US3630846A

US3630846A - Antibiotic production using streptomyces kitasatoensis

Info

Publication number: US3630846A
Application number: US871372A
Authority: US
Inventors: Toju Hata; Akihiro Matsumae; Satoshi Omura; Jinnosuke Abe; Tetsuo Watanabe
Original assignee: KITASATO INST AND TOYO JOZO KK
Current assignee: KITASATO INST AND TOYO JOZO KK; KITASATO INSTITUTE AND TOYO JOZO KK
Priority date: 1967-09-01
Filing date: 1969-10-07
Publication date: 1971-12-28
Anticipated expiration: 1988-12-28

Abstract

Manufacture of new antibiotic substances by the cultivation of a micro-organism belonging to Streptomyces kitasatoensis. The antibiotic of the invention has the general structural formula:

WHEREIN WHEN R1 represents hydrogen, R2 represents acetyl, propionyl, butyryl or where when R1 represents acetyl, R2 represents acetyl, propionyl, butyryl or isovaleryl.

Description

United States Patent [72] inventors Toju Hata;

Akihiro Matsumae; Satoshi Omura, all of Tokyo; Jinnosuke Abe, Takata-gun; Tetsuo Watanabe, Tokyo, all of Japan [21] Appl. No. 871,372 [22] Filed Oct. 7, 1969 [45] Patented Dec. 28, 1971 [73] Assignees The Kitasato Institute;

Toyo Jozo Kabushiki Kaisha Original application Sept. 1, 1967, Ser. No. 665,059, now Patent No. 3,535,309, dated Oct. 20, 1970. Divided and this application Oct. 7, 1969, Ser. No. 871,372

[54] ANTIBIOTIC PRODUCTION USING STREPTOMYCES KITASATOENSIS Watanbe: Chemical Abstracts, Vol. 55, 1961, p. 10329 ad Primary Examiner-Joseph M. Golian Attorney-Jacobs and Jacobs ABSTRACT: Manufacture of new antibiotic substances by the cultivation of a micro-organism belonging to Streptomyces kitasatoensis. The antibiotic of the invention has the general structural formula:

wherein when R, represents hydrogen, R represents acetyl, propionyl, butyryl or where when R, represents acetyl, R, represents acetyl, propionyl, butyryl or isovaleryl.

fiATENIEU mama 3,630, 45 sum 01 or 14 PI G.

WAVE NUMBER (CM 4000 3600 3200 2800 2400 2000 I900 I800 I700 I600 I500 I400 I300 I200 H00 I000 900 800 700 IATENIEUBECZBISII 3630.846 SHEET U2UF 14 F i G. 2

WAVE NUMBER (CM-I l 4000 3600 3200 2800 2400 2000 I900 I800 I700 I600 I500 I400 I300 I200 Il00 I000 900 800 700 PATENTEB IIEC28 l97I 31 30345 sum on or 14 FIG. 4

WAVE NUMBER (CM"') 4000 3600 3200 2800 2400 2000 I900 I800 I700 I600 I500 I400 I300 I200 IIOO I000 900 800 700 PAIENIED utcza Ian saw us or 14 :.28 55552 w 83 009 com.

8 INVENTORZ 8 4311/ Harri OON m GE

ATTORNEY PATENIEDUEC28I97I 31530 46 sum as [If 14 WAVE NUMBER (cw) I 4000 3600 3200 2800 2400 2000 I900 I800 I700 I600 I500 I400 I300 I200 IIOO I000 900 800 700 j g-aw ATTORNEY PATENTEB DEE28l97l 3,630,846

.- SHEET UBUF 14 FIG. 8

PATtNIEnnzczelsn 31630.846

SHEET user 14 ATTORNEY PATENIEuutc-zsasm $530,845

' sum 110514 FlG.

ATTORNEY PATENIEB um xsm FIG. l2

sum 12 0F 14 INVENTOREJ ATTORNEY mammaczalsn 3530.846

' SHEET 13UF 14 ANTIBIOTIC PRODUCTION USING This application is a divisional application of Application Ser. No. 655,059 filed Sept. 1, 1967, now U.S. Pat. No. 3,535,309 ofOct. 20, 1970.

