GB1602318A - Antibiotics c-14919 e-1 and e-2 - Google Patents

Abstract

The invention relates to the antibiotics C-14919 E-1 and E-2, which have antimicrobial and cytostatic properties, and to a process for their preparation by culturing a strain of the genus Nocardia, preferably of the novel strain Nocardia No. C-14919, which produces these antibiotics. According to the invention, the antibiotic C-14919 E-1 is also prepared by oxidation of the antibiotic C-14919 E-2, and the antibiotic C-14919 E-2 is also prepared by reduction of the antibiotic C-14919 E-1.

Description

(54) ANTIBIOTICS C-14919 E-1 AND E-2 (71) We, TAKEDA YAKUHIN KOGYO KABUSHIKI KAISHA, also known as TAKEDA CHEMICAL INDUSTRIES LIMITED, of 27 Doshomachi 2-chome, Higashiku, Osaka, Japan, a joint stock company of Japan; do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to novel Antibiotics C-14919 E-1 and E-2, and to a method of producing them.

We have collected many soil and other samples and performed a screening research for antibiotics on the microorganisms isolated from such samples. Our research shed light on the fact that certain of those microorganisms were able to produce novel antibiotic agents, the fact that such microorganisms belonged to the genus Nocardia and the fact that, by cultivating any of those microorganisms in a suitable nutrient medium, it was possible to have said antibiotic accumulated in the cultured broth. The novel antibiotics were designated as Antibiotics C-14919 E-1 and E-2, respectively. The above findings provided the impetus for further research which has resulted in the development of this invention.

This invention is, therefore, concerned with: (1) Antibiotic C-14919 E-1 and Antibiotic C-14919 E-2; and (2) A method of producing Antibiotic C-14919 E-1 and/or E-2 characterized in that said method comprises cultivating an Antibiotic C-14919 E-1 and/or E-2 producing strain of the genus Nocardia in a culture medium to cause said strain to elaborate and to accumulate Antibiotic C-14919 E-1 and/or E-2 in the resulting broth and harvesting said Antibiotic C-14919 E-1 and/or E-2 from the broth.

Throughout this specification, Antibiotic C-14919 E-1 and Antibiotic C-14919 E-2 will sometimes be referred to briefly as C14919 E-1 and C-14919 E-2, respectively.

The new Antibiotic C-14919 E-1 and/or E-2-producing microorganism 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-1 and/or E-2.

The microorganism, which we have isolated, is one of the preferred microorganisms which can be employed according to this invention.

The aforementioned microorganism is actinomycete 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 is a novel type of microorganism of the genus Nocardia as will be seen from the following description.

A) Microbiological Characteristics of Strain No. C-14919 The microbiological characteristics of Strain No. C-14919 were investigated by procedures analogous to those proposed by Schirling & Gottlieb [International Journal of Systematic Bacteriology 16, 313-340 (1966)j. The results of cultivation at 280C over 21 days are as follows; 1) Morphological characteristics The vegetative mycelium extends far, developing into branches, both on agar and in liquid media. Many of the hyphae measure 0.8 to 1.2 llm in diameter 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 jimx 200-1500 llm) on which the aerial growth takes place. Many of the aerial mycelia are flexuous or straight, with a loosely spiral-like configuration being found on a few occasions.

Microscopic examination of aged cultures reveals that, only in a few cases, does the conidia-like form occur in chains, while the cell suspensions obtained from the surfaces of such cultures, as viewed under a microscope, contain elongated ellipsoidal (0.8-1.2 x 3.7-6.7 Fm) and ellipsoidal (0.8-1.2 x 1.0-2.0 Rm) bodies resembling conidia.

Electron-microscopic examination showed that these bodies had smooth surfaces.

2) The constituents of the cell The strain was inoculated into a modified ISP No. 1 medium and cultured at 280C for 66 to 90 hours under shaking culture, at the end of which time the cells were collected and rinsed. By the method of B. Becker et al. [Applied Microbiology 12, 421(1964)] and the method of M.P. Lechevalier et al. [Journal of Laboratory and Clinical Medicine 71, 934(1968)], the above cells were examined for diaminopimelic acid and sugar composition.

The former was found to be the meso-form, while spots were in evidence which corresponded to galactose and arabinose.

