .. . ` .. " -....... i ... ` .. ...`..... ........ ; ....... ` ......... ...~.. ,.~.. ~ ................... .....
; ` ,.. ...... ` . ;. .. . ......... ` ... . .. `...... ... .. ~. "` .` `.`"................. .
, ..... ` ` .... ~ .;. " ` .` .`:, . ~ , . .` ` `. . .. ,,`. . .

1(~8~441 CH~ CH3 ~ OH ~ C/H ~fH

CO-NH-fH-CO-NH-CH-CO-N CH-fO -fH-CH3 1~CH3 :
I H~
fO ICO
CH-IH-CO-lCH-NH-CO-fH - N-CH3 :
~ CH3 ~H3 fH-CH3 ~-CH3 and dep~ipeptide antibiotic neoviridogri~ein III of the . formula CH~ ~CH3 OIH
OH IH

CO-NH-fH-CO-NH-CH-CO-N CH-fO
~H-CH3 ~-CH3 ~H2 '' co 1 ~ ~
. H- ~ -CO-fH-NH-CO-fH - N-CH3 : ~ ~ CH3 1~-CH3 ' ' ' '~:

I ~ 18,374-F ~5-10~
-viridogrisein and griseoviridin which comprises culti-vating Streptomyces sp. P8648 (FERM P3562; ATCC
31289) under aerobic conditions, at a t;emperature com-prised between 18 and 37C, in an aqueous nutrient medium containing assimilable sources of carbon, assim-ilable sources of nitrogen, and essential mineral salts, at a pH comprised between 6 and 9, until a substantial antibiotic activity is imparted to the medium, and recover-ing the fermentation product from the medium.

The invention also provides a method for the selective production of the desired neoviridogrisein component(s) by supplementing some pertinent amino acid(s~, more particularly, proline to incréase neoviridogriseins I and II, and alpha-amino-n-butyric acid to increase neo-viridogriseins I and III, during or before fermer,tation.

Neoviridogriseins I, II and III of the present invention are valuable antibiotics which are highly active against various pathogenic microorganisms including Gram--positive bacteria and mycoplasma, and, accordingly, find utility in human and veterinary medicine. More practically, the antibiotics of this invention can be used as thera-peutic drugs for the treatment of infectious diseases caused by Gram-positive bacteria and mycoplasma, for example, StaPhylococcus aureus, Stxeptococcus pvogenes, Diplococcus pneumoniae, MYcoplasma gallisepticum, Myco-plasma fermentans, Mycoplasma agalactiae, etc.

In this invention, the unmodified term "neo-viridogrisein" i~ used to generically designate depsi-peptide antibiotics called neoviridogriseins; that is, 18,374-F ~6-'~; .
'`~ ~'1 ' _7_ it means not only a member in the group of neovirido-griseins I, II and III and viridogrisein (=neovirido-grisein IV), but also a mixture of any two or more members selected from the said group.

The new depsipeptide antibiotics of the inven-tion are produced by a new strain of Streptomyces called Streptomyces sp. P8648 (FERM-P 3562; ATCC 31289) together with viridogrisein and griseoviridin. This microorganism has been isolated from a soil sample collected near the Kuzuryu dam in Fukui-ken.

Generally speaking, the microorganism of this invention elongates colorless, short aerial mycelia ~rom well-branched (single bxanching) substrate mycelia.
Spore chains with smooth sur~ace are formed in a loose loop on top of aerial mycelia. Neither whirl nor ascos-pore is observed. The cultural characteristic~ o~ this microorganis on various agar media are described as follows-(1) Sucrose-nitrate agar 20 Growth : Poor Aerial mycelium : Thin, white aerial mycelia occasionally formed Reverse : Colorless to grayish white Soluble pigment : None (2) Glucose-asparagine agar Growth : Abundant Aerial mycelium ~ Little or none. When formed, white Reverse : Pale yellowish white to light yellow Soluble pigment : None ., ' " '. ' .

18,374-F ~7-. ~ ' .
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(3) Glycerin-asparagine agar Growth : Moderate Aerial mycelium : Little ox none. When formed, white 5 Reverse . Pale yellow to grayish yelIow Soluble pigment : None (4) Yeast extract-malt extract agar Growth : ~bundant ..
10 Aerial mycelium : ~White to white with grayish tinge Reverse ~ Light yellow, later turning to brownish gray 15 Soluble pigment : None or rarely slight brown (5) Starch agar Growth : Moderate Aerial mycelium s None or little. When formed, white Reverse : Pale yellow with light grayish tan in the center of colonies Soluble pigment : None 25 Starch hydrolysis : Poor (6) Tyrosine agar Growth : Moderate Aerial mycelium : None or a few spats of white aerial myceliul occasionally observed Reverse : Grayi.sh yellow to light .:
yellowish tan Soluble pigment : Initlally pale purple to light reddish brown, .-lO days later turning to -pale brown. Little melanoid pigment formed :
:: ;.
.~ .

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18,374-F 5 8-.. ..

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1(~8~41.

(7) Nutrient agar Growth : Good Aerial mycelium : Thin, white Reverse : Pale yellow ~.
5 Soluble pigment : None (8) Oatmeal agar Growth : Good Aerial mycelium : White to grayish white - Reverse : Grayish yellow to ~ight reddish brown with grayish Soluble pigment None The optimum growth temperature for the micro-organism of this invention is in the range of 25-35C.
Though the growth is very poor, the microbe can grow even at a temperature beyond the said temperature range like 10C or 45C. But, it cannot grow at a temperature o~ 52C.

This actinomycete liquefies gelatin in glucose--peptone-gelatin medium; weakly. hydrolyzes starch in starch-inorganic salts agar; and peptonizes skimmed milk .
without coagulation. ~ -Production of melanoid pigment is occasionally observed in tyrosine agar, but not in peptone-yeast 25 extract-iron agar and tyrosine-yeast extract broth. ...
Carbon source assimilation pattern of this microorganism is as..follows (in Pridham-Gottlieb's medium): -. ' ~.
".

1~,374-F -9-~B
;. .. . .. .. ...... .... ... . ... ...
., . . .. , . ., ... , .. , . . ... . , .. ., . " .. , .. , . . . .... ~ .. .... . . . . .. ..