This invention relates to new antibiotic agents and further to a process for preparing the same. More particularly, it is concerned with a novel antibiotic substance as expressed generally by:

was changed whiteto yellow or light tan yellow. Ordinarily the primary and secondary whirls will grow in an amount that ranges from a few to many, however sometimes whirl formation becomes progressively degenerated until none can be detected. Sometimes, few spirals of hyphae were observed. Cultural findings: (UCzapsk's agar:

Growth:

Aerial mycelia; Soluble pigment;

JOURNAL, 1953, Vol -VI, No. 3, p. 109-112. These properties and particularswere also disclosed in Japanese Pat.Publication No. 2299/1955, later matured in Japanese Pat. No. 214984.

Several of us investigated into the antibiotic leucomycin and found that it can be divided generally into two groups, or more particularly into leucomycin A-group and leucomycin B- group and further that the former comprises two kinds of substances: leucomycin A and A,; and the latter comprises leucomycin B,, B B and B.,, as disclosed in Japanese Pat. Publication Nos. 18750/1960 and 98/1961 which were matured later in Japanese Pat. Nos. 278,546 and 278,547, respectively.

It is the main object of the present invention to provide several new effective antibiotics different from those denoted above leucomycin A, B, A,, A B B B and B and called A;,, A A A A A and A, compounds briefly denoted throughout the present specification and particularly and definitely pointed out by the general formula disclosed hereinbefore.

Furthermore, this present invention provides a novel process for the preparation and isolation of these new an- The new antibiotic agents described hereinbefore are formed during the cultivation under carefully controlled conditions by the micro-organisms Srreptomyces kitasatoensis which had been deposited at the Northern Utilization Research and Development Division, U.S. Department of Agriculture, Peoria, 111., USA, and added to its collection of micro-organisms as NRRL 2,486 and 2,487. The description of the organisms are set forth in the following.

Morphological findings A mycerium was formed with the display of slightly curved hyphae on Czapeks agar media though development of aerial mycelium was poor.

Cultivating on starch, asparagine or tyrosine agar, thin grayish white aerial mycelia grewafter several days, sometimes cottony or flocculous aerial mycelia appeared. In some instances the aerial myceliurn grew slowly and sparsely. Later the color of the aerial mycelia on starch or asparagine agar (2 Krainskys agar:

Growth;

Aerial mycelia; Soluble pigment;

yellow to yellowish brown, penetrating into the medium. none to grayish white.

none to Yellowish.

yellowish white, center raised, penetrating into the agar, spreading thin edge.

thin, grayish white or yellow.

none to light yellowish.

In the course of incubation small cottony patches which turned from whiteto light tan yellow appeared and whole sur- -.face was covered by them.

(3) Nutrient agar:

Growth;

Aerial myceria;

Soluble pigment;

(4) Glucose agar:

Growth;

Aerial mycelia;

Soluble pigment;

(5) Starch agar:

Growth;

Aerial mycelia;

Soluble pigment; (6) Tyrosine agar:

Growth;

Aerial mycelia; Tyrosirrase;

brown restricting with raised center, folds from center to edge.

none

brown.

brown to dark brown, penetrating into agar.

very faint, grayish or mouse gray. brown.

colorless to yellow or yellowish brown, penetrating into medium, center raised and brown.

thin

In the course of cultivation, white cottony or fiocculous aerial mycelia grew rapidly, and the whole surface of the medium was covered soon, and tinged with yellowish color.

none or slight.

brown to dark brown, penetrating into medium.

grayish white and thin. later cottony. positive Sometimes tyrosinase production was negative in some strains, giving faint yellowish coloring of medium. Otherwise abundant production of grayish black pigment was observed.

(7) Potato plug:

Growth; yellowish brown and wrinkled.

Aerial mycelia; none or white, thin, sometimes became cottony and light tan yellowish.

Color of plug; unchanged or light brown or (8) Carrot plug:

sometimes blackening to show a chromogenic type.

Color of ring;

brown.

(ll) Egg medium:

Growth; grayish grown. Aerial mycelia; thin, spreading, grayish white, became cottony.

Soluble pigment; purple.