3) Characteristics on taxonomical media The strain gives comparatively good growth on various media, with the vegetative mycelium being colourless to pale yellow in initial phases of culture and light yellowish tan to yellowish tan in later phases. Moreover, substantially no soluble pigments were produced 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 media are set forth in Table 1.

TABLE 1 Cultural characteristics of Strain No. C-14919 on taxonomical media (A) Sucrose nitrate agar Growth (G): Poor, colourless to light yellow Aerial mycelium (AM): None Soluble pigment (SP): None (B) Glycerol nitrate agar G: Poor, colourless to light yellow; coremia-like bodies formed.

AM: Scant, Lt Ivory (2cay SP: None (C) Glucose asparagine agar f G: Moderate, colourless to Brite Yellow (3na)*; coremia-like bodies formed.

AM: Scant, white to Lt Ivory (2ca)* SP: None (D) Glycerol aspargine agar G: Moderate, white to light orange yellow; coremia-like bodies formed.

AM: Scant, Lt Ivory (2ca)* SP: None (E) Starch agar G: Moderate, Lt Ivory (2ca)* to Lt Wheat (2ca)*; coremia-like bodies formed AM: Abundant, Lt Ivory (2ca)* SP: None (F) Nutrient agar G: Moderate, colonial Yellow (2ga)* to Brite Maize (31a)*; coremia-like bodies formed AM: Moderate, white to Pearl Pink (3ca)* SP: None (G) Calcium malate agar G: Moderate, light yellow to Colonial Yellow (2ga) *; coremia-like bodies formed AM: Moderate, light yellow to yellow SP: None Other characteristic: Calcium solubilize (H) Yeast extract-malt extract agar G: Moderate, Amber (3inc)*; coremia-like bodies formed AM: Moderate, white SP: None (I) Oatmeal agar G: Moderate, Amber (3inc) to Brite Yellow (3na)*; coremia-like bodies formed AM: Moderate, light yellow SP: None (J) Tyrosine agar G: Moderate, Amber (31c)B; coremia-like bodies formed AM: Scant, light yellow SP: None (K) Potato plug G: Moderate, Lt Amber (3ic)*; coremia-like bodies formed AM: None SP: The plug becomes pale yellowish tan (L) Peptone yeast extract iron agar G. Moderate, light yellowish tan to light orange yellow AM: None SP: None * The colour codes were according to the Color Harmony Manual, 4th ed.

(Container Corporation of America, 1958).

4) Physiological characteristics Physiological characteristics of the strain are shown in Table 2 just below. Temperature range for growth: 12"C to 38"C. The temperature range in which good aerial mycelium growth occurs on agar (ISP No. 2) is 20 to 32"C.

TABLE 2 The physiological characteristics of Strain No. C-14919 Temperature range for growth: 12"C-38"C Temperature range for sporulation: 20"C-28"C Liquefaction of gelatin: Positive Hydrolysis of starch: Positive Reduction of nitrates: Positive Peptonization of milk: Positive Coagulation of milk: Doubtful positive Decomposition of casein: Positive Production of melanoid pigments: Negative (peptone yeast extract iron agar), negative (tyrosine agar) Decomposition of tyrosine: Positive Decomposition of xanthine: Negative Decomposition of hypoxanthine: Negative Tolerance to lysozyme: Positive Tolerance to sodium chloride: 2% 5) Utilization of various carbon sources The utilization of various carbon sources was investigated using a medium described in Pridham and Gottlieb [Journal of Bacteriology 56, 107(1948)]. The resulting 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-Arabinose - Melibiose + D-Glucose + + i-Inositol D-Galactose + + D-Sorbitol D-Gructose +++ D-Mannitol + L-Rhamnose + Glycerol + D-Mannose + Soluble starch + Sucrose + + Control Lactose + Maltose + Trehalose + Note: +++: Luxuriant growth ++: Good growth Growth +: Poor growth No growth 6) Other characteristics The cells were harvested by the procedure previously described at (2) above and DNA was prepared by a procedure analogous to that of J. Murmar et al. [Journal of Molecular Biology, Vol. 3, 208, 1961]. The G-C content of the DNA was found to be about 71 mole %.

Gram-staining of the vegetative mycelium of this strain produced a positive reaction.

