CA1104078A - Antibiotics c-14919 e-1 and e-2 - Google Patents

Abstract

Abstract of the Disclosure Novel Antibiotics C-14919 E-1 and E-2 are produced by cultivating a microorganism of the genus Nocardia. The Antibiotics C-14919 E-l and E-2 are useful primarily as disinfectants.

Description

i ~ ~ 4~!7 8 ~ :;

This invention relates to novel Antibiotics C-14919 E-l . and E-2, and a method of producing the same.
The invention is based on the discovery that certain ~ .
microorganisms lsolated from soil and other samples and belonging to the genus Nocardia are able to produce novel antibiotic agents.
It was further discovered that by cultivating these microorganisms , : .
~ m a suitable nutrient medium, it was possible to have said~anti-:
biotics accumulate in the cultured broth. The above novel anti-biotics were designated as Antibiotics C-14919 E-l and E 2~ re-spectively.

This invention, therefore, provides ~ ~ ~1) Antlblotic C-14915 E-l and Antibiotic C-14919 E-2;

;- and (2~ A process for produc mg Antibiotic C-14919 E-l -~ and/or E-2 which comprises cultivating an Antibiotic C-14919 E-l and/or E-2-producing stra m of the genus Nocardia in a ~, culture medium to cause said strain to elaborate and accumulate ~,. ..
` Antibiotic C-l4919 E-l and/or E-2 ln the resultant broth -~ and harvesting said Antibiotic C-14919 E-l and/or ~-2 '.:,~::
.

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from the broth.
Throughout this speci.fication~ Antibiotic C-14919 F.-l and Antlbiotic C-14919 E~2 will sometimes be referred to briefly as C~14919 E~1 and C 14919 E~2 respectively.
The new Antibiotic C-~14919 E-l and/or E 2-producin~ microor~anism which is employed herein according to this invention may be any strain belonging to the genus Nocardia which is able to elaborate and to accumulate the Antibiotic C 14919 E-l and/or E-2.
The microorganism, which we have isolatedy is one o~ the p.re~erred microorganisms which can be employed according to this inventionO
The aforementioned microorganism is ~ctinomycete Strain No. C-14919 (hereinafter sometimes referred to briefly as Strain No. C-14919~ which is an actinomycete organism discovered in the course of our screening for antibiotic producers and which i.s a novel type of microorganism of the genus Nocardia as wlll be seen from the following description.
A) Microbiological Characteristics o~ Strain No. C-14919 The microbiological characters of Strain No. C-14919 were investigated by procedure~ analogous with those proposed bD Schirling & Gottlieb [International Journal of Systematic Bacteriology 16~ 313-340 (1966)~.
The results of cultivation at 28C over 21 days are . .
~' : .

,, . . - . ~ :. ~

as follows~
1) Morphologlcal characters The vegetative mycelium extends far~ developing into branchesg both on agar and in li~uid media. Many of the hyphae measure o.8 to 1.2 ~m ln dlameter and the hyphae are fragmented depending on cultural conditions.
The strain gives good growth on various taxonomical media, with the aerial mycelium superimposed on the vegetative mycelium, although it forms coremia like bodies (50-200 ~mx 200-1500 ~m)on which the aerial growth takes place. Many of the aerial mycelia areflex-UOU5 or straight, with a loosely ~piral like configuratlon ; being found on a few occasions. Microscopic examination of aged cultures reveals that only in few cases the conidia like form occur in chains, while the cell suspensions obtalned from the surfaces of such cultures, as viewed under a microscope) contains elongated ellipsoidal ~o.8 1.2 x 3.7-6.7 ~m) and ellipsoidal (0.8-1.2 x 1.0-2.0 ~m) bodies resembling conidia.
Electron microscopic examinations showed that these bodies had smooth sur~aces.

2) The constituents of the cell The strain was inoculated into a modified ISP
No. 1 medium and cultured at 28C for 66 to 90 hours under shalcing culture, at the end of which time the

- 3 131~ ) 7~

cells were collected and rinsed. By the method of B.
Becker et al. [Applied Microbiology I29 421 (1964)] and the method of M.P. Lechevalier et al. ~Journal o~

Laboratory and Clinical Medicine 71~ 934 ( 1g68 ) ~, the above cells were examined for diamlnopimelic acid and sugar composition. The ~ormer was ~ound to be the meso-form, while spots were in evidence which corres-ponded to galactose and arahinose.
3) Characteristlcs on taxonomical media The strain gives comparatively good growth on various media, with the vegetative mycelium being colorless to pale yellow in lnitial phases of culture and light yellowish tan to yellowish tan in later phases. Moreover, substantially no soluble pigments were produGed in the taxonomical media used for the present strain. The aerial mycelium is powdery and , grows moderately, being white to yellow or light yellowish tan.
.:
~ The characteristics of the strain in various . .
~ taxonomical medla are set forth in Table 1.
.-'~1' ' '~ Table 1~ CulturaI characteristics of Strain No~ C-14919 ' ~ on taxonomical media - (A) Sucrose nitrate agar ' Growth(G): Poor~ colorless to light yellow ; Aerial mycelium(AM): None -` Soluble pigmenttSP): None " .
-~ - - 4 -.