. ~ . . .. ...... .. . . .

lQ~

--10-- ,, , Positive : D-Xylose, D-glucose, D-fructose L-rhamnose, D-mannitol Slightly positive : Sucrose 5 Negative : L-Arabinose, i-inositol, raffinose In relation to the production of the known peptide and non-peptide macrolide antibiotics like mikamycins A and B, virginiamycins, ostreogrycins, etamycin, vernamycins, viridogrisein, griseoviridin and pristinamycins, the following microorganisms should be compared with Streptomyces sp. P8648:
Streptomyces qriseus NRRL 2426 griseoviridus NRRL 2427 sp., etamycin producer con~anensis ostreogriseus mitakaensis ;:
loidensis ' The available information on the cul-tural and physiological characteristics of the said microorganisms shows clear differences between the streptomycete claimed in this invention and the above-mentioned ones. Fo:r .
éxample, Streptomyces griseus NRRL 2426 differs in that ~ 25 it belongs to the Section Rectiflexibiles with straight ; . or slightly wavy spore chains while the microorganism :~
of this invention is included in the Section Spirales; :~
that the former produces gray to yellowish gray aerial ..
mycelia on yèast:extract-malt extract agar while the latter produces white to grayish white aerial mycelia;
and that the former utilizes L-arabinose while the latter does not. Streptomyces ~., etamycin producer which has . .
:

18:j374-F -10-~ !
L ~-~

1~8~L41 been specified in Antibiotics annual 1954-1955, pages 728-732, can be differentiated from the microorganism of this invention in the assimilation pattern of carbon sources and the cultural characteristics on Czapek agar, glucose-asparagine agar and nutrient agar.

Streptomyces conqanensis s]hows clear differences in the morphological characteristics of spores. Among the above-listed microorganisms, Streptomyces qriseovlridus NRRL 2427 looks most similar to the streptomycetes of this invention. The results of the taxonomical comparison between the said two type cultures are summarized in the following table:

streptomYces griseoviridus Stre~tomvces NRFE~E~---- sp~. P 86~8 Pale orangish yellow to White to grayish yellowish pink with gray white aerial mycel-Color of tinge on yeast extract- ium poorly formed aerial -malt extract agar, oat- or most ISP media.
20 mycelium meal agar, starch agar White aerial mycel-and glycerin-asparagine ium abundantly agar. formed on yeast extract-malt extract agar.
; 25 Grayish yellow to oliv- Pale yellow or ish brown or blackish light yellow to - brown on yeast extract- grayish brown on Color or -malt extract agar, oat- most ISP media.
substrate meal agar, starch agar 30 mycelium and glycerin-asparagine agar.
No melanoid pigment No melanoid pig-formed. No other formed. No other Soluble pigment usually pigment usuaIly 35 pigment observed, but rarely observed, but yellow pigment poorly rarely brown pig-formed. ment slightly formed.

18~,374-E

!

4~

Utilization L-arabinone +++ L-arabinose of carbon D-fructose i D-fructose +++
sources sucrose - sucrose .
As apparent from the preceedi.ng table, clear differences have been confirmed between Streptomyces griseoviridus NRRL 2427 and the streptomycete of this invention in the morphological and cult;ural characteris-tics and the utilization pattern of carbon sources. In addition, when both are fermented under identical con-10 ditions, the microorganism of this invention can produce ; i neoviridogriseins I, II and III as well as viridogrisein (neoviridogrisein IV) and griseoviridin, while the type .:
culture of Streptomyces qriseoviridus NRRL 2427 produces only viridogrisein and griseoviridin, but not neovirido-griseins I, II and III.

From the above described results, the micro-organism employed in this invention has been concluded to be a new species of Streptomyces and named Streptomyces :~
sp. P8648. The type culture of this microorganism has :
been deposited with Fermentation Research Institute, Agency of Industrial Science and Technology, with the deposition number of FERM-P No. 3562. It has also been deposited with the American Type Culture Collection where it was assigned the code number ATCC 31289.

It may be well understood to those skilled in the art that this invention is not limited to the particu-lar microorganism which has been specified above and filed as FERM-P No. 3562 to Fermentation Research Institute, but includes all those spontaneous and arti- -icial mutants derived from the said microorganisim which are capable of producing the new antibiotics, neovirido-grisein I, II and III.

18,374-F -12-~r ~ l ..... l . . . . ~

8~9~1

-13-Fundamentally speaking, the new antibiotics of this invention are produced by inoculating and propagating Streptomyces sp. P8648 in a suitable mledium under aerobic conditions at a temperature in the range of 18-37C for a period of 2-14 days whereby the accwmulated antibiotics are recovered from the fermentation broth and purified by conventional methods.

The preferred embodiments of the processes according to this invention will be illustrated in more details in the following:

For the fermentation of the microorganism of this invention, employable are all kinds of media which have been well known as media for Streptomycetes. For example, preferable carbon sources o the medium are ;lS glucose, glycerin, starch, de~trin, oatmeal, molasses, fat and oil and the like. As a suitable nitrogen source for the purposes of this invention are listed soybea~
meal, cotton seed meal, meat extract, peptone, dry yeast, corn steep liguor, yeast extract, casein and its hydroly-sate and inorganic salts such as ammonium sulfate and ammonium nitrate. If desired, minor growth factors may be added to the medium. They include vitamins, aminoacids, organic and inor~anic salts such as calcium carbonate, ~`
~sodium chloride, potassium chloride, sodium phosphate, potassium phosphate and magnesium sulfate.
.
The new antibiotics of this invention can be produced by fermentation in traditional vessels such as a shake flask, a jar fermenter and a tank fermenter, but, from the economical viewpoint, the submerged culti-vation under forced aeration will be most ad~antageousin an industrial scale. The fermentation is desirably .

-I8,374-F -13-.:
1~ ' ' ~L3 "

8~441

14-carried out under aerobic conditions at a tempera~ure in the range of 25-35C. When a shake flask or a tank --fermenter is employed, the production of neovirido-griseins reaches a peak usually in 2-10 days. The pH
value during fermentation may change beyond the phy-siological range, depending on the kind of medium employed. It is more desirable to adjust and maintain the pH value during fermentation in the range of 6-9.
Usually the pH of the medium is adjusted to 6.5-8.5 :
before inoculation.

As described above, the microorganism of this invention produces a mixture of neoviridogriseins I, II
III and IV and griseoviridin. It is possible to change the composition of neoviridogriseins in the fermentation broth by a suitable combination o carbon and nitrogen sources in the medium without the specific addition o~ ;
either a free aminoacid or an organic acid. But it will be more profitable from the viewpoint of industrial production to adjust the content of neoviridogriseins I, II and/or III in the fermentation broth by adding to the medium the pertinent constituent amino acid(s) in free form during fermentation, depending on the circum-stances and the demand. It goes without saying that the composition of neoviridogriseins in the fermentation broth may be varied appropriately by selecting sponta-neous or artificial mutants derived from the type culture of the streptomycete of this invention; by adjusting fermentation conditions like temperature, pH and aeration;
and/or by adding to the medium physiologically active agen~s such as enzyme inhibitors and promoters. One of the preferred embodiments of methods for se~ective produc-tion of particular neoviridogrisein components consists of feeding the pertinent constituent amino acid(s), 18,~74-F -14-~1 '' .'.