The above characteristics of strain No. 14919 were compared with the descriptions in S.A. Waksman's "The Actinomycetes Vol. 2" [The Williams and Wilkins Co., 1961]; R.E.

Buchanan and N.E. Gibbons, "Bergey's Manual of Determinative Bacteriology, 8th ed.", 1974; and other similar literature. While this strain was thought to belong to Group III of the genus Nocardia, failure to find among the known species any species having the characteristics so far described led us to conclude that it was a novel species of microorganism.

The present Strain No. C-14919 has been deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology (FERM), Japan, under FERM-P No. 3991; at the Institute for Fermentation, Osaka (IFO), Japan under the accession number of IFO 13723, and at The Americal Type Culture Collection (ATCC), Maryland, U.S.A. under the accession number of ATCC 31280.

While Strain No. C-14919 is a novel species of the genus Nocardia as just mentioned, it is liable, as are microorganisms generally, to undergo variations and mutations, either spontaneously or under the influence of a mutagen. For example, the many variants of the strain which are obtainable by the irradiation of the parent with X-rays, gamma rays, and ultraviolet light, etc., by monocell isolation, by cultivation on media containing various chemicals, or by other mutagenic treatments, as well as the mutants spontaneously obtained from the parent strain, may not be substantially considered to represent any other distinct species but, rather, any of such variants and mutants which are still able to elaborate C-14919 E-l and/or E-2 may be invariably utilized for the purposes of this invention. Thus, for example, subjecting 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 colourless or yellowish green, or orange red, mutants which produce abundant white mycelia are no mycelium at all, or mutants whose hyphae are liable to fragmentation.

The culture medium employed in the practice of this invention may be a liquid or a solid medium, provided it contains nutrients which the strain may utilize, although a liquid medium is preferred for high-production runs. The medium may contain carbon and nitrogen sources which Strain No. C-14919 may assimilate and digest, inorganic matter, trace nutrients, etc. As examples of the carbon sources there may be mentioned glucose, lactose, sucrose, maltose, dextrin, starch, glycerol, mannitol, sorbitol, and fats and oils (e.g. soybean oil, lard oil, chicken oil, etc.). The nitrogen sources may be for example meat extract, yeast extract, dried yeast, soybean meal, corn steep liquor, peptone, cottonseed flour, spent molasses, urea or ammonium salts (e.g. ammonium sulphate, ammonium chloride, ammonium nitrate or ammonium acetate. The medium may further contain salts of sodium, potassium, calcium or magnesium, metal salts of iron, manganese, zinc, cobalt or nickel, and organic acid salts such as acetates or propionates. Further, the medium may contain, as added, various additives such as aminoacids (e.g. glutamic acid, aspartic acid, alanine, lysine, methionine or proline), peptides (e.g. dipeptides or tripeptides), vitamins (e.g. B1, B2, nicotinic acid, B12 or C) or nucleic acids (e.g. purine, pyrimidine and the corresponding derivatives). For the purpose of adjusting the pH of the medium. there may be added an inorganic or organic acid, an alkali, a buffer or the like. Suitable amounts of oils and fats and of surfactants may also be added as "antiforms".

The cultivation may be conducted by any of the stationary, shake, aerobic submerged and other cultural methods. For high production runs, submerged aerobic culture is preferred. While the conditions of culture depend on the condition and the composition of the medium, the strain used, the cultural method and other like factors, it is normally preferred to carry out incubation at 20 to 320C with an initial pH near neutral. A temperature of from 23 to 26"C in an intermediate stage of cultivation, with an initial pH of 6.0 to 7.0 is particularly desirable. While the incubation time is also variable according to these factors, it is advisable to continue the incubation until the titer of the desired antibiotic elaboration product is maximal. In the case of shake culture or aerobic submerged culture in a fluid medium, the time required normally ranges from 72 to 192 hours.

The pH suited to the production of the antibiotics is near neutral, the optimum range being pH 6.5 to pH 7.0. 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 composition. Thus, the yield of the antibiotics increases considerably as medium ingredients, e.g. inorganic phosphates are added in amounts greater than are necessary for the growth of the strain, i.e. 1000 to 7000 ppm., or certain assimilable carbohydrates which are ready to form acids, e.g. glucose or mannitol, are employed as sources of carbon, together with suitably chosen sources of nitrogen.