( l2) Serum:

Growth; brownish yellow. Aerial mycelia; none. Soluble pigment; brown.

( l3) Blood:

Growth; dark brown. Aerial mycclia: none Hcmolysis: positive.

(14) Nitrate reduction: positive.

(l5) Starch hydrolysis: positivev (l6) Cellulose decomposition: negative.

(l7) Hydrogen sulfide production: positive.

(18) Utilization of carbon sources:

Utilized; glucose, starch, dextrin, glycerol,

galactosc, maltose, trechalose, sucrose, inositol, sorbitol, sodium succinate, sodium citrate, sodium acetate.

Not utilized; xylose, rhamnose, rafl'lnose, lactose,

arabinose, mannosc, mannitol, inulin dulcitol, fructose, salicine, eskulin.

Above-described morphological characteristics of Streptomyces kitasatoensis were mainly classified into four types of colony form including natural or induced mutant strains, named type A, B, C, D, respectively.

Type B resembles the original type.

Type D shows irregular form.

Thickness of colony:

Type A Type B Type C Type A and C; none Type B; poor Aerial mycelium:

Foldings:

The cultivation of the organisms Streptomyces kitasaloensis may take place in a variety of liquid culture media. Media which are useful for the production of the novel antibiotic may include an assimilable source of carbon such as lactose, maltose, dextrine, molasses, starch, glucose, corn syrup, sugar, glycerine, fatty acids such as stearic acid, palmitic acid, acetic acid, propionic acid, etc., an assimilable source of organic and inorganic nitrogen such as corn steep liquor, yeast or its extract, meat extract, meat juice, peptone, milk, cottonseed oil, casein hydrolysate, amino acids, soybean powder, etc., and such as inorganic nitrate, ammonium salts and the like. These nitrogen sources are used separately or in combination. This applies to the carbon sources. The aforementioned carbon and nitrogen sources are only illustrative, and thus have been set forth in no limiting sense for the invention. The media further include the source of inorganic anions and cations,

such as potassium, sodium, calcium in the form of phosphate,

carbonate, chloride, etc. Trace elements such as boron, molybdenum, copper, nickel, cobalt, iron, etc., especially in the form of, for instance, ferroand ferric chloride, sulfate, those of cuproand cupricompounds, or the like, may be added to the media.

For the culture of streptomyceteous micro-organisms belonging to Streptomyces kitasatoensis, either the liquid culture process or the solid culture process may be employed. For the industrial purpose, however, the submerged aeration culture is most advantageous.

For carrying out the culture of the organisms for the preparation of crude antibiotics according to the invention, the culturing temperature is kept generally at 20-40 C., preferably 25-35 C. The culturing period, when the aeration is employed, extends generally to 2-10 days. When the culture broth reveals a highest potency in antibiotic performance, the

culture should naturally be ceased. In order to separate and purify the crude antibiotic products from the broth, various conventional processes commonly employed for this purpose, such as solvent extraction, adsorbent treatment, distribution chromatography, crystallization from solvents, and the like techniques, may be employed.

In order to prepare a solvent extract containing the crude antibiotics, the filtrate of the culture broth of the streptomycetous organisms, upon removing the mycels off, is subjected to extraction generally at a pH of 7 to an alkalinity, preferable pH 7.5-9, with use of a water-immiscible organic solvent, such as alcohol, for instance, butanol, propanol or the like; ketone such as methyl ethyl ketone or the like; ester such as ethyl acetate, butyl acetate or the like; chloroform; benzene; xylol; toluol, etc. The extract is mixed with a proper quantity of acidic water of pH 2-4 so as to control the aqueous liquid to pH 2-4, in order to transfer the effective antibiotics to the aqueous phase which is then mixed with a water-immiscible organic solvent while adjusting the pH to 7.5-9.0 This adjustment may be carried out in advance of, in the course of, or, after the mixing step. This transfer operation is repeated several times until sufficiently condensed crude antibiotics are contained in the final organic solvent solution which is then subjected to a distillation in vacuo or a condensing step. In this way, an antibiotic composition which includes A A A A A A and A compounds can be separated. Or alternatively, an addition of petroleum ether, the antibiotics can be sedimented. In a still another way, the final condensed solution containing the effective antibiotics may be subjected to lyophilization for the desired separation of the crude products.