3l ` : ~

15-alpha-amino-n-butyric acid and/or proline during fermen-tation. More particularly, the addition of proline during ~ermentation increases the percentage of neoviridogriseins I and II in the total amount of neovirido~riseins I, II, III and IV, which are more potent in antimicrobial activi-ties then neoviridogriseins III and IVo This is also true of alpha-amino-n-butyric acid. The amount of neovirido-griseins I and III can be selectively increased by feed-ing alpha-amino-n-butryic acid to the rnedium before inocu-lation or during fermentation. As the microorganismof this invention produces protease durin~ growth, pro-teinaceus material which contains the said pertinent constituent amino acid(s) may be added instead of the free amino acid(s). For e~ample, proline can be sub-stituted py casein or corresponding hydrolysates fromacid hydrolysis such as, ~or instance, casamino acids.

The new antibiotics, neovirido~riseins I, II and III an~ viridogrisein can be isolated from the fermentation broth by conventional methods based on their physico-chemical properties as depsipeptide antibiotics. If necessary, neoviridogriseins may be recovered from the fermentation broth together with griseoviridin as a neoviridogriseins-griseoviridin mixture. When they are prepared for feed additive or veterinary drug use, a crude mixture of neovirido-griseins and griseoviridin will be more advantageous ~rom the economical viewpoint.

Neoviridogriseins and griseoviridin in the fermentation broth can be extracted with a water-immis-cible organic solvent. For example, ethyl acetate, butylacetate, n-butanol, methylene chloride, chloroform, and the like are suitable for extraction of neoviridogriseins ' 18,374-F -15-;
`' ;~ ' ' . ~ ... . . ~ . , . . . .. ., . . . . - . .. .

-16-and griseoviridin at one time. When it is more desirable to selectively extract neoviridogriseins without griseo-viridin, preferred organic solvents are methyl isobutyl ketone, benzene, toluene and other aromatic hydrocarbons.
As the mycelium substantially contains no neovirido-griseins and the extractable lipid in cells may often interfere with subse~uent purification steps, it is more profitable to extract the said antibiotics with an organic solvent from the filtered broth or centri-~0 fuged broth together with the water wash.

The solvent extract of neoviridogriseinsand/or griseoviridin can be further isolated and puri-fied in a number of different ways. For example, adsorp-tion and elution processes with active carbon, Amberlite~
XAD-4 and 7 (Rohm & Haas Co.), ion exchange resins such as Amberlite~ IR-120 (Rohm and Haas Co.) and Dowex~
50W-X2 (The Dow Chemical Company); gel filtra~ion with Sephadex~ LH-20 (Pharmacia Fine Chemicals AB) and its equi~alents; adsorption chromatography on alumina and silica gel, etc. can be conveniently combined for iso-lation and purification. In addition, countercurrent ; distribu1:ion with a suitable solvent system may work well for the said purposes.

Neoviridogriseins I, II and III as well as viridogrisein are amorphous white solids and are soluble in methanol, ethanol, n-propanol, isopropanol, 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; hardly ' 18,374-F ~16-, . 3~
' `':- - ~
.... '' ' ' '~

:, - ': '. , . ' ., .. ~ . , ' , , . ' ', ' ' ' ,'' '' " ' . ' ' ', ' ' ' ' ` ' ' ',

-17- -soluble in ~7ater and ethyl ether; and practically insoluble in petroleum ether and hexane. They are stable in aqueous solution for at least one month at a temperature in the range of 25-37C; and for 30 minutes at 60C, at a pH of 2-9. The melting points of the antibiotics were determined in a Kofler apparatus and the following results were obtained:
Neoviridogrisein I : not determined II : 93C
III : 115C
Viridogrisein ~ 140C

In the following decision of characterizing data for these compounds, reference will be made to Figures 1 to 12 wherein:
1~ Figuxe 1 is the W spectrum of neoviridogrisein (NVG I) in methanol Figure 2 is the W spectrum of neoviridogrisein II
(NVG II) in methanol Figure 3 is the W spectrum of neoviridogrisein III
(NVG III) in methanol Figure 4 is the W spectrum of neoviridogrisein IV
(NVG IV, VG) in methanol Figure 5 is the W spectrum of neoviridogrisein I -(NVG I) in methanol/NaOH O.lN
Figure 6 is the W spectrum of neoviridogrisein II
(NVG II) in methanol/NaOH 0.1N
Figure 7 is the W spectrum of neoviridogrisein III
(NVG III) in methanol/NaOH 0.1N
Figure 8 is the W spectrum of neoviridogrisein IV
(NVG IV, VG)~in methanol/NaOH 0.1N
Figure 9 is the I.R. spectrum of neoviridogrisein I
(NVG I)-KBr tablet

18,374-F 17-. .
~e :

4~ :
ol8-Figure 10 is the I.R. spectrum of neoviridogrisein II
(NVG II)-KBr tablet ;
Figure 11 is the I.R. spectrum of neov:iridogrisein III
(NVG III)-KBr tablet Figure 12 is the I.R. spectrum of neov:iridogrisein IV
(NVG IV, VG)-KBr tablet The ultraviolet absorption spectra of neovirido-griseins I, II, III and viridogriseins are reproduced in Figures 1-8 wherein Figures 1-4 show the U.V. spectrum of neoviridogrisein I, II, III and viridogrisein re.spec-tively when registered in methanol and Figures 5-8 the U.V. spectrum o the same substances in the same order registered in 0.1N NaOH-methanol. The E1/Cm value of neoviridogrisein at their maxima is as follows:
In neutral methanol, Neoviridogrisein I : 305 nm(65) II : 305 nm(88) III : 305 nm(90) Viridogrisein : 305 nm(90) In 0.1 N NaOH-methanol, Neoviridogrisein I : 340 nm(70) II : 340 nm(84) III : 340 nm(96) Viridogrisein : 340 nm(96) The infrared absorption spectra of neovirido-griseins I, II, III and viridogrisein, in a KBr tablet are shown in Figures 9-12 respectively. The character-istic peaks and shoulders are observed at the following wave numbers:

':
18,~374-F -18-: . .

~ , . . . : -..... .