Antibiotics C-14919 E-1 and E-2, thus elaborated by the present strain, normally occur extracellularly but there are cases in which the cells contain from 10 to 30 percent of the total yield of the antibiotics. The potency titration of the antibiotic agents can be accomplished by the cylinder method or the paper disk method, which employs Candida albicans IFO 0583 as an assay organism and TSA (Trypticase soy agar, BBL) as an assay medium.

The antibiotics occur in the filtrate and in cells which may be obtained from the cultured broth. Thus, the broth is filtered or centrifuged to recover the cells and the filtrate or supernatant independently. The potency of the filtrate is assayed directly and that of the cells is assayed after they have been stirred with the same volume of 70% acetone-water as the filtrate at 20"C for one hour. The assay method is that described hereinbefore, the assay organism being Candida albicans.

Because C-14919 E-1 and E-2, which are produced in the fermentation broth, are lipophyl and neutral substances, they can be conveniently recovered by separation and purification procedures which are normally employed for the harvesting of such microbial metabolites. For example, there may be employed a procedure which utilizes the difference in solubility between the antibiotic and impurities, a means which utilizes the difference between the antibiotic and impurities in the adsorptive affinity of various adsorbents such as activated carbon, macroporous non-ionic resins, silica gel and alumina, or a procedure for removing the impurities by means of ion-exchange resins. These procedures may be used alone, in suitable combinations or applied in repetition. Since, as aforementioned, C-14919 E-1 and E-2 occur in both the filtrate and cells, any of the following procedures may be adopted: (1) The culture broth as such, before separation of the cells, is extracted with a solvent, or (2) the cells and the filtrate or supernatant as obtained by filtration or centrifuging are independently extracted with solvents.

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 be mentioned organic solvents which are immiscible with water, such as fatty acid esters, e.g. ethyl acetate or amyl acetate; alcohols, e.g. butanol; halogenated hydrocarbons, e.g.

chloroform; and ketones, e.g. methyl isobutyl ketone. The extraction is carried out in the neighbourhood of neutrality and, preferably, is accomplished with ethyl acetate from the broth preadjusted to pH 7. After the extract has been washed with water and concentrated, C-14919 E-2 is obtained as crude crystals. The filtrate remaining after the separation of the crude crystals is rich in E-1.

The extraction from the cells may be accomplished by using a mixture of water with a water-miscible organic solvent, such as a mixture of water with a lower alcohol (e.g.

methanol or ethanol) or a mixture of water with a ketone (e.g. acetone or methyl ethyl ketone); or by means of a water-immiscible organic solvent such as a halogenated hydrocarbon (e.g. methylene dichloride). It is, however, advantageous to employ 70% acetone-water. Thus, the cells are reconstituted with the same volume of 70% acetonewater as the corresponding filtrate, and the mixture is stirred at room temperature for 3 hours. By this procedure, all the C-14919 E-1 and E-2 occurring in the cells are extracted.

The solvent extracts are pooled and the solvent, e.g. acetone, is removed under reduced pressure. The aqueous solution is extracted with ethyl acetate as in the case of the filtrate and the solvent layer is washed with water and concentrated under reduced pressure.

Hexane or the like is added to the concentrate to precipitate the active components. A mixture of C-14919 E-1 and E-2 can thus be obtained by centrifuging or filtration.

Separation of the mixture into components may be accomplished by adsorption chromatography on various adsorbents. For this purpose, various carriers commonly used for the adsorption of antibiotic substances can be employed. For example, adsorbent resins, silica gel or alumina may be mentioned. A mixture of water and a water-miscible organic solvent such as a lower alcohol or lower ketone may be used to elute the antibiotics from such adsorbent resins. As the lower alcohol these may be mentioned methanol, ethanol, propanol or butanol, and the lower ketone may be for example, acetone or 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 (trade mark) HP-10 (Mitsubishi Kasei). The column is first washed with 40% methanol-water and, then, elution is carried out with 60% methanol-water, whereby the C-14919 E-2 fraction is obtained. Elution with 90% methanol-water yields the C-14919 E-1 fraction. Each fraction is concentrated under reduced pressure and, after the addition of methanol, allowed to stand. The procedure yields crystals of C-14919 E-1 and crystals of C-14919 E-2 from the corresponding fractions. Where silica gel is used as the adsorbent, development can be started using a nonpolar solvent with the addition of a polar solvent, e.g. methanol, in small increments, whereby C-14919 E-1 and E-2 are eluted. The crystals of C-14919 E-1 are recrystallized 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-1 and E-2, which can thus be obtained, are convertible to each other by reversible oxidation-reduction. Thus, whilst the reduction of C-14919 E-1 with a reducing agent (e.g. hydrosulfite, ascorbic acid, NaBH4 or zinc with acetic acid) yields C-14919 E-2, oxidation of C-14919 E-2 with an oxidising agent (e.g. ferric chloride, nitric acid, AgO2 MnO2 or air) yields C-14919 E-1.