The condensed solution may be alternatively treated so that it is adjusted to pH 7.0-9.0 so as to precipitate the effective antibiotics in the form of their free bases.

The thus-obtained products represent the following properties:

Appearances: white powder Melting point: 1 28-145 C.

Elemental analysis: C: 60.00%; H: 8.80%; N: 1.68%, as

found. Infrared absorption spectrum (using KBr tablet, is shown in FIG. 1.

Ultraviolet absorption spectrum:

M'ifi' 231 mp. E, 3%,; 353

Molecular weight (by titration): ca. 800

Optical rotation: [a] :53(C=l chloroform).

This antibiotic composition is soluble in lower alcohols such as methanol, ethanol and the like; esters such as ethyl acetate, butyle acetate and the like; ketones such as acetone, methyl isobutyl ketone and the like; benzene; chloroform and the like; difficult to dissolve in water; insoluble in petroleum ether. I

Color reactions on the new antibiotic composition are as follows:

negative: Adamkiewiczs reaction; biuret reaction: Milon reaction; xanthoprotein reaction.

positive: Anthrone reaction; Schriwanoff's reaction;

Molischs reaction; conc, sulfuric acid reaction; tetrazolium reaction.

When it is desired to isolate several constituent antibiotic compounds A;,, A A A A A and A from the said composition, the latter is further treated by the chromatography technique using silica gel, cellulosic material, activated alumina or the like, by the countercurrent distribution technique, or the like.

As an example, the antibiotic composition is adsorbed by a proper quantity of silica gel, activated alumina or the like material which is commonly used in the conventional chromatographic processing and then dissolved in a combination solvent system, for instance, benzene/acetone, benzene/methanol or the like. By further treating the solvent system as conventionally, each of the constituent compounds can be isolated in the form of white prisms, with exception of A A, and A which are in the form of white crystalline cumpound l ll): d chloroform) p A, compound [01], -s2 c=1,

Analysls chloroform) A, compound: found: C: 60.94%; H: 8.36%; A. compound 1 1 -S6 (C=l,

N: l.75% 5 chloroform) theoretical: C: 60.92%; H: 8.40%; A, compound 91 65 .0 (C: l .3,

N: l.69%, based upon chloroform) our proposed formula: A compound 1 58.3 (C -1.8, u u u chloroform) A, compound: found: C: 60.80%; H: 8.13%; A compound -65.l (0 L3,

N: l.65% l0 chloroform) theoretical: C:60.50%; H: 8.30%; v l f K th l) N: 1.72%. based upon our A, compound 6:70 proposed formula: A, compound A, compound 6.69 A, compound: found: C: 60.98%; H: 8.83%; A compound 6J2 15 A, compound 6.73 theoretical: C: 60.66%; H: 8.50%; A! compound N: l.8l%, based upon A compound 75 our propose formula: CaHuNO Ultraviolet absorption spectrum in u C: 60.5%; '17; methanol: A. compound found N l 7m, H 8 l0 231432. 713

theoretical: c: 60.06%: H: 8.19%; A ;'j ;23 1, E}{,,, :325

N: l.75% based upon 1 our proposed formula: 5")t$22"1232, 2 370 m u u A m6thuuo1:23 1% 5 A, compound: found: c: 60.10%; H: 8.40%; 6 1 p N: r9096 A,)\::* ':232, El imillo theoretical: C: 60.22%; H: 3.88%; 25

N: l.85%, based upon a $l"' Elam-380 ocurkproNpgsed formula: A ::2 ;232 1512 3395 S! u l A, compound: found: c: 59.71%; a: 812%; Infrared abSOYRUOTI Spectrum N: Lao'ib Those taken with use of carbon tetrachloride solutions are g 3 22a 3 shown in FIGS. 1-7, respectively in the order of A A comuse upon pounds. our proposal formula. c,,n,,uo., Nuclear magnetrc resonance spectrums are shown in FIGS. compound= found: 831%: 8-14, respectively, in the order of A -;A compounds.

theoretical H 8 27%- solubility N: 1.88%, hased upon The following table I shows respective solubility of the our theoretical formula: respective compound: C H NO TABLE I.NOMINATION OF COMPOUNDS Compound Solvent A3 A A5 A5 A1 A3 A0 Methanol. Ethanol Ethyl acetate Found molecular weights determined method are as follows:

A compound: 835:] 5;

A, compound: 78km;

A, compound: 770x15;

A, compound: 760$ 10.