= 19- , Neoviridogrisein I (KBr tablet) 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. l Neoviridogrisein II (KBr tablet) 3320, 2950, 2920, 2820, 2800, 1745, 1670(sh.), 1630, 1600(sh.), 1575, 1515, 1460(sh.), 1445, 1405, 1390, 1365, 1330(sh.), 1295, 1275, 1240, 1200, 1195, 1160, 1130, 1095 and 1065 cm. 1 10 Neoviridogrisein III (KBr tablet) . .
3335, 2960, 2940, 2870, 1750, 1670(sh.), 1660(sh.), 1635, 1590(sh.); 1515, 1450, 1405, 1390(sh.), 1370, 1340(sh.), 1300, 1245, 1200, 1160(sh.), 1130, llO0 and 1065 cm. 1 In the thin layer chromatography systems indicated below, neoviridogriseins I, II and III
viridogrisein and griseoviridin have the ~ollowing Rf values: :
(1) TLC plate: Pre-coated TLC plate Silica -20 Gel~ 60F-254, E. Merck, :~
Darmstadt ..
Solvent Benzene:methanol = 5:1 Neoviridogrisein IRf-0.66 II0.62 IIIRf=0.59 Viridogrisein 0.55 Griseoviridin 0.20 (2) TLC plate: Same as ~1) Solvent: Chloroform:methanol=30:1 ~
30 Neoviridogrisein IRf=0.39 --II0.32 III0.19 ..

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Viridogrisein 0.18Griseoviridin 0.02 For the analysis of the constituent amino acids, each neoviridogrisein component was hydrolyzPd in 6N HC1 overnight at llO~C and the resulting hydroly-sate was evaporated to dryness. After even a trace of hydrochloric acid was removed by repeated evaporations,: -amino acids in the hydrolysate were determined by thin layar chromatography (Eastman Chromagram~ sheet 13254 cellulose with fluorescent indicator, Eastman Kodak Co.;
solvent system:n-butanol/acetic acid/water=4/1~1), high voltage paper electrophoresis (Toyo Filter paper No. 51A, .
Toyo Roshi Kaisha, Ltd.; buffer system:ormic acid/acetic acid/water=2S/75/900, pH-1.8; 60V/cm at 0C for 30 minutes) lS and auto-amino acid analysis (Hitachi auto-amino acid analyser ~LA-5, Hitachi, Ltd.). The presence of the following amino acids was confirmed:
Neoviridogrisein I : threonine leucine proline Neoviridogrisein I : alpha-amino-n-butyric acid sarcosine phenylsarcosine ~,N-dimethylleucine 25 Neoviridogrisein II : threonine leucine proline alanine ...
: - sarcosine phenylsarcosine -~,N-dimethylleucine 1&,374-F 20-,- - . .- , , ~

',: : ' ' l~B~49tl Neoviridogrisein III : threonine leucine hydroxyproline alpha-amino-n-butryic acid sarcosine phenylsarcosine ~,N-dimethylleucine Viridogrisein : threonine ~-leucine hydroxyproline alanine sarcosine phenylsarcosine ~,N-dimethylleucine .
15The presence of 3-hydroxy-pycolinic acid was confirmed by mass spectrometry and thin layer chromato-graphy as follows:

An authentic sample of viridogrisein and each ; of neoviridogriseins I, II, III and IV were hydrolyzed ;20 overnight in 6N HCl at 110C to give the hydrolysates as described above. Each hydrolysate showed only one W -~absorbing spot with the same Rf value ~mder the indicated conditions.
(1) Silica gel TCL ;
TLC plate : Pre-coated TLC plate Silica Gel~
60 F-254, E. Merck, Darmstadt Solvent : Chloroform:methanol=2:1 Rf : 0.46 (2) Cellulose TLC
TLC plate ~: Eastman Chromagram~ sheet 13254 cellu~
Iose with fluorescent indicator, Eastman Kodak Co.
:'.

; 18,374-F - -21-1.
B

. ~ . . . . .. . .... .. . . ~, ... . . .

Solvent : n-Butanol:acetic acid:water =4:1:1 Rf : 0.62 The molecular weight of these ~ntibiotics was determined by direct insertion into a mass spectrometer.
Neoviridogrisein I : 876 II : 862 III : 892 Viridogrisein : 878 For the study on the chemical structure of neo-viridogriseins I, II and III, these three new antibioticsand viridogrisein were hydrolyzed overnight in 0.1 N
NaOH at room temperature and then methylated with diazo-methane before mass spectrometry according to the method o Compernolle et al. (Organic Mass Spectromekry, Vol. 6, pages 151-166, 1972). The structure of neoviridogriseins I, II and III is concluded from the available information described above to be as follows:

18,374-F ~22-. .
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Neoviridogrisein I .

CH~ /CH3 .;
O~ fH /CH2 CO-NH-~H-CO-NH-CH-CO-N CH-~O
-CH3 ~ 3 I f ~.
CO CO
CH-IH-CO-CH-NH-CO-ClH - N-CH3 CH3 CH2 fH-CH3 ~H3 CH-CH3 CH3 ;
` ':' Neoviridogrisein I~

C~ f H3 H fH /

O-NH-fH-CO-NH- H-CO-N - CH-fO
fH-CH3 ~-CH3 ICH~ ' .', .
CO . ~0 , .
CH-NH-CO-fH-NH-CO-f~--~ ~-CH3 ... .

~ IH3 CH3 ICH-C}33 . , CH3 ~ , : ~ 13,374-F ~23-, , , , ~ , - ;, , . . .; "1 ,. . . ,, ... ., .- , . , .. - .. .

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~ -24-.

Neoviridogrisein III

CH~ / H3 ~H
~ OH fH C ~ :

N ~ 8H2 fH2 ~H2 CO-NH-fH-CO-NH- H-CO-N H-CO
fH-CH3 1-CH3 f 1~2 fo fo fH- ~ -CO-IH_NH_CO_lH---N_CH3 H3 CH2 ~H CH3 1H3 ~H-CH3 As can be seen rom the preceeding structures of neoviridogrisein~ I, II and III, the antibiotics of the invention are a group of new depsipeptide antibiotics homologous to viridogrisein. The identity of neovirido-grisein IV with viridogrisein and etamycin was confirmed by mass spectrometry, thin layer chromatography, W and IR spectromekry and amino acid analysis, using authentic ~samples o viridogrisein and etamycin.