The physical and chemical properties of the novel antibiotics C-14919 E-1 and E-2 crystals obtained in the above manner are as follows (Table 4).

Reference should also be made in this connection to Figures 1 and 2 of the accompanying drawings which are graphs of variations om tramsmittance relative to wave number (cm TABLE 4 C-14919 E-1 C-1491.9 E-2 2) m.p. 187"C (decomp.) 148"C (decomp.) 2) Appearance yellow crystals pale yellow crystals (needles or prisms) (needles or prisms) 3) Solubility Insoluble in: Insoluble in: petroleum ether, petroleum ether, hexane, water hexane Sparingly soluble in: Sparingly soluble in: diethyl ether, diethyl ether, benzene benzene, water, chloroform Soluble in: Soluble in: ethyl acetate, ethyl acetate, chloroform, butanol butanol, methyl methyl isobutyl isobutyl-ketone, ketone, ethanol, ethanol, acetone, acetone, methanol methanol Readily soluble in: Readily soluble in: dimethylsulfoxide dimethylsulfoxide 4) Acid, neutral a neutral substance a neutral substance or basic 5) Elemental C 65.31, 65.05, 64.85 C 62.32, 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, 22.45 0 20.81, 20.81 6) Empirical C30~32H42~48N208-9 C3O-32H44-50N2 formula 08~9-xH20 (x = 0-1) 7) Ultra-violet absorption spectrum #maxMeOH.N-HCl(9:1)(nm)(E1%1cm) 274(455),240(sh),397(43) 255(295) ,308(sh) BMa H(nm)(E, ) 274(455) ,240(sh) ,397(43) 255(290)308(sh).

#maxMeOHN-NaOH(9:1)(nm)(E:1%1cm) 236(585) ,265(500) ,550(56) 236(505,265(420) ,550(50) 8) Infrared absorption Figure 1 Figure 2 spectrum (KBr) Dominant peaks (cm-l) Dominant peaks (cm-l) 3430,3340,2950 3480,3250,2980 2910,1740,1692 1685,1625,1598, 1660,1645,1605, 1472,1390,1370 1500,1375,1315 1315,1207,1090, 1120,1100,1085, 1065,1042,1030 1060,1025 9) Specific rotation [a]D5 + 350 + 10 [a]25 + 62 + 4 (C=0.5,methanol) (C=0.5,methanol) 10) Colour reactions Negative: Negative: ninhydrin reaction, ninhydrin reaction, Ehrlich reaction, Ehrilich reaction, Peptide reaction, peptide reaction magnesium acetate and magnesium reagent and 1% acetate reagent iron chloride-1% ferricyanide(1:1) Positive: reagent 1% iron chloride -1% ferricyanide Potassium per- (l:l)reagent(blue) manganate reagent, discoloured Potasium per manganate reagent, discoloured Number Number of of 11) (ppm) J(Hz) protons (ppm) J(Hz) protons Nuclear 0.63 d 6.5 3H 0.63 d 7 3H magnetic resonance 0.94 d 6.5 3H 0.82 d 7 3H spectrum (100 mega- 0.96 d 6.5 3H 0.89 d 7 3H cycles in dimethyl- 1.4 s 3H 1.16 s 3H sulfoxide 1.88 s 3H 1.55 m 1.4-1.8 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 3.13 s 3H 3.43 s 3H 3.24 s 3H 3.3 m 3.2 m 4.5 s 1H 3.39 s 3H 5.21 d 10 1H 4.54 d 5 1H 5.28 d 1H 4.64 d 8 1H 5.59 dd 8.10 1H 5.02 d 8 1H 6.22 t 11 1H 5.21 d 10 1H 6.32 s (disap- 1H 5.86 t 10 1H pears by ad- 5.98 s. like 1H dition of D2O) 6.23 s (disap- 1H pears 6.41 d 2 1H by ad dition 6.81 d 2 1H of D2O) 6.95 d 11 1H 6.35 d 2.5 1H 9.3 s (disap- 1H 6.45 d 2.5 1H pears by ad- 7.51 s (disap- 1H dition pears of D2O) by ad dition of D2O) 8.64 s ( do. ) 1H 8.85 s ( do. ) 1H 12) Stability Room temp.: Room temp.: Stable in neutral Stable under neutral and weakly and weakly acid acidic solutions; conditions; Unstable in basic Unstable under basic solution. conditions.