A, compound Butyl acetate:

enzene Chlorotorm do do ..do d0 do ..do Do.

aterfl Dlfl'erence. Difference Difference. Dlfierence. Difference Dlfierencm Difference.

Petroleum ether Insoluble. Insoluble- Insoluble. Ins0luble Insoluble- Insoluble. Insoluble.

y the titration Color reactions of A, to A, compounds are just same as disclosed hereinbeforc with respect to the antibiotic composition, as follows:

negative: Adamkiewiczs reaction: biuret reaction: Milon reaction; xanthoprotein reaction.

A, compound: 820:5; A. compound: 801110; A compound: 790x10;

D positive: anthrone reaction: Schriwanoffs reaction; Meltmg Polms Molischs reaction; conc. sulfuric acid reaction; tetrazoli- A, compound l20-l2l9 C. P um z g l I A,compound l26-l27C: p ol'mu A, compound n5-n7c. According to our experimental determlnation, we propose r g the following general formula covering the aforementioned compoun compound c new antibiotic compounds A -A o e A,compound 121 C. CH0 CH2 /CH3 H N H H M11 y -H H 0 l OH H\ H H -0CH3 H H 1 -H E/ o 1: H I 0 on 0H: 0 H on,\ Optical rotation H H i/ H b t-551cc. (c=1, chloroform) CH3 wherein R represents hydrogen and R, represents acetyl, propionyl, butyryl: or wherein R represents acetyl and R represents acetyl, propionyl, butyryl and isovaleryl.

A clear understanding may be obtained by reference to the following table I].

TABLE II i 25 522223201 d r t t t l Radical R; Radical R1 A CMHWNOH CCH3 --CCH1Cfi g H CH3 A CnHuNOa Same (u3CHr-CH2CHa A CNH NO -H Samoasabovc.

A. CmHuNO -C-CH; CCH:CH:

I; ll

A Ci H NOn H Same as above.

A; CavHuNOis CCH3 C-CH2 II II A; CuHeiNOu H Same as above.

These properties of A}, A A A A-,, A and A compounds serve to distinguish those from other known macrolide antibiotics such as spiramycin, carbomycin, erythromycin, methymycin and oleandomycin, because of having different chemical structures.

Of those antibiotics denoted in the prior art, leucomycin A 8,, B B and B are different from the compounds A -A in their physicochemical properties.

Antimicrobial spectra of the novel antibiotic compounds are shown in the following table 111.

Table IV Toxicity. Toxicity, Composition i.v., L0,, per oral. LD or compounds mgJkg. mgjkg.

Composition A A. 650 l,000 A Compound 650 1,000 A. Compound 500 1,000 A, Compound 500 1,000 A Compound 500 1,000 A, Compound 500 l,000 A, Compound 500 l.000 A. Compound 500 l,000

EXAMPLE 1 Preparation of activity in shaker bottles One hundred milliliters of an aqueous culture medium comprising peptone 0.5%, meat extract 0.5%, glucose 2%, common salt 0.3%, dry yeast 0.3%. and calcium carbonate 0.3% were introduced into a SOD-milliliter Sakaguchi flask, adjusted to pH 7, sterilized at 120 C. with steam at 15 p.s.i. for about minutes, inoculated with Streptomyces kitasatoensis and then reciprocally shake cultured at 27 C. for 72 hours.