Neoviridogrisein I, II and III have a broad antimicrobial spectrum against bacteria, mycoplasmas, actinomycetes and rickettsiae in laboratory test. More exactly, they display a remarkable activity in vitro against the usual and resistant strains of Staphylococcus aureus, as well as strains of Streptococcus pyogenes, Diplococcus pneumoniae, Sarcina lutea, Bacillus subtills, . .
~ 18,37~-F -24-, ~ ..
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4~1 , ~25-mycoplasma gallisepticum, MYcoplasma pulmonis, Mycoplasma fermentans and Mycoplasma a~alactiae. The minimal inhibi- ..
tory concentrations of the new depsipeptide antibiotics of this invention were determined separately and together with viridogrisein and griseoviridin on various micro-organisms by the tube dilution method. The results are shown in the following tables:

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~.~3&1~41 As shown in the table of MIC values listed above, neoviridogrisein II is more active than neovirido-grisein IV, that is, viridogrisein. This MIC experiment was based on the two-fold dilutions. To differentiate neoviridogrisein II and viridogrisein in their antibiotic activity, the MIC determination was repeated with a far lower dilution rate. The subsequent table indicates that neoviridogrisein II is 2-3 times more active than ~iridogrisein.

Table 2: Comparison of neoviridogrisein II
with viridogrisein ~ ' Microorganism MIC(mcq/ml) NV*II VG**
Staphylococcus aureus 209 P 0.078 0.133 (EM, CM, SM, PC, TC)r 0.125 0.334 (TC, CP, PC) 0.094 0.334 Bx~1633(PC)r 0.125 0.267 Ru~sell(PC)r 0.125 0.267 Smith 0.125 0.267 Medium: brain heart infusion broth *NV = neoviridogrisein **VG = viridogrisein When the minimal inhibitory concentrations of '~' neoviridogriseins I, II and III were tested in the pres-ence of griseoviridin, the synergism was observed between the neoviridogrisein member and griseoviridin, as is the case of viridogrisein and griseoviridin. Therefore the synergistic phenomenon of the new antibiotics of this ''~
invention with griseoviridin was studied in more detail 30 with the,varied ratios of the neoviridogriseins mixture '' to griseoviridin. The obtained results are shown in the following table: -. .

18,374-F o28-.
I`B
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~)B~44~.

Table 3: Synergism of neoviridogriseins mixture with griseoviridin Neoviridogriseins : Griseoviridin MIC*(mcg/ml~
mixture 100 : 0 0.313 : 10 0.156 .
e 20 0.125 : 30 0.125 : 40 0.094 : 50 0.07~
: 60 0.094 : 7a 0.125 : 80 0.250 : 90 0.250 1~5 0 : 100 0.250 *Test microorganism : Sarcina lutea Tube dilution method with brain heart in~usion broth.

From the above table it results that the synergistic action of the neoviridogriseins mixture with griseoviridin was most significant at the ratio of 50:50; that is, a 1:1 mixture of neoviridogriseins and griseoviridin is 3-4 times more active than neo- :
viridogriseins or griseoviridin only.

The following Table 4 reports the high ln vitro activity of neoviridogrisein II against various Mycoplasma strains as well as the superior synergism shown by a mixture neoviridogrisein II-griseoviridin in comparison with a mixture viridogrisein-griseo-viridin. The MIC were determined by the dilution method.

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Table 4 Microorqanism Medium NV II VG GV NV II+GV* VG+GV*
Mycoplasma gallisepticum 5 KP13 (1) O.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 fermentans (2) <0.05 ~0.05 <0.05 <0.05 <0.05 10 Mycoplasma agalactiae PG 2 (2) 0.39 0.78 3.13 <0.05 0.39 Medium (1): PPLO enrichment broth (Eiken, Japan) (2): PPLO broth (Chanock's medium; Difco) *Mixing ratio: 50/50 NV II, VG, GV: see Table 1 As illustrated above, the novel neovirido-griseins I-III of the present invention display a remarkable activity against gram-positive backeria and ;
mycoplasma strains both alone or ln various admixtures with viridogrisein (neoviridogrisein IV) and griseoviridin.
It has been found that the mutual weight proportions of neoviridogriseins I-III, viridogrisein (neoviridogrisein IV) and griseoviridin in a mixture may vary wi~hin very wide limits, but the surprising antimicrobial and anti--mycoplasma activities are still retained. As a repre-sentative though not limitative example, a mixture having the following percent composition (by weight):
Neoviridogrisein II 27%
Neoviridogrisein IV (viridogrisein 23%
Griseoviridin 50%
was tested in vitro against StrePtococcus mutans, a microorganism associated with dental caries and peri-dontal diseases. The test was carried out in Todd 18,374-F -30 '1 ' ~:. ., . .. , . , ~ . :; . ., .. ; . ..
: ,, . : ~ . " . , ,. :: . . : .

4ill ~31-Hewitt Broth (Difco) with 0.5% TC lactalbumin hydroly-sate (Difco). Initial organism count approximately 3 x 10 organisms per ml. Culture tubes were incubated anaerobically at 37C for 48 hours. The minimum inhibi-tory concentration (MIC) and minimum bactericidal con-centration were both found at a neoviridogrisein con-centration of 1.0 part per million (ppm).

In another representative though not limi-tative example, the same mixture was tested in vitro against Treponema hyodysenteriae, a swine dysentery organism. The mixture was tested as dilutions in blood agar at concentrations of 100, 50, 10, 5, 1, 0.5 and -:
0.1 ppm. Plates were inoculated with a swab and incu-bated 4 days at 42C. The MIC was determined to be 0.5 ppm. In a further representative though not limi-tative example, a mixture having the following percent composition (by weight):
Neoviridogrisein II 25%
Neoviridogrisein IV (viridogrisein~ 25%
20 Griseoviridin 50% ;
was tested in vitro against several Mycoplasma strains.

The minimum inhibitory concentrations (MIC) were found to be as follows:
~train MIC
Mycoplasma gallisepticum KP 13 0.07 Mycoplasma pulmonis 0.20 Mycoplasma fermentans 0.05 Mycoplasma agalactiae 0.05 These types of neoviridogriseins-griseo-viridin mixtures are also useful in the treatment of ., .
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animals suffering from infectious diseases caused bythe above pathogenic bacteria.

As mikamycins A and ~ have been known to be ver~ effective as feed additive, the new antibiotic compounds of this invention were subjected to the animal feed test. Neoviridogriseins as mixture were added to the chicken feed at a rate of 2-20 ppm and fed to male chickens for 10 weeks. ~ompared with the control group of chicken which received the same feed without neovirido-griseins, the neoviridogrisein-fed chickens were superior in the increase rate of body weight and the feed effi-cency. Thus, neoviridogriseins of this invention have been proved very useful as feed additive.