80"C, lhr.: 80"C, 1Hr.: unstable unstable 13) Thin layer chromato graphy Adsorbent: Silica Gel, Spot Film(Tokyo Kasei) Chloroform-methanol (9:1) 0.85 0.50 Chloroform-methanol (19:1) 0.77 0.22 ethyl acetate acetone (9:1) 0.80 0.50 Ethyl acetate methanol (9:1) 0.85 0.78 Antimicrobial activity The antimicrobial activities of Antibiotics C-14919 E-1 and E-2 were assayed by the serial dilution method using Trypticase-soy agar (BBL), glucose nutrient agar and glycerol nutrient agar as test media (Table 5). In addition, the activities against Tetrahymena pyriformis W were determined by the serial dilution method using the assay medium Proteosepeptone 20 g, yeast extract 1 g, glucose 2 g, distilled water 1000 ml, 1 M-phosphate buffer (pH 7.0) 10 ml].

TABLE 5 C-14919E-1 C-14919E-2 Assay organisms IFO No.

MIC (llg/ml) MIC(Ilg/ml) *Escherichia coli K-12 3301 > 100 > 100 Proteus vulgaris 3045 > 100 > 100 Pseudomonas aeruginosa 3080 > 100 > 100 Salmonella typhimurium 12529 > 100 > 100 Alcaligenes faecalis 13111 > 100 100 Serratia marcescens 3046 > 100 > 100 Bacillus pumilus 3813 50 100 Bacillus subtilis 6633 3134 50 100 Bacillus subtilis PC1219 3513 100 > 100 Bacillus cereus 3514 > 100 > 100 Bacillus megaterium 12108 50 50 Bacillus brevis 3331 50 50 Staphylococcus aureus 209P 12732 > 100 > 100 Sarcina lutea 3232 50 50 Micrococcus flavus 3342 50 50 Candida albicans 0583 25 6.25 **Candida tropicalis 1400 > 100 > 100 Candida pseudotropicalis 0617 100 100 Candida utilis 0619 > 100 > 100 Candida parapsilosis 1396 50 25 Candida krusei 1395 > 100 > 100 Candida albicans 0583 50 25 Cryptococcus neoformans 0410 > 100 > 100 Saccharomyces cerevisiae 0209 100 100 Microsporum canis 7863 > 100 > 100 Microsporum cookei 8303 > 100 > 100 Aspergillus niger 4066 100 100 Trychophyton rubrum 5467 > 100 > 100 Ho rmoden drum pedrosoi 6071 > 100 > 100 Penicillium chrysogenum 4626 50 25 ***Mycobacterium avium > 100 > 100 Mycobacterium phlei > 100 > 100 **** Tetrahymena pyriformis W 10 10 Note: Medium * Trypticase-soy agar ** Glucose nutrient agar Glycerol nutrient agar * * * * Proteose-peptone solution Toxicity The assumed LD50 as determined by intraperitoneal administration of C-14919 E-1 and E-2 in an acute toxicity test using mice as test animals was as follows.

C-14919 E-1 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-1 and 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.

Antitumour activity The therapeutic activity (intraperitoneally administered for a consecutive days) of C-14919 E-1 and E-2 against P388 leukemia in mice (1 x 106 cells/animal, mouse, intraperitoneally 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 antitumour agents.