Next, 1 liter of a fermentation medium comprising soybean powder 3%, cone steep liquor 2%, starch 2%, glucose l%, sodium chloride 0.3%, calcium carbonate 0.5%, potassium phosphate dibasic 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05% and manganese sulfate 0.0003% was equally divided and introduced into 10 Sakaguchi flasks having each a volumetric capacity of 500 milliliters, thus each flask containing 100 milliliters of said medium. After sterilization, l milliliter of the incubated and cultured medium in the foregoing stage was transferred to each of said prepared flasks which are then incubated on a reciprocating shaker at 27 C. for 96 hours. The broth gathered from all the flasks contained a mix- TABLE III Minimum inhibitory concentration vlml.

Comp. a-

Test organism Comp. A4- A A5" A-r- As- Anso Staphylococcus aureus FDA 2091 0.15 0.15 0.08 0. 3 0.15 0.3 0.6 0. 2 Staphylococcus albus 5.0 2 5 2. 5 2. 5 5.0 5. 0' 10 2. 5 Staphylococcus aureus LM, EM R 10 10 10 10 10 10 10 10 Microcaccus jlavus 0.3 0.3 0. 15 0.6 0.15 0.3 0. 6 0.3 Bacillus aubtilis PC1219. 0. 6 1. 25 0. 3 1. 25 0. 3 1. 25 2. 5 0. 6 Bacillus anthracis 1.25 1.25 0.3 1. 5 0.6 1. 25 1.25 1.25 Mucobacterium ATCC 607. 10 10 10 10 10 10 10 10 M ycobactcrium avium 10 10 10 10 10 10 10 10 Nocardia astcroides 2. 5 0.6 2. 5 0.6 2. 5 5 10 '2. 5 Escherichia coli NIH.1 10 10 10 10 10 10 10 10 Escherichia coli B 10 10 10 10 10 10 10 10 Salmonella typhosa H 901 W 10 10 10 10 10 10 10 10 Salmonella lyphimurium 10 10 10 10 10 10 10 10 Shiaella dysentcrr'ae E-1 5 5 5 5 5 10 10 5 Shigella flezncri 15-20 5 5 5 5 5 10 10 5 Shigella 807l7li E-33. 5 5 5 5 5 5 10 5 Vibrio comma type A. 0.8 0.8 0. 8 0.8 0.8 0.8 0.8 0. 8 Stre tococcua hcmolytz 0.15 0.3 0. 15 0.3 0.15 0. 6 1. 25 0.3 Dip ococcus pneumoniae I 0.08 0.15 0.08 0.3 0.08 0.15 0.3 O. 08 Corynebacterium diphtheriae 0. 04 0.15 0. 04 0.3 0.08 0.3 0.6 0. 04 Ncisseria aonarrhoeae 0. 6 0. 6 0. 3 0.6 0.6 1. 25 5.0 0. 6 I'Icmophilua influenza. 0.15 0.15 0.15 0.3 0.15 0.3 0.6 0. 2 Pscudam'nas aeruglnoaa 10 10 10 10 10 10 10 10 Proteus vulgaria 0X19 10 10 10 10 10 10 10 0 Klebaiclla pneumoniac PCI 602 10 1O 5 10 10 10 Y 10 10 Piricuralia oryzae 10 10 10 10 10 10 10 Altcrnaria sowbinettii. 10 10 10 10 10 10 10 10 Microsporium aypsecum" 10 10 10 10 10 10 10 10 Trichophg ttm asteroidca- 10 10 10 10 10 10 10 10 Candida albicans 10 10 10 10 10 10 10 10 Saccharomyces cereal 10 10 10 10 10 10 10 10 Aapergillus uiqar 10 10 10 10 10 10 10 0 Pcm'cillium glaucum. 10 10 10 10 10 10 10 10 Cryptococcus neoforma s. 10 10 10 10 10 10 10 Sporotrium sicnkt'L... 10 10 10 10 10 10 10 Torula utilis 10 10 10 10 10 10 10 1 Resistant strain for Lcucomycin and Erythromycin.

Acute toxicity, LD in mice of the novel antibiotic composition and compounds, as detennined by intravenous and per oral doses, is shown in the following table lV:.

ture of the new antibiotics, or A A A A A A and A compounds, 550 mg./ml. when convertedly calculated to the corresponding amount of the normal standardized leucomycin. it was then filtered off for. removal of the mycelia.