A1BO the compositions containing one or more o~ the neoviridogriseins I-III, optionally in admixture with viridogrisein (neoviridogrisein IV) and griseo-viridin, wherein the mutual weight proprotions of the components may vary within very wide limits, proved to be very useful as feed additive.

As a representative though not limitative example, a mixture having the following percent com-position (by weight):
Neoviridogrisein II 27%
Neoviridogrisein IV (viridogrisein) 23%
Griseoviridin 50%
was tested in vivo by incorporation in the diet of growing chicks. One-day old cockerels, 15 birds per treatment were used. The chicks were maintained for 11 days on a feed containing about 55% rye grain, supplemented with vitamins, minerals, fat and protein sources. The high ~18,374-F -32-! , .
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content of rye grain normally gives poor to moderate growth, and this diet is a standard one used for screening growth promoters and antibiotic feed additives.
Penicillin (100 ppm) was used as a posltive control. The data is tabulated below. Feed/Gain ratio is grams feed consumed per gram of weight gained. Body weight ratio is the ratio of chick body weight at the end of the 11 day study to the initial body weight. The last column is the average gain per bird (in grams).

10 TREATMENT DIET FEED BODY (GMS) CONC GAIN WEIGHT AVG
PPM RATIO RATIO B.W. GAIN
Rye Control --- 1.334 4.696 156.47 Penicillin 100 1.197 5.012 163.20 15 Tested mixture 100 1.256 4.789 161.66 Tested mixture 50 1.283 4.892 163.47 Tested 20 mixture 25 1.204 5.226 174.67 ".

In the following, this invention will be further illus-trated by preferred examples, but should not be construed as limited by those examples.

EXAMPL~ 1 .
Seed culture medium consisting of soybean meal 0.5%, Pharmamedia (Traders Oil Mill Co.) 0.5%, oatmeal 0.5%, dry yeast 0.5% and beet molasses 0.5% was adjusted to pH 6.5 and distributed in a 50 ml. amount in a 250 Erlenmeyer flask. After autoclaving at 120C for 15 minutes, a loopful amount of Stre~tomyces sp. P8648 on an ISP-2 agar slant was inoculated and the flask incubated at 28C for 48 hours on a rotary shaker.

.
~ 18,374-F 33-',:~) "
, ~ . .. . .

.....

Two milliliters of the said seed culture was transferred into a 500 ml. Erlenmeyer flask contaiIling 100 ml. of `
the following fermentation medium:
Soybean meal 0.5%
5 Peanut meal 0.5%
Oatmeal 0.5% (pH 6.5 prior to autoclaving) Dry yeast Q.5% - -Beet molasses 0.5%
and cultivated at 28C for 96 hours on a rotary shaker 10 at 200 r.p.m. (radius of circle 3.5 cm.). The culture broth was collected from 12 flasks and filtered to give a clear broth filtrate. The obtained cake on the filter was washed with 100 ml. of water. The water washing and the broth filtrate were combined. The antibiotic activity of this solution (pH 8.3~ was determined to be 23.0 mm on a nutrient agar assay plate of Sarcina lutea when the standardized disc assay was carried out with a 8 mm paper disc. Eight hundred milliliters of the said a~ueous solution was twice extracted with 200 ml. each of n-butanol, and the butanol extracts were combined and evaporated to dryness under reduced pressure to yield 90 mg. of crude powder of neovirido-griseins and griseoviridin. This crude powder was mixed with a small amount of silica gel and applied on a silica gel column (Wako-Gel~ C-100, Wako Pure Chemical Industries, Ltd.; (1.5 x 25 cm.). The silica gel column was eluted first with 300 ml. of a benzene--acetone mixture (5:1), and then with a benzene-acetone `
mixture (2:1). Ten gram fractions were collected on an automatic fraction collector. Active fractions of Nos.
25 to 35 were combined and evaporated to dryness to provide 30 mg. of neoviridogrisein mixture (consisted of neoviridogriseins I, II and III and viridogrisein).

~, ~
18,374-F 34-:
~ 7 ~

. . , ~ , . . . . . ., . :..... . ;.:
. - . . . . .....

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.:. ~... : . . , . : .... : . ., .. . - . . : :. .

:.
: ' o In addition, evaporation of active fractions of Nos.
45-54 to dryness gave a crude powder, the antibiotic activity of which corresponded to griseoviridin by TLC.
These two preparations were subjected to thin layer 5 chromatography under the indicated conditions. The antimicrobial activity was detected on a nutrient agar assa~ plate of Sarcina lutea.
T~C plate: Pre-coated TLC plate Silica Gel~ 60 F-254, E. Merck, Darmstadt 10 (1) Solvent: Chloroform:methanol = 20:1 Neoviridogriseins Rf = 0.45 Griseoviridin 0.05 (2) Solvent : Benzene:acetone = l:1 Neoviridogriseins Rf = 0.55 15 Griseoviridin 0.13 Two hundred milliliters of the 48 hour-old culture of StreptomYces sp. P8648 in the same culture medium as in Example 1 was inoculated into a 15 liter 20 stainless steel jar fermenter containing 10 liters of the same seed culture medium as in Example 1 cultivated at 27-28C for 96 hours under forced aeration of 5 liters/
min. of sterile air. The agitation during cultivation was per~ormed at 200 r.p.m. with an impeller, the radius 25 of which is about a fourth of the diameter of the jar fermenter. At the end o fermentation, the mycelia and solids we~e removed by filtration. The obtained broth filtrate was adjusted to pH 6.0 and extracted four times with 2 liters each of ethyl acetate. The ethyl acetate 30 extracts were combined, dried over anhydrous sodium sul-fate and evaporated to dryness under reduced pressure to .

18,374-F 35-t ~ . . . . . ` - - :

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,: '' ' ' ' ~,' ' , . . , ' . . .
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yield 700 mg. of crude neoviridogriseins and griseo-viridin. The recovered crude powder of neovirido-griseins and griseoviridin was dissolved in a small amount of methanol and charged on a Sephadex~ LH-20 column; Pharmacia Fince Chemicals AB; 1~3 x 50 cm.).
Ten milliliter fractions were collected with methanol as an eluting solvent. Neoviridogrise:ins were located in fraction Nos. 22-29. These fractions were collected, concentrated to dryness and further purified by silica gel column chromatography (Silicar~ CC-7 Special;
Mallinckrodt Chemical Works; (1.5 x 20 cm.). The elution was run with a mixture of chloroform and methanol (30:1).
Eight 5 gram fractions from fraction No. 5 to fraction No.
12 were combined and evaporated to dryness under reduced pressure to yield 25 mg. of white powder of neovirido-griseins. The percent composition of neoviridogriseins I, II and III and viridogrisein in this powder was as follows:
Neoviridogrisein I : 15%
Il : 20%
III : 20%
~iridogrisein : 45%
Griseoviridin was found by TLC in fraction Nos. 30-34 of the said Sephadex~ LH-20; Pharmacia Fine Chemicals AB.
These active fractions were combined, evaporated to dry-ness under reduced pressure and crystallized in warm methanol to give 30 mg. of needle crystals of griseoviridin. The identity of these crystals with ~riseoviridin was proved by TLC and other physico-chemical determina-tions.
' ~, The same fermentation as described in Example lwas carried out for 96 hours except that the fermentation : .
18,374-F ~36-. . ~ ,.
, ~ .