By formulating the present Antibiotic C-14919 E-1 or E-2 into an aqueous solution containing 10 to 100 Fg/ml of aqueous ethanol (e.g. containing 5% ethanol), it can be used e.g. for the disinfection of bird cages, laboratory equipment and human hands.

The following examples are further illustrative but by no means limitative of the invention, 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 "milliliter(s)", and "%" is based on "weight/volume" unless otherwise noted.

Example 1 Strain No. C-14919 (ATCC 31280, IFO 13723; FERM-P No. 3991) was inoculated onto a tyrosine agar medium, followed by incubation at 28"C for 240 hours. The well-grown mycelium was suspended in a 0.6% solution of sodium glutamate and stored in a refrigerator. A 1 part by volume-portion of 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 glucose, 30 parts of soluble starch, 10 parts of corn steep liquor, 10 parts of soybean flour, 5 parts of peptone, 3 parts of sodium chloride and 5 parts of calcium carbonate/water 1000 parts by volume, pH 7.0). The inoculated medium was incubated on a r pressure 1 kg/cm2, 48 hrs. A 10,000 parts by volume portion of the resulting preculture was used to seed a 200,000 parts by volume tank of stainless steel charged with 100,000 parts by volume of a fermentation medium (5% of glycerol, 2% of corn steep liquor, 2% of yeast extract, 2% of KH2PO4, 0.5% of MgCl2 and 0.1% of CaCO3, pH 6.5) and sterilized. The initial phase of fermentation was carried out under the following conditions: temperature 28"C, 100,000 parts by volume/min. aeration, 180 r.p.m. (1/2 DT), internal pressure 1 kg/cm2, 24 hours, whereby the microorganism was allowed to grow well. Thereafter, the cultivation was further carried out for 114 hours under the same conditions as above except that the incubation temperature was 24"C and the stirring was effected at 200 r.p.m. (/2 DT). At the end of the above period, the mycelia were separated from the filtrate by filtration. The filtrate contained 380 llg/ml of activity, while the titer in the mycelia was 90 llg/cells in one part of broth. The potency titrations were carried out by the cylinder method or the paper disc method using Candida albicans IFO 0583 as an assay organism and C-14919 E-1 as a standard test material.

Example 2 The procedures described in Example 1 were duplicated up to the seed culture stage. The cultivation was carried out in a 200,000 parts by volume tank of stainless steel charged with 100,000 parts by volume of a fermentation medium (5% of mannitol, 3% of dried yeast, 0.5% of MgCl2 and 0.1% of CaCO3, pH 6.5) and sterilized. The initial phase of cultivation was conducted at 26OC, 100,000 parts by volume/min. aeration, 180 r.p.m. (1/2 DT) and internal pressure 1 kg/cm2 for 18 hours. Thereafter, the cultivation was further continued for 106 hours at 24"C and 200 r.p.m. ( DT) and under otherwise the same conditions as above. At the end of the above cultivation time, the activity of the filtrate was 310 llg/ml and that of the cells was 100 jigkells in 1 ml of broth.

Example booth obtained in Example 3 The culture broth obtained in Example 1(95,000 parts by volume) was thoroughly admixed well with 2000 parts of Hyflo-Supercel (trade mark), (Johns Manville Products, U.S.A.). The mixture was filtered on a filter press to yield 85,000 parts by volume of filtrate and 31,000 parts of moist cells. The filtrate (85,000 parts by volume) was extracted with 30,000 parts by volume of ethyl acetate under stirring and the procedure was repeated twice. The ethyl acetate layers were pooled, washed twice with 30,000 parts by volume portions of water, dried by the addition of 500 parts of anhydrous sodium sulphate and concentrated to 200 parts by volume under reduced pressure. The concentrate was allowed to stand in the cold room and the crude crystals of C-14919 E-2 were recovered by filtration (8.2 parts). The filtrate was further concentrated to 50 parts by volume, followed by the addition of 300 parts by volume of petroleum ether. The resulting precipitate was recovered by filtration. The resulting crude crystals (38 parts) were dissolved in 50 parts by volume of methanol and, after the addition of 5 parts of silica gel (Merck, West Germany, 0.05-0.2 mm), the methanol was distilled off under reduced pressure. The residue was put onto the top of a column of 500 parts by volume of silica gel (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 (1:1), 1000 parts by volume of ethyl acetate and 1000 parts by volume of ethyl acetate-methanol (30:1), the eluate being collected in 100 parts by volume fractions. C-14919 E-1 was eluted from the column in fractions No. 13 to No. 16, while C-14919 E-2 was eluted in fractions No. 21 to No. 23. The C-14919 E-1 fractions (400 parts by volume) were pooled and concentrated and, after the addition of 80% aqueous methanol, allowed to stand in the cold room. The resulting crystals were collected by filtration. This procedure yielded 10 parts of crystals. The C-14919 E-2 fractions (300 parts by volume) were also pooled, concentrated to 100 parts by volume and allowed to stand in the cold room. The crystals were recovered and dried. By this procedure there was obtained a 6.8 parts crop of C-14919 E-2 crystals.

Separately, 31,000 parts of the moist cells were extracted twice with 40,000 parts by volume portions of 70% acetone water and the resulting extracts were pooled and concentrated under reduced pressure to remove the acetone. The aqueous extract was diluted with water to 30,000 parts by volume and passed through a prepared column of 1,000 parts by volume of Diaion (trade mark) HP-10 (Mitsubishi Kasei), whereby the active component was adsorbed. The column was washed with 2,000 parts by volume of water and 2,000 parts by volume of 40% methanol-water in the order mentioned, followed by elution with 2,500 parts by volume of 60% methanol-water and 2,500 parts by volume of 90% methanol-water in that order. The eluate was collected in 250 parts by volume fractions and the fractions Nos. 3 and 4 were combined, concentrated under reduced pressure and allowed to stand in the cold room. The resulting crude crystals of C-14919 E-2 were collected by filtration and dried (1.8 parts). The fractions Nos. 13, 14 and 15 were pooled, concentrated under reduced pressure and, after the addition of 80% methanol-water, allowed to stand in the cold room. The resulting crude crystals of C-14919 E-1 were collected by filtration and dried (5.8 parts).

Example 4 The filtrate obtained in Example 3 (85,000 parts by volume) was extracted twice with 30,000 parts by volume portions of ethyl acetate as in Example 3 and the ethyl acetate layers were pooled, washed with 30,000 parts by volume of water and concentrated under reduced pressure down to 500 parts by volume. 4,000 parts by volume of methanol were added to the concentrate, followed by the addition of 4,000 parts by volume of water and 3,000 parts by volume of hexane. After stirring, the bottom layer was separated. A solution of 20 parts ferric chloride in 8,000 parts by volume of water was added to the bottom layer, and, with occasional stirring, the mixture was allowed to stand at room temperature for 5 hours, at the end of which time it was cooled. The resulting crude crystals of C-14919 E-1 were collected by filtration and dried (18 parts). The filtrate was further extracted with 5,000 parts by volume of ethyl acetate. The ethyl acetate solution was washed with water and concentrated. Upon the addition of petroleum ether to the concentrate, 13 parts of crude powders of C-14919 E-1 were obtained. As in Example 3, the crude powders were chromatographed on a colum (200 parts by volume) of silica gel (Merck, West Germany, 0.05 to 0.2 mm) using the solvents mentioned. 7.2 parts of crude C-14919 E-1 crystals were obtained by the above procedure.

Example 5 The C-14919 E-1 crystals (0.400 part) obtained in Examples 3 and 4 were dissolved in 40 parts by volume of ethyl acetate and, in a separatory funnel of 100 parts by volume capacity, 30 parts by volume of water were added. Then, with 0.500 part of sodium hydrosulphite (Na2S204), the solution was shaken intensively. The mixture was allowed to stand and the bottom layer was discarded. The upper layer was then thoroughly shaken with 30 parts by volume of water and 0.500 part of sodium hydrosulphite (Na2S204). The bottom layer was discarded and the ethyl acetate layer was thoroughly washed with water and dried by the addition of anhydrous sodium sulphate. After drying, the ethyl acetate layer was concentrated to dryness under reduced pressure at a 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 filtrate was allowed to stand in the cold room.

The resulting C-14919 E-2 crystals were collected by filtration and dried. m.p. 147-148"C (decomp.) Example 6 The C-14919 E-2 crystals (