Eight hundred milliliters of the filtrate was adjusted to a pH of 8.5, extracted twice with benzene, 300 milliliters, and the benzene phase was further extracted twice with an aqueous hydrochloric acid, 150 milliliters, of pH 3.5. The aqueous phase was controlled to pH 8.5 and extracted twice with 100 milliliters of benzene. The benzene extract was dehydrated over anhydrous sodium sulfate and dried freezingly. In this' way, a mixture of antibiotics A to A was obtained in white powder.

Yield: 400 milligrams.

EXAMPLE 2 The last benzene extract obtained in the foregoing example was further extracted with 50 milliliters of aqueous hydrochloric acid, pH 3, and the aqueous phase was separated off and adjusted to pH 9 so as to cause enough sedimentation of the antibiotics. Other treating conditions and procedures were substantially same as employed in the foregoing example. The antibiotics were in the form of white powder.

Yield: 340 milligrams.

EXAMPLE 3 Large-scale production of antibiotics An aqueous culture medium comprising peptone 0.5%, meat extract 0.5%, glucose 2%, sodium chloride 0.5%, dry yeast 0.3% and calcium carbonate 0.3%, made up in a volume of 100 milliliters, and introduced into a 500-milliliter Sakaguchi flask, adjusted to pH 7, sterilized as usual manner, inoculated with Streptomyces kirasatoensis and then cultured at 27 C. for 72 hours with reciprocal shaking.

The fermented broth was inoculated under completely aseptic conditions to 20 liters of a fermentation medium comprising soybean powder 3%, starch 2%, glucose 1%, sodium chloride 0.5%, ammonium sulfate 0.3%, dryyeast 0.5%, calcium carbonate 0.3%, potassium phosphate dibasic 0.1% and urea 0.0l% and contained in a 30-liter jar fermenter already sterilized.

The fermentation mixture was incubated at 27 C. for 50 hours while agitating by an impeller revolving at 250 revolutions per minute and being forced through with sterile air at a rate of 20 liters per minute.

The broth was then transferred aseptically to a 400-liter stainless steel fennenter tank and mixed evenly with 200 liters of fermentation medium of the same composition as that disclosed above and contained therein.

The mixture in the tank was further incubated at 27 C. for 85 hours while being agitated by an impeller rotating at 200 revolutions per minute and aerated with sterile air at a rate of 200 liters per minute. At the end of the incubation, the broth contained a mixture of the aforementioned seven kinds of antibiotics, 1,200 mg./ml., when 'convertedly calculated to the corresponding quantity of the normal and standardized leucomycin. One hundred and sixty liters of the filtrate obtained from the broth after centrifuging removal of the mycelia were adjusted to pH 8 and extracted twice with 50 liters of butyl acetate. The separated butyl acetate phase was adjusted to pH 3 by the addition of 30 liters of aqueous hydrochloric acid of pH 3, agitated strongly and the separated aqueous phase was conditioned to pH 8.5, and then extracted twice with 8 liters of butyl acetate. The butyl acetate phase was extracted with 6 liters of aqueous hydrochloric acid of pH 3.5. The aqueous phase, after adjusted to pH 9, was extracted twice with 6 liters of benzene. The benzene phases were collected together and subjected to a condensation under vacuo. ln this way, l30 grams of the antibiotic mixture were obtained in the form of white powder free base.

The potency: 1,150 mgjmg.

EXAMPLE 4 Preparation and isolation of compound A, Two hundred and fifty milligrams of the white powder product were dissolved in 1 milliliter of benzene and the solution was poured into the top end of a chromatocolumn, having a diameter of l centimeter and-filled with 10 grams of silica gel in benzene. Then, the column was washed with benzene thoroughly. Next, a benzene/acetone mixture, ratio being 5:1, was led to pass through the column at a flow rate of H4 v.v.h. (which means a rate of hourly flow corresponding to a quarter of the quantity of the silica gel). The eluate was dividingly collected in a large number of tubes in the quantity of l milliliter per tube, using a fraction collector. Upon investigating the composition of the content of each tube relying upon the thin layer chromatographic method, it was found that the tubes No. 15-25 contained considerable amounts of the compound A,

The contents of the above-specified tubes were collected together and the solvent was distilled off under vacuo. The residual was dissolved in benzene at 60 C., and left overnight in a ice-cooled chamber. The crystalline product, compound A;,, was obtained as free base in white prisms.