37~

medium was composed of soybean meal 0.5%, Pharmamedia 0.5%, oatmeal 0.5%, dry yeast 0.5%, beet molasses 0.5%
and DL-alpha-~mino-n-butyric acid 0.1% (pH 6.5). '~he fermentation broth was collected from 13 flasks and filtered. The filtered liquid was extracted with n-butanol (300 ml. each, twice). The removal of n-butanol from the extracts left about 70 mg. of crude powder of neoviridogriseins and griseoviridin. This crude powder was analyzed by silica gel thin layer chromatography followed by bio-autography on Sarclna lutea as the test organism. The TLC plate employed in this assay was a product of E. Merck, Darmstaclt (Pre-coated TLC plate Silica Gel~ 60 F-254). The obtained Rf values and the solvent systems were as follows:
Chloroform:methanol20:1 30:1 40:1 Neoviridogrisein IRf-0.60 0.39 0.20 II0.56 0.32 0.16 ; III 0.50 0.19 0.13 Viridogrisein 0 43 0.18 0.10 Griseoviridin 0.05 0.02 0.00 ; EXAMPLE 4 Fifty milliliters of the seed culture medium i conkaining 0.3% beef extract, 0.5% tryptone (Difco Labox-; 25 atories), 0.1% glucose, 2.4% soluble starch, 0.5% yeast extract, 0.4% calcium carbonate and 0.5% soybean meal -(pH 7.0) was distributed in a 250 ml. Erlenmeyer flask and autoclaved at 120C for 15 minutes. Spores of Streptomyces sp. P8648 on an agar slant were seeded in the said flask and shake-cultured at 25C for 3 days to supply the seed culture. The fermentation medium 18,374-F 37-', ' . .

.
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- . , . : , ~0~ 4~L

was composed of 0.5% soluble starch, 2.0% glucose, 1.0% Pharmamedia, 0.5% oatmeal, 0.5% corn steep liquor, 0.05% dipotassium phosphate and 0.05% magnesium sulfate (pH 6.5). Fifty milliliters of this fermentation medium was placed in a 250 ml. conical flask and autoclaved at 120C for 15 minutes. The size of the inoculum was 2%
~v/v). The fermentation flask was inoc:ulated with the above described seed culture and incubated at 25C on a rotary shaker. Twenty-four or 48 hours after inocu-lation, a sterilized solution of casamino acid (DifcoLaboratories) or proline (pH 7.0) was added to a final concentration of 0.4% and the fermentation was continued.
Four days after inoculation, the fermentation broth was -filtered and the broth filtrate was extracted twice with an e~ual volume of ethyl acetate. The ethyl acetate extracts were combined and evaporated to dry powder under ~educed pressure. This crude powder was taken in ethyl acetate and an aliquot amount o the solution was quanti- ;
tatively spotted on a silica gel TLC plate. The T1C
plate was developed in a solvent system of chloroform and methanol (100:1) and the solvent was evaporated off in the air. The same TLC plate was again developed in the said solvent system of chloroform and methanol (100:1). Each component of neoviridogriseins was located under ultra-violet light (3650 A), scraped off from the TLC plate and suspended in a known volume of methanol. After the si:Lica gel was removed by decantation, the amount of the antibiotics in the extracts was determined by the W -assay method, know-ing that the epsilon value at 305 nm is 8,000. The results of the W-assay are as follows: ~ -"'" ~

: .
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Amino acid added None Casamino acid Proline Neoviridogrisein I2% 2% 2%

Viridogrisein 85 55 35 About 10 liters of the 23 hour-old seed culture was prepared in a jar fermenter under the same condition as described in Example 2. The fermentation medium (600 liters) comprising 0.5% soybean meal, 0.5% Pharmamedia, 0.5% oatmeal, 0.5% dry yeast, 0.5% beet molasses and 0.1% DL-alpha-amino-n-butyric acid (pH adjusted to 6.5 prior to autoclaving) was steam-sterilized at 120C
15 for 15 minutes in a 1400 liter stainless steel tank fermenter and cooled down to 28C. To this tank fermenter, the above-mentioned seed culture (lG liters) was added and cultivated at 28C for 75 hours under forced aeration with stirring at 180 r.p.m. (by means -of a double impeller; radius of circle, ~ of the diameter of the tank fermenter~, the sterile air being fed at 300 liters/minute through a sparger from the bottom of the tank. At the end of fermentation, the broth was filtered through a filter press. The broth filtrate was extracted twice with 150 liters each of n-butanol. The n-butanol extracts were combined, washed with a small volume of saturated NaCl solution, and concentrated to 2 liters in a rotary evaporator.
At this stage, 20 grams of silica gel (Wakogel3 G-100, Wako Pure Chemical Industries, Ltd.) was added and the concentration in a rotary evaporator was further con tinued to complete dryness. The obtained antibiotics--silica gel mixture was suspended in a small amount of :
18,374-F -39-..... .
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chloroform and put on top of a silica gel column ~Wakogel~
C-100; Wako Pure Chemical Industries, Ltd); ~6.5 x 75 cm.).
The elution of the antibiotic activities was carried out stepwise first with 7 liters of chloro.Eorm, then with 10 liters of a mixture of chloroform and methanol (50:1); and finally with methanol. Fraction Nos. :L6-29 (300 g/fraction) which were found bio-active on Sarcina lutea (neoviridogriseins) were collected and concentrated to dryness under reduced pressure to yield a crude powder of neoviridogriseins.
This crude powder was dissolved in a small volume of methanol and passed through a Sephadex~ LH-20 column;
Pharmacia Fine Chemicals AB; (7.0 x 45 cm.), each fraction (100 g.) being eluted with methanol. About 15 grams of crude powder of neoviridogrisein mixture was recovered from fraction Nos. 6-15 after the solvent was removed by evaporation.
Fraction Nos. 50-60 of the above described silica gel column contained griseoviridin. A similar purification procedure with Sephadex~ LH-20; Pharmacia Fine Chemicals AB;
(column size 7.0 x 45 cm.) as used for the neoviridogrisein mixture was repeate~ to provide 4 grams of crude griseoviridin.