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(B) Glyceroi nitrate agar G : Poor~ colorless to light ~ellow9 coremia like bodies formedO
AM: Scant, Lt Ivory (2ca)*
SP: None (C) Glucose asparagine agar G : Moderate, colorless to Brite Yellow (3na)*;
coremla like bodi.es formed.
AM: Scantgwhite to ~t Ivory (2ca)*
SP: None (D) Glycerol aspargine agar ~ Moderate, white to light orange yellow~ coremia like bodles formed.
AM: Scant~ Lt Ivory (2ca)*
SP: None (E) Starch:agar G : Moderateg Lt Ivory ~2ca~* to Lt Wheat (2ea)*~
.coremla like bodies formed AM: Ab~ndant3 Lt Ivory (2ca)*
.~ SP: None ,~
. ~F) Nutrient agar G : Moderate, Colonial Yellow (2ga)* to 9rite Maize (31a~ coremla like bodies formed AM: Moderate~ white to Pearl Pink (3ca)~
: SP: None (G) Calcium maIate ag~r G : Moderate~ light yellow to Colonial Yellows (2ga)* 3 coremia like bodies formed , , " ,i", " ~,; " :, , " ":

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AM: Moderate~ light yellow to yellow SP None Other character: Calcium solubllize (H) Yeast extract-malt extract agar G . Moderake, Amber (3nc)*~ coremia like bodies formed AM: Moderate, white : SP~ None (I) Oatmeal agar G : Moderate, Amber~3nc)* to Brite Yellow (3na)*; coremia like bodles formed AM: Moderateg light yellow : SP: None ~J) Tyrosine agar G : Moderate, Amber (31c)*3 coremia like bodies ~ formed ;~ AM: Scant., light yellow SP: None (K) Potato plug .
;; G : Moderate~ Lt Amber ~3iC)*3 coremia like bodies ~ .formed : AM: ~one SP: The plug becomes pale yellowish tan (L) Peptone yeast extract ~ron agar G : Moderate~ light yellowith tan to light orange yellow AM: None SP: None * The color codes according to Color Harmony Manualg 4th ed. (Container Corporation of America, 1958)o

4~ Physiological characters P~lysiological characters of the strain are shown in Table 2. Temperature range for growth: 12C to 38C
The temperature range in which good aerial mycelium growth occurs on agar (ISP No. 2) ls 20 to 32C.
Table 2 The physiological characters of Strain No. C 14919 Temperature range for growth: 12C-38C
Temperature range for sporulation: 20C-28C
Lique~action of gelat~n: Positive I-lydrolysis of starch: Positive :
: Reduction of nitrates: Positive . Peptonization of milk: Positive , Coagulation of milk: Doubtful positive : Decomposition Or casein: Positive . Production of melanoid pigments:

Negative ~peptone yeast extract iron agar~
negative (tyrosine agar) Decomposit.ton of tyrosine: Positive ~ Decomposition of Xanthine: Negative .~ Decomposition of hypoxanthine: Negative Tolerance to lysozyme: Positive Tolerance to sodium chloride: 2%

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5) Utilization of various carbon sources ~ le utillzation of various carbon sources was investigated usin~ a medlum descri.bed i~ Pridham and Gottlieb ~Journal of Bacteriolo~y 56, 107(1948)~.
The resultant spectrum is shown in Table 3.
Table 3 The utilization of carbon sources by Strain :
No. C-14919 Source of carbon Growth Source of carbon Growth D--Xylose + Raffinose L-./Lrabinose ~ ~elibiose ~ ;

D-~lucose ~ i~Inositol D-Galactose ~ D~Sorbitol D-Fructose +~ D~Mannitol -~

L-~hamnose + Glycerol +

D Mannose + Soluble starch +

Sucrose ~ Control Lactose +
1altos~ +
~rehalose +

Note: ~+: Luxuriant growth ~: Good growth ~: Growth +: Poor growth -: No ~rowth ~ 3

6) Other characteristics The cells were harvested by the procedure previously described in 2) and DNA was prepared by a procedure analogous to that of J. Murmar et al~ CJournal of Molecular BiOlogyg VolO 3g 208g 1961]. The G-C
content of the DNA was ~ound to be about 71 mole %.
Gram-staining of the vegetative mycelium of this strain produced a positive reaction~
The above characteristics of straln No. C-14919 were compared with the descriptions in S.A. Waksman's The Actlnomycetes Vol. ~7 [The Williams and Wilkins Co.g 1961]g R~Eo Buchanan and N.E. Gibbonsg"Bergey~s Manual of Determinative Bacterlologyg 8th ed!', 1974~

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and other literatureS While this strain was thought to belong to Group m of the genus Nocardia, failure to ~ind any species having the characters so far described among the known species led us to conclude that lt was a novel species o~ microorg~nisms.
The present Strain No. C-14919 has been deposited at Fermentation Research Instltuteg Agency of Industrial Science and Technology (FERM), Japan under RM-P No. 3991; at Institute for Fa~nent.~tion,Osaka . .:
(IFO) 9 Japan under the accession number o~ IFO 13723, and at The American Type Culture Collection (ATCC~, Marylandg U.S.A. under the accession number of : ATCC 31280.

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~ !hile Strain No. C-14919 is a novel species of the genus Nocardia as ~ust mentioned~ it is liable9 as are microorganisms generally~ to undergo variations and mutations, either spontaneously or under the influence of a mutagen. For example, the many variants o~ the strain which are obtainable by irradiation of the parent with X~rays~ gamma rays, and ultraviolet lightg etc., by monocell isolation, by cultivation on media containing various chemicals~ or by other mutagenic treatments, as well as the mutants spontaneously obkained from the parent strain, may not be sub~tantially consldered to represent any other distinct species but, rather, any of such variants and mutants whlch is still able to elaborate C-14919 E-l and/or E-2 may be invariably utilized for the purposes of this invention. By way of example, sub~ecting Strain No. C-14919 to various mutagenic treatments yields mutants which produce soluble pigments from light yellow to light yellowish tan or tan, mutants whose substrate mycelia are colorless or yellowish green9 or orange red, mutants whlc~l produce abundant white mycelia or no mycelium at all9 or mutants whose hyphae are liable to fragmentation.
The culture medium employed in the practice of this invenkion may be whichever of a liquid or a solid medium, only if it contains nutrlents which the strain may utilize~ although a liquid medium is preferred for high-production runs~ The medium may conta~n carbon and nitrogen sources which Strain No. C-14919 may assimilate and digest~ irlorganic matter, tr~ce nutrients, etc. As examples of said carbon sources may be mentioned glucose, lactose, sucroseg maltose, dextrina starch~
glycerol~ mannitolg sorbitol, and fats and oils (e.g.
soybean oil~ lard oll, chlcken oil~ etc.~. The nitrogen sources may for example be meat extract3 yeast extract, dried yeast~ soybean meal, corn steep liquor, peptone9 cottonseed flour, spent molasses9 urea~ ammonium salt (e.gO ammonium sulfate~ ammonium chloride, ammonium nitrate 9 ammonium acetate~ etc.) and so forth. The medium may further contain salts o~ sodium, potassium, calcium~ magnesium, etc., metal salts of iron, manganese, zinc3 cobalt, nickel, etc., and organic acid salts such as acetates and propionates.
Further9 the medium may contain, as added, various amino acids (e.g. glutamic acid3 aspartic acid~ alanine, lysine, methionine, proline, etc.)g peptides ~e.gO
dipeptides, tripeptides~ etc.), vitamins (e.g. Bl, B2 nicotinic acid, B12, C, etc.), nucleic acids (e.g.
purine, pyrimidine and the corresponding derivatives) and so forth For the purpose of adjustin~ the pH
of the medium~ there may be added an inorganic or organic acid, alkali~ buffer or the like. Suitable '' ' '' ' ' " '' '' . ` :', `, ' ' ' ' ' ,' ~ 9~ ~

amounts of oils and fats, surfactants, etc. may also be added as antiforms.
The cultivation may be conducted by any of the stationary 9 shake 9 aerobic submerged and other cultural methods. ~or high production runs9 submerged aerobic culture is of course preferred. While the conditions o~ culture, of course, depends on the condi~ion and composition of the medium~ the strain used, cultural method and other factors 9 lt is normally preferred to carry out incubation at 20 to 32C with an initial pH near neutral. Particularly desirable is a temperature from 23 to 26C in an lntermediate stage of cultivation,with an initial pH of 6.0 to 7Ø
While the incubation time also is variable according to said factors, it îs advisable to continue the incubation until the titer of the desired antibiotic elaboration product is maxlmal. In the case of shake culture or aerobic submerged culture ln fluid mediumgthe time required normally ranges from about 72 to 192 hours.
The p~f suited for the production of the antibiotics is near neutral 9 the optimum range being pH 6.5 to pH 7Ø The yield can be increased by controlling the medium pH in the course of cultivation~ by the addition of an acid or alkali or by selecting the proper medium composltion. Thus 9 the yield o~ the antibiotics increases considerably as medium ingredients, inorganic .

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phosphates are added in an amount more than necessary for growth of the strain. ~.e. 1000 to 7000 ppm., or certain assimilable carbohydrates which are ready to form acids, e.g. glucose~ mannitol, etc.~ are employed as sources of carbon~ together with suitably selected sources of nitrogen.
Antibiotics C-14919 E 1 and E ~g thus elaborated by the present strain, normally occur extracellularly but there are cases in which the cells contain about 10 to 30 percent o~ the total yields o~ the antibiotics.
The potency titration of the antlblotic agents can be accomplished by the cyllnder method or the paper disk method 3 which employs Candida albicans IF0 0583 as an assay organism and TSA (Tryp~icase soy agar, B~L) as an assay medium.
The antibiotics occur in both the filtrate and cells which may be ~btained from thecultured broth.

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Thus~ the broth is filtered or centrifu~ed to recover the cells and the filtrate OI' supernatant independently.
The potency of the ~iltrate is assayed directly and that of the cells is assayed after they are stirred with the same volume of 70% acetone-water as the filtrate at 20C for one hour. The assay method is the one described hereinbefore, the assay organism being Candida alblca_ .
Because C-14919 E-l and E-2, which are produced . ~

~ r~8 in the fermentation broth~ are lipophyl and neutral substancespthey can be conveniently recovered by separation and purification procedures which are normally employed for the harvest of such microbial metabolites. For example, there may be employed a procedure which utilizes the difference in solubility between the antibiotic and impuri~y, a means which utilizes the difference in adsorptive affinity between the antibiotic and impurity of various adsorbents such as activated carbon, macroporous non-ionic res1ns, silica gel~ alumina, etc.~ a procedure of removing the impurities by means of ion exchange resins and so forth. These procedures may be used sin~ly~ in a suitable combination or applied in re~etition. Since, as aforementionedg C-14919 E-1 and E-2 occur in both the filtrate and cells~ whichever of the following procedures may be adopted. (1) The culture broth as suchg before separation of the cells, is extracted with a solvent or (2) the cells and the filtrate or supernatant as obtained by filtration or centrifugation are lndependently extracted with solventsO
Where the cells and filtrate are independently extracted, the following procedure may be followed with advantage. As the solvents suited for extraction from the filtrate, there may ~e mentioned organic solvents immiscible with water, such as fatty acid : ,: ,. ,, . . : .

esters, e.g. ethyl acetate~ amyl acetate~ etc.;
alcohols, e.g. butanolg halogenated hydrocarbons, e.g. chloroform; and ketones~ e.g. methyl isobutyl ketone. The extraction is carried out in the neighborhood of neutrality andg preferably~ is accomplished with ethyl acetate from the broth ~read~usted to pH 7.
After the extract is washed with water and concentrated3 C 14919 E 2 is obtained as crude crystals. The filtrate remaining after separation of said crude crystals is rich in E-l.
The extraction ~rom the cells may be accomplished using a m~xture of water with a water-miscible organic solventg such as a mixture of water with a lower alcohol (e.g~ methanol, ethanolg etc ) or a mixture of water with a ketone(e.g~ acetone~ methyl ethyl ketone~ etc.), or by means of a water-immiscible organic solvent such as a halo~enated hydrocar~on (e.g. methylene dichloride)O
It is, however, advantageous to employ 70% acetone-water.
Thus~ the cells are reconstituted with the same volume of 70% acetone-water as the corresponding filtrate, and the mixture is stirred at room temperature for 3 hours. By this procedure~ all the C-14919 E-l and E-2 occurring in the cells are extracted.
The solvent extracts are pooled and the solvent, e.gO acetone, is removed under reduced pressure. The aqueous solution is extracted with ethyl acetate as in )7~3 the case of the filtrate and the solvent layer is washed with water and concentrated under reduced pressure. ~o the concentrate is added hexane or the like to precipitate the ac~ive components~ A mixture of C~14919 E~l and E-2 can thus be obtained by centri-fu~ation or filtration. Separation of the mixture into components may be accomplished by adsorptlon chromatograp~y on various adsorbents. For this purpose3 various carriers commorlly used for adsorption of antibiotic substances can be employedO For example, adsorbent resins, silica gel, alumina3 etc. may be mentioned. For elutin~ the antibiotics from such adsorbent resins~ use is made of a mlxture of water and a water-miscible organic solvent such as a lower alcohol or lower ketone. As said lower alcohol may be mentioned methanolg ethanol) propanol, butanol~
etc. and said lower ketone may for example be acetone, methyl ethyl ketone. Esters such as ethyl acetate may also be employed, A typical procedure may be as follows.
The crude product is dissolved in 40% methanol-water and adsorbed on a column of Diaion HP-10 (Mitsublshi Kasei). The column is first washed with 40% methanol-water and, then, e].ution is carried out with 60% methanol-water3 whereby the C-14919 E-2 fraction is obta~ned.
Elution with 90% methanol water yields the C-14919 E~l fraction. Each fraction is concentrated under .

'7~

reduced pressure and, after addition o~ methanol, allowed to stand. The procedure yields crystals o~ C-14919 E-l and crystals of C-14919 E-2 from the corresponding fractions. ~here silica gel is used as said adsorbent, development can be started using a nonpolar solvent wi~h the addition of a ' polar solvent, e.g. methanol, in small increments, whereby C-14919 E-l and E-2 are eluted. The crystals of C-14919 E-l are recrystalli~ed from ethyl acetate, methanol, aqueous methanol or the like, and the C 14919 E-2 may also be recrystallized from similar solvents.
; C-14919 E-l and E-2, which can thus be ob~ained, are convertible 2~ 10 to each other by reversible oxidation-reduction. Thus~ whilst reduction of C-14919 E-l with a reducing agent (e.g. hydrosulfite, ascorbic acid, ; NaBH4, and zinc with acetic acid) yields C-14919 E-2, whereas oxidation of C-14919 E-2 with an oxidizing agent (e.g. ferric chloride, nitric acid, AgO2, MnO2, and air) yields C-14919 E-l.
The physical and chemical properties of the novel antibiotics C-14919 E-l and E-2 crystals obtained in the above manner are set out in Table 4. In this table, reference is made to ~igures 1 and 2 of the accom-panying drawings which represent the infrared absorption spectra ~KBr) of the new antibiotics C-14919 E-l and E-2 respectively.

T_ e C`~14919 E-lC~-14919 E-2 . .. ... _ . _ _ 1) m.p. 187C (decompO)148C (decompO) . ~ _ _~
2) Appear~ yellow crystals pale yellow crystals - ance (needles or prisms) (needles or prisms) . _. , . . ~
Insoluble in: Insoluble in:
3) Solu- petroleum etllerg petroleum ether, :~ bili~y hexane, water hexane ~: Sparingly soluble in: Sparingly soluble in:
di.ethyl ether~ diethyl ether, benzene benzene~ water~
.~ Soluble in: chloro~orm ethyl acetate~ Soluble i.n:
chloroform~ butanolg ethyl acetate, . methyl isobutyl butanol~ methyl : ketone J ethanol, isobutyl-ketone, acetone, methanol ethanol, acetone, Readily soluble in: methanol dimethylsulfoxide Readily soluble in:
dimethylsulfoxlde _ _ _ .. , ............ _ 4) Acidg .
neutral a neutral substance a neutral substance or basic _ . _ ___ 5) Ele- C 65~31, 65.o5 3 64.85 C 62.329 62.07 : analysis H 7.71, 7.58, 7.62 H 8.58, 8.43 % N 5.01, 4.95~ 5~01 N 4.82, 4.78 0 21.37, 22.83, 22O~5 O 20.~1, 20.~1 _ ~ _ . .
~ormula C30-32H42-48N2 8-9 C30_32H44_50N2 _ _ 8-9 xH2O

7) Ultra- _ ~
violet r absorption ~ spectrum ;~ MeOH N-HCl(9~ nm)~El ) 274~455),240~sh~, 255t295) ~max lcm 397(43) 308(sh) ~Me ~nm)~E ) 274(455),240~sh) 255(290) ; max lcm 397~43) 308~sh) MeOH N-NaOH(9:1)(nm)~E1% ) 236~585),265(500), 236(505),265~420) ~max cm 550(56) 550(50~
_

8) Infrared absorption Fig. 1 Figo 2 spectrum (KBr) Dominant -1 Dominant 1 ~ -- peaks ~cm ) peaks (cm ) ,. ,.
, 3430,334~,2950, 3480,3250,2980, , 29lO,1740,16g2, 168~,1625,1598, -~ 1660,1645,1605, 1472,1390,1370, 1500,1375,1315, 1315,1207,1090, 1120,1100,1085, 1065,1042,1030, , I060,1025 , ., .- _ _ _ ~ _ .

9) Specific rotation ~ ~DS + 350 + 10 ~ ~]D +~62 + 4 ` (C=0.5,methanol) (C=0.5,methanol) ,: _ .
103Color reactions Negative: Negative:
ninhydrin reaction, ninhydrin reaction, Ehrlich reaction, Ehrlich reaction, Peptide reaction, Peptide reaction magnesium acetate and magnesium acetate reagent and 1% reagent iron chloride-1%
; ~ ferricyanide(l:l) Positive:
reagent 1% iron chloride '1% ferricyanide Potassium per- (l:l)reagent(blue) manganate reagent, discolored Potassium per-manganate reagent, ~ discolored : ~ : _ _ _ :,~ :
- 19 - i' ~ A
.~ .

Num-j Num-~
: ber ber pro- pro~
11~ ~(ppm) _ J(Hz) tons ~(ppm) J~Hz) tons Nuclear 0.63 d 6.5 3H 0.63 d 7 3H
magnetic 0 94 d 6.5 3H 0.82 d 7 3H
resonance .
~ spectr~lm 0.96 d 6O5 3H V.89 d 7 3II
1~ (100 mega~ 104 S 3H 1.16 s 3H
:~ cycles) in ~ dimethyl- 1.88 s 3H 1.55 m : . sulfoxide 1O4~1.8 m 1.83 s 3H
2.2-2.4 m 2.1-2.2 m 3.0-3.2 m 2.65 m 3.23 s 3H 2.86 m . 3.27 s 3H 3fl3 s 3M
3~43 s 3H 3.24 s 3H
3.3 m 3.2 m 4.5 .s lH 3O39 s 3H
5.21 d 10 lH 4.511 d 5 lH
5.28 d lH 4.64 d 8 lH
5.59 dd &,10 lH 5.02 d 8 lH
6.22 t 11 lH 5.21 d 10 lH
6.32 s (dlsap~ lH 5.. 86 t 10 lH
pears 5.98 s.like lH
dition 6.23 s (disap- lII
of D20) pears 6 41 d 2 lH bditidn 6.81 d 2 lH o~ D20) 6.95 d 11 lH 6.35 d ~.5 lH
. 9.3 s (disap~ lH 6.45 d 2.5 lH

PdYiatid2nO) 7.51 s (disap- lH

of' D20 ) 8,64 s ( do. ) lH
_ . _ _ ~ 8.85 s ( do. ) lH

- 2~ -3t7~

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~ , _ . .......................... . ...... _ _~
12) Stabillty Room temp.: Room temp.:
Stable in neutral Stable under neutral ; and weakly acidic and weakly acid solutions; conditions~
Unstable in basic Unstable under basic solution. conditions.
80Cg lhr.: 80C~lhr.:
unstable unstable ~: 13) _ _ ~ _ Thin layer chromato-graphy Adsorbent:
Silica Gel, Spot ~ilm(Tokyo Kasei) Chloroform-methanol (9:1) 0.85 0.50 Chloroform-methanol tl9:1) 0.77 0.22 Ethyl acetate-acetone (9:1) o.80 0.50 Ethyl acetate-methanol (9:1) o.85 o~7 .
_ Antimicrobial activity The antimicrobial activities of Antibiotic~ C~ 919 E 1 and E-2 were assaye~ by the serial dilution method using Trypticase~soy agar(~BL)~glucose nutrient agar and glycerol nutrient agar as test media (Table 5). In addltlon, the activities against Tetrahymena ~yriformis W were determined by the serial dilution method using the assay medium [Proteose-peptone 20 g~ yeast extract 1 g, glucose 2 g, distilled water 1000 ml, 1 M-r q.~

phosphate bu~fer (pH 7.0) 10 ml].
Table 5 ~ _ _ Assay organisms T~0 No : .O MIC(~g/ml) MIC(~/ml) _ _. _ _ _ _ Escherichia coli K-l23301 >100 ~100 Proteus vulgaris 3045 >100 ~100 Pseudomonas aeruginosa 3080 >100 >100 Salmonella typhimurium 12529 >100 >100 Alcaligenes faecalis13111 ~100 100 Serratia marcescens 3046 ~100 >100 Bacillus pumilus 3813 50 100 Bacillus subtilis 6633 3134 50 100 Baeillus subtilis PCI219 3513 100 >100 Bacillus cereus 3514 >100 >100 Baeillus megaterium 12108 50 5o Baeillus brevis . 3331 50 5o Staphyloeoeeus aureus 209P . 12732 >100 >100 Sareina lutea . ' 3232 50 : 50 Microcoeeus flavus 3242 50 5o Candida albieans 0583 25 6.25 _ . _ _ _ _ _ . . .
**Candida tropicalis 1400 >100 >100 Candida pseudotropieaIis 0617 100 100 Candlda utilis 0619 > 1 oo >Ioo _ .

~ -- - -- --Candida parapsilosis 1396 50 25 Candida krusei 1395 >100 ~100 Candida albicans 05~3 50 25 Crypkococcus neoformans 0410 ~100 >100 Saccharomyces cerevi~iae 0209 100 100 Microsporum canis 7863 >100 >100 Microsporum cookei ~303 ~100 >100 Asperglllus ni~er 4066 100 100 Trychophyton rubrum 5467 >100 ~100 Hormodendrum pedrosoi 6071 ~100 >100 ~enlcillium chrysogenum _ 4626 5 25 ~ ... . ~ .,_ _ *~*Mycobacterium avium >100 >100 Mycobacterium phlei >100 >100 ~ ~ ._~ ,_ ___ ~ ****Tetrahymena pyriformis W _ . 10 _ Note: Medlum * Trypticase-soy agar ** Glucose nutrient agar *~* ~lycerol nutrient agar **** Proteose-peptone solution Toxicity The assumed LD50 as determined by intraperitoneal administration of C-14919 E~l and E-2 in an acute toxicity test using mice as test animals are as follows.

. ~ .. , ., ~ .. ... .. ~ . " .. ..

C-14919 E~l 50-100 mg/kg C-14919 E-2 25-100 mg/kg As will be seen from the antimicrobial spectrum of Table 5~
C-14919 E-l ~ E-2 display activity against Gram-positive bacteria, fungi and yeasts. Therefore, the antibiotics are of use as germicides or disinfectants against pathogenic bacteria or fungi of the same species as the assay organisms.
By formulating the present Antibiotic C-14919 E-l or E-2 into an aqueous solution containing 10 to 100 ~g/ml of aqueous ethanol ~e.g. containing 5% ethanol), it can be used for the disinfection of bird cages, laboratory equipment, human hands, etc.
Antitumour activity The therapeutic activity ~intraperitoneally administered for consecutive days ) of C-14919 E-l and E-2 against P388 leukemia in mice ~1 x 10 cells/animal, mouse, itraperitoneally transplanted) was investigated.
The antitumour activity in terms of the extension of life spans was 144% at the dose level of 5 mg/kg/day.
Thus, since Antibiotics C-14919 E-l and E-2 have a life-extending effect upon tumour-bearing mammals ~e.g.
mouse), they are expected to be useful as an~itumour agents.
The following examples are further illustrative but by no means limitative of the invention9 wherein "part(s)~' is based on weight unless otherwise noted and the relationship between "part(s)" and "part(s) by volume" corresponds to that between "gram(s)" and '1m111iliter(s)"9 and "%" is based on "weight/volume"
unless otherwise noted.
E~ample 1 Strain No. C~14919 (ATCC 31280~ IF0 13723; F~F~ P
No. 3991) was inocu~ated on the tyrosine agar medium~
~ollowed by incubation at 28C for 240 hours~ The well-grown mycelium was suspended in a 0.6% solution o~
sodium glutamate and stored in a refrigerator. A
1 part by volume-portion o~ the mycelial suspension was used to inoculate a 2000 parts by volume fermenter containing 500 parts by volume of a seed culture medium (20 parts of glucoseJ 30 parts o~ soluble starch~

10 parts of corn steep liquo~g 10 parts of soybean ~lour, 5 parts of peptone~ 3 parts o~ sodium chloride and 5 parts of calcium carbonate/water 1000 parts by volume, pH 7.0). The inoculated medium was incubated on a reciprocating shaker at 28C for 48 hoursO This culture (500 parts by volume) was used to inoculate a 50000 parks by volume tank of stainless steel charged with 30000 parts by volume of a seed culture medium.
This seed culture was carried out under the following conditions: 28C, 30000 parts by volume/min. aeration7 ::

4~

280 r.p.m. ~l/2 DT)~ internal pressure l kg/cm , 48 hrs. A 10000 parts by volume portion of the resultant preculture was used to seed a 200000 parts by volume tank of stainless tank charged with lO0000 parts by volume of a fermentation medium (5% of glycerolg 2% of' corn steep liquor~ 2% of yeast extract~ 2%
of KH2PO4~ 0.5% of MgC12 and 0.1% of CaCO39 pH 6.5 and sterilized~ The initial phase of fermentation was carried out unde the following conditions:
temperature 28C~ lO0000 parts by volume/min. aerati.on~
180 r~p.m. ~1/2 DT)~ internal pressure :L kg/cm2g 24 hours, whereby the microorganis~l was allowed to grow well.
Thereaf'ter~ the cultivation was ~urther carried out ~or 114 hours under the same condltions as above except that the incubatlon temperature was 2~iC and the stirring was effected at 200 r.pOm. (l/2 DT). At the end of the above period 9 the mycelia were separated from the filtrate by filtration. The filtrate contained 380 ~g/ml of activity9 while the titer in the mycelia was gO ~g/cells in one part of brothO The potency tltrations were carried out by the cylinder method or the paper disc method using Candida albicans IFO 0583as an assay organism and C-14919 E-l as a standard test material.
Example 2 The procedures descrlbed in Example l were '~

71~

duplicated up to the seed culture stage, Then, the cultivation ~as carried out in a 200000 parts by volume tank of stainless steel charged with 100000 parts by volume of a fermentation medium (5% of mannitol~3~ o~
dried yeast~ 0.5% of MgC12 and 0.1% of CaCO39 pH 6.5) and sterilized, The initial phase of cultlvation was conducted at 26C, 100000 parts by ~olu~e/min. aeration, 180 r.p.m. (1~2 DT) and internal pressure 1 kg~cm2 for 18 hoursO Thereafter9 the cultivation was further continued for 106 hours at 24C and 200 r.p.m. (1/2 DT) and under otherwise the same condi.tions as above. At the end of the above cultivation time, the activity of the filtrate was 310 ~g/ml and that of the cells was 100 ~/cells in 1 ml o~ broth.
Examp]e 3 m e culture broth obtained in Example 1 (95000 part~
by volume) was admixed well with 2000 parts of Hyflo-Supercel (Johnes Manville Productsg U.S,A.). The m~xture filtered on a ~ilter press to obtain 85000 parts by volume of filtrate and 31000 parts of moist cells.
The filtrate (85000 parts by volume) was extracted with 30000 parts by volume of ethyl acetate under stirring and their procedure was repeated twice. The ethyl acetatelayers were pooledg washed twice with 30000 part,s by volume portions of water9 dried by the additlon of 500 parts o~ anhydrous sodium sulfate a~d concentrated . , .. . .... ,.. : . : : : . ~

to 200 parts by volume under reduced pressureO The concentrate was allowed tc stand in the cold rosm and the crude crystals of C-14919 E-2 were recovered by filtration (8.2 parts). The filtra~e was further concentrated to 50 parts by volume~ followed by addition of 300 parts by volume of petroleum ether. The resultant precipitate was recovered by ~iltration.
The resultant crude crystals (38 parts) were dissolved in 50 parts by volume of methanol and a~ter the addition of 5 parts of silica gel (Merck~ West Germany, 0.05-O.2 mm)g the methanol was distilled off under reduced pressure. The residue was put onto the top o~ a column of 500 parts by volume silica ~el ~same as above). The column was washed with 500 parts by volume of hexane and elution was carried out with 1000 parts by volume of hexane~ethyl acetate (1~ 1000 parts by volume of ethyl acetate and 1000 parts by volume of ethyl acetate-methanol ~30 1)9 the eluate belng collected in 100 parts by volume fractlonsO C-14919 E~l was eluted from'~he column in fraction~ No, 13 to No. 16, while C-14919 E-2 was eluted in fractions No. 21 to No. 23. The C-14919 E~l fractions (400 parts by volume) were pooled and concentrated and~ ~ter the addition of 80% aqueous methanol~ allowed to stand in the cold room~ The resultant crystals were collected by filtration. This procedure y~elded 10 parts crystals.

7~

The C-14913 E-2 ~ractions (300 parts by volume) were also pooled~ concentrated to 190 parts by volume and allowed to stand in the cold room. The crystals were recovered and dried~ By this procedure was obtained a 6.8 parts crop of C 14919 E-2 crystals, Separatelya 31000 parts of the moist cells were extracted twice with 40000 parts by volume portions Or 70% acetone water and the resultant extracts are pooled and concentrated under reduced pressure to remove the acetone. The aqueous extract was diluted with water to 30000 parts by volume and passed through a prepared column of 1000 parts by volume Diaion HP-10 (Mitsubishi Kasei) 9 whereby the active component was adsorbedO The column was washed wlth 2000 parts by volume of water and ~000 parts by volume of 40~ methanol-water in the order mentioned~ followed by elution with 2500 parts by volume of 60% methanol~water and 2500 parts by volume of 90% methanol-water in that orderO The eluate was collected in 250 parts by volume fractions and the fractions NosO 3 and 4 were combined, concentrated under re~uced pressure and allowed to stand in the cold room.
The resultant crude crystals of C~14919 E~2 were collected by ~iltration and dried (1.8 parts). The fractions Nos. 13, 14 and 15 were pooled, concentrated under reduced pressure andg after the addition of 80%
methanol-water~ allowed to stand in the cold room~ The d~

resultant crude crystals of C-14919 E-l were collected by filtration and dried (508 parts).
~ le 4 The filtrate obtained in Example 3 ~85000 parts by volume) was extracted twiGe with 30000 parts by volume portions of ethyl a,cetate as in Example 3 and the ethyl acetate layers were pooledg washed with 30000 parts by volume of water and concentrated under reduced pressure down to 500 parts by volume. To the concentrate was added 4000 parts by volume o~ methanol, followed by addition of 4000 parts b~ volume of water and 3000 parts by volume of hexane. After stirring~
the bottom layer was separated. To the bottom layer was added a solution of 20 parts ferric chloride in 8000 parts by volume of watera and with occasional stirring, the mixture was allowed to stand at room temperature for 5 hoursg at the end of which time it was cooled. The resultant crude crystals of C-14919 E-l were collected by ~iltration and dried (18 parts).
The filtrate was further extracted with 5000 parts by volume of ethyl acetateO The ethyl acetate solution was washed with water and concentrated. Upon addition of petroleum ether to the concentrate 13 parts of crude powders of C-14919 E-l were obtained. As in Example 3, the crude powders were chromatographed on a column (200 parts by volume) of siïica gel (Merck~ West Germanyg .

0.05 to 0.2 mm) using the solvents mentioned. By the above procedure was obtained 7.2 parts of crude C-14919 E-l cryskals.
Example 5 The C-14919 E-l crystals (0.400 part) obtained in Examples 3 and 4 were dissolved ln 40 parts by volume of ethyl acetate andg in a separatory funnel of 100 parts by volume capacity, 30 parts by volume o~ water was added. Then~ with 0.500 part of sodlum hydrosulfite (Na2S20LI ) a the solution was shaken intensively. The mixture was allowed to stand and the bottom layer was discarded. Then~ the upper layer was shaken well with 30 parts by volume of water and 0.500 part of sodium hydrosulfite (Na2S2O4)~ The bottom layer was discarded and the ethyl acetate layer was washed well with water and dried by the addition of anhydrous sodium sulfate.
After drying~ the ethyl acetate layer was concentrated to dryness under reduced pressure at low temperature.
The residue was dissolved by the addition of 10 parts by volume of methanol and filtered. Following the addition of 40 parts by volume of water, the flltrate was allowed to stand in the cold room. The resultant C-14919 E~2 crystals were collected by ~iltration and dried. m.p. 147-148C (decomp~) The C-14919 E-2 crystals (o.LIoO part~ obtained in . -,... . .... . .

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

Example 3 were dissolved in 60 parts by volume of methanolg ~ollowed by addition o~ 32 parts by volume of a 1% aqueous solution of ferric chloride. The mixture was allowed to stand at room temperature for 1 hour and the resultant C--14919 E-l crystals were collected by filtration~ washed with 20% methanol-water and dried. m.p. 186 187C (decomp.) Example 7 The crude crystals of C-14919 E-l (4 parts) obtained accordlng to Example 3 were clissolved in 500 parts by volume of methanol and9 after filterin~g 200 parts by volurne of water was added. The mixture was allowed to stand in the cold room and the resultant purified crystals of C-14919 E-l were collected by filtratlon and dried (208 parts). m.p. 187-188C
(decomp.) Similarlyg 3.5 parts of the crude C-14919 E~2 crystals obtained by the procedure of Example 3 were dissolved in 100 parts by volume of methanol andg after filterlng,500 parts by volume of water was added. The mixture was cooled and the resultant purified crystals of C-14919 E-2 were collected by flltration (2.1 parts).
m.p. 148C (decomp.) : -. ~ .: : . . . .
, . . . .

Claims (6)

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

1. A process for producing Antibiotic C-14919 E-l and/or E-2 which comprises cultivating an Antibiotic C-14919 E-l and/or E-2-producing strain of the genus Nocardia in a culture medium to cause said strain to elaborate and accumulate Antibiotic C-14919 E-l and/or E-2 in the resultant broth and harvesting said Antibiotic C-14919 E-l and/or E-2 from the cultured broth.

2. A method as claimed in Claim 1, wherein the microorganism is Nocardia No. C-14919 (ATCC 31280; IFO 13723; FERM-P No. 3991).

3. A method as claimed in Claim 1, wherein the antibiotic recovered is Antibiotic C-14919 E-l.

4. A method as claimed in Claim 1, wherein the antibiotic recovered is Antibiotic C-14919 E-2.

5. A process as claimed in Claim 4, wherein the resultant Antibiotic C-14919 E-2 is converted to Antibiotic C-14919 E-l by subjecting Antibiotic C-14919 E-2 to oxidation.

6. Antibiotics C-14919 E-l and E-2, which have the following properties.
(a) Antibiotic C-14919 F.-l i) m.p. 187°C (decomp.) ii) Appearance: yellow crystals (needles or prisms) iii) Solubility:
Insoluble in: petroleum ether, hexane, water Sparingly soluble in: diethyl ether, benzene Soluble in: ethyl acetate, chloroform, butanol, methyl isobutyl ketone, ethanol, acetone, methanol Readily soluble in: dimethylsulfoxide ii) Acid, neutral or basic: a neutral substance iii) Elemental analysis:

C, 65.31; 65.05; 64.85 (%) H, 7.71; 7.58; 7.62 N, 5.01; 4.95; 5.01 O, 21.37; 22.83; 22.45 vi) Empirical formula C30-32H42-48N2O8-9 vii) Ultraviolet absorption spectrum:
: 274(455), 240(sh), 397(43) : 274 (455), 240(sh.), 397(43) : 236(585), 265(500), 550(60) viii) Infrared absorption spectrum (KBr):
Dominant peaks (cm-1) at:
3430, 3340, 2950, 2910, 1740, 1692, 1660, 1645, 1605, 1500, 1375, 1315, 1120, 1100, 1085, 1060, 1025 ix) Specific rotation: + 350° + 10° (c=0.5, methanol) x) Color reactions Negative ninhydrin,Ehrlich, peptide and 1% iron chloride-1% ferricyanide (1:1) reactions (b) Antibiotie C-14919 E-2 i) m.p. 148°C (decomp.) ii) Appearance: Pale yellow crystals (needles or prisms) iii) Solubility:
Insoluble in: petroleum ether, hexane Sparingly soluble in: dlethyl ether, benzene chloroform, water Soluble in: ethyl acetate, butanol, methyl isobutyl ketone, ethanol, acetone, methanol Readily soluble: dimethylsulfoxide iv) Acid, neutral or basic: a neutral substance v) Elemental analysls:

C, 62.32; 62.07 (%) H, 8.58; 8.43 N, 4.82; 4.78 O, 20.81: 20.81 vi) Empirical formula: C30-32H44-50N208-9.xH2O
vii) Ultraviolet absorption spectrum:
: 255(295), 308(sh) : 255(290), 308(sh) : 236(505), 265(420), 550(50) viii) Infrared absorption spectrum (KBr):
Dominant peaks (cm-1) at:
3480, 3250, 2980, 1685, 1625, 1598, 1472, 1390, 1370, 1315, 1207, 1O90 1065, 1042, 1030 ix) Specific rotation: + 62° ? 4° (c=0.5, methanol) x) Color reactions Negative ninhydrin, Ehrlich and peptide reactions;
Positive 1% iron chloride-1% ferricyanide (1:1) reaction (blue) when prepared by the process of Claim 1 or 2, or by an obvious chemical equivalent thereof.