Yield: 30 milligrams.

EXAMPLE 5 Isolation of Compound A;

Four grams of the product white powder obtained in example 3 were subjected to a countercurrent distribution using a solvent system of benzene/chloroform/methanol/l/l5 molar citric acid buffer, pH 4.9(mixing ratio by weight 20:6:20z8), and transferred 300 tubes, each of which collected 10 milliliters of the treated solvent. Upon investigation into the tube contents relying upon the silica gel thin layer chromatography, it was found that the tubes specified as No. 65-80 contained considerably the compound A;,. These tube contents were then collected together and treated as before, so as to isolate the free base of compound which was in the form of white powder.

Yield: 350 milligrams.

Thus-obtained compound A; was dissolved in 5 milliliters of ethylether and dropped into 5 milliliters of 2-percent solution of tartaric acid in ethylether with stirring. Precipitated compound A tartrate was washed with ethylether and dried after isolation.

Yield: 400 milligrams.

EXAMPLE 6 Isolation of Compound A,

Among the tubes treated in the foregoing example 4 the contents of those specified as No. 28-35 were collected together and distilled off under vacuo for removal of the solvent. The residual white powder was dissolved in l milliliter of benzene and poured into the top end of a chromatocolumn, 6- millimeter diameter, filled with 5 grams of silica gel mixed with a proper quantity of benzene, and then a solvent mixture of benzene and aceton, 4:l, was led to pass through the column as rechromatography. The resultant solvent solution was then distilled off and the residual was recrystallized from hot benzene. The thus obtained crystalline compound A, free base was in the form of white prisms.

Yield: 20 milligrams.

EXAMPLE 7 Isolation of Compound A The contents of the tubes specified as No. -1 15 in example 5 were collected together and treated in the usual method as before. In this way, the compound A, in white powder was obtained. Yield: 294 milligrams.

EXAMPLE 8 Isolation of Compound A The contents of the tubes specified as No. 45-68 in example

Claims

2. The process according to claim 1 wherein said micro-organism is cultivated under submerged aerobic conditions.
3. The process according to claim 1 wherein the culture broth is extracted at pH 7- 9 using a water-immiscible organic solvent, the extract is acidified to pH 2- 4, whereby the antibiotic mixture is in the aqueous layer, the aqueous layer adjusted to pH 7.5- 9, extracted with a water-immiscible organic solvent and said alternate acidification and basification followed by extraction with said water-immiscible organic solvent, are repeated until the mixture of antibiotics in the form of their bases, is effectively concentrated in the water-immiscible organic solvent.
4. The process according to claim 3 wherein the solvent extract containing the concentrate of said antibiotics is freed of the solvent and lyophilized.
5. The process according to claim 3 wherein said solvent extract containing the mixture of antibiotics is mixed with petroleum ether whereby the mixture of antibiotics precipitates.
6. A process for the production of an antibiotic of the formula as described in claim 1 which comprises cultivating a micro-organism belonging to Streptomyces kitasatoensis in an aqueous nutrient medium at 25- 35* C. for 2- 10 days under aerobic condition containing an assimilable source of carbon and nitrogen and essential inorganic cations and anions until substantial antibiotic activity is imparted to said medium, recovering the mixture of antibiotics wherein R1 and R2 are as defined therein from the broth, and fractionating the mixture to recover the said antibiotic.
7. The process according to claim 6 wherein the culture broth is extracted at pH 7- 9 using a water-immiscible organic solvent, the extract is acidified to pH 2- 4, whereby the antibiotic mixture is in the aqueous layer, the aqueous layer adjusted to pH 7.5- 9, extracted with a water-immiscible organic solvent and said alternate acidification and basification followed by extraction with said water-immiscible organic solvent, are repeated until the mixture of antibiotics in the form of their bases, is effectively concentrated in the water-immiscible organic solvent.
8. The process according to claim 7 wherein said solvent extract containing the mixture of antibiotics is fractionated by silica gel partition chromatography.