For final purification, preparative thin layer chromatography with a silica gel TLC plate was employed.
One gram of the crude neoviridogrisein mixture prepared in Example 5 was dissolved in 2 ml. of ethyl acetate and applied in bandwise fashion on ten silica gel TLC
plates (Pre-coated TLC plate Silica Gel~ 60 F-254);
E. Merck, Darmstadt. These TLC plates were first developed with a solvent system of chloroform and methanol - ;
(50:1). After the solvent was evaporated off in the air, the s~id TLC plates were subjected to a second development with a solvent system of chloroform and methanol (25:1).

18j3~7~-F -~0-~ D ~ .

~ .... . . . . . ; . . . . .. .... . .
. . . . . . . . .

.
~41-Neoviridogriseins I, II and III and viridogrisein were marked on the TLC plates under ultra-violet light (3650 A); Blak Ray~ W L-22, Ultra-Violet Products, Inc.
and scraped off the elution. Each neoviridogrisein com-ponent was eluted with a small amount of methanol andevaporated to dryness. The recovered amount of each neoviridogrisein component in pure sta1e was as follows:
Neoviridogrisein I : 16.7 mg (less pure, oily) - II : 11.1 mg (white powder) 10 III : 15.2 mg (white powder) Viridogrisein ~ 30.0 mg (white powder) About 5 mg. each of the neoviridogriseins was hydrolyzed in 6 N HCl at 110C for 36 hours in a sealed tube and subjected to thin layer chromatography, paper chromato-graphy, high voltage paper electrophoresis and autoaminoacid analysis. The presence o the ollowing constituent compounds was ~ound in each component:
Neoviridogrisein I : 3-hydroxy-picolinic acid threonine leucine proline sarcosine ~,N-dimethylleucine ~-amino-n-butyric acid phenylsarcosine Neoviridogrisein II : 3-hydroxypicolinic acid threonine leucine proline sarcosine ~,N-dimethylleucine alanine phenylsarcosine 18,374~-F ~41-!
. [~) ~. ~
- -. , . ~ . : . . .
; . . . ,. ~ . .

.
.
. .
~ . ~ .. . . .

Neoviridogrisein III : 3-hydroxy-picolinic acid threonine leucine hydroxyproline sarcosine -~,N-dimethylleucine a-amino-n-]butyric acid phenylsarcosine Viridogrisein o 3-hydroxypicolinic acid .
threonine leucine hydroxyproline sarcosine ~,N-dimethylleucine alanine phenylsarcosine .

In addition, the identity of neoviridogrisein IV
with viridogrisein was further confirmed by IR, W, NMR ~ :
and mass spectrometry, thin layer chromatography, hydroly-sate analysis and antimicrobial spectrometry. On the other hand, the griseoviridin preparation obtained in Example 5 was crystallized in warm methanol to yield needle crystals.
Then a part of the needle crystals were compared and identi-:.
ied with an authentic preparation of griseoviridin by IR, W, NMR and mass spectrometry, thin layer chromatography, elementary analysis and other physico-chemical properties.

::~

. .
18,374-F . ~42- .
.

"

. .. . . . . , . ... -.. .. . .. . . - .

Claims (11)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for preparing depsipeptide anti-biotic neoviridogrisein I of the formula depsipeptide antibiotic neoviridogrisein II of the formula and depsipeptide antibiotic neoviridogrisein III of the formula viridogrisein and griseoviridin which comprises cul-tivating Streptomyces sp. P8648 (FERM-P3562, ATCC
31289) under aerobic conditions, at a temperature comprised between 18° and 37°C, in an aqueous nutrient medium containing assimilable sources of carbon, assimil-able sources of nitrogen, and essential mineral salts at a pH comprised between 6 and 9, until a substantial antibiotic activity is imparted to the medium, and recovering the fermentation product from the medium.

2. The process of Claim 1 wherein griseoviridin is separated from the fermentation product.

3. The process of Claim } wherein the fermen-tation is carried out in the presence of alpha-amino-n--butyric acid and/or natural sources containing alpha--amino-n-butyric acid.

4. The process of Claim 1 wherein the fer-mentation is carried out in the presence of proline and/or a natural source containing proline.

5. A process for preparing depsipeptide antibiotic neoviridogrisein I of the formula which comprises cultivating Streptomyces sp. P8648 (FERM-P3562; ATCC 31289) under aerobic conditions, at a temperature comprised between 18° and 37°C, in an aqueous nutrient medium containing assimilable sources of carbon, assimilable sources of nitrogen, and essential mineral salts, at a pH comprised between 6 and 9, until a substantial antibiotic activity is imparted to the medium, and recovering the fermentation product from the medium and isolating neoviridogrisein I.

6. A process for preparing depsipeptide anti-biotic neoviridogrisein II of the formula which comprises cultivating Streptomyces sp. P8648 (FERM-P3562; ATCC 31289) under aerobic conditions, at a temperature comprised between 18° and 37°C, in an aqueous nutrient medium containing assimilable sources of carbon, assimilable sources of nitrogen, and essential mineral salts, at a pH comprised between 6 and 9, until a substantial antibiotic activity is imparted to the medium, and recovering the fermentation product from the medium and isolating neoviridogrisein II.

7. A process for preparing depsipeptide anti-biotic neoviridogrisein III of the formula which comprises cultivating Streptomyces sp. P8648 (FERM-P3562; ATCC 31289) under aerobic conditions, at a temperature comprised between 18° and 37°C, in an aqueous nutrient medium containing assimilable sources of carbon, assimilable sources of nitrogen, and essential mineral salts, at a pH comprised between 6 and 9, until a substantial antibiotic activity is imparted to the medium, and recovering the fermentation product from the medium and isolating neoviridogrisein III.

8. The antibiotic substance neoviridogrisein I
whenever prepared by the process of Claim 5.

9. The antibiotic substance neoviridogrisein II whenever prepared by the process of Claim 6

10. The antibiotic substance neovirido-grisein III whenever prepared by the process of Claim 7.

11. An antibiotic composition of matter pro-duced by Streptomyces sp. P8648 (FERM-P3562; ATCC 31289) consisting of neoviridogrisein I, neoviridogrisein II, neoviridogrisein III, viridogrisein and griseoviridin whenever prepared by the process of Claim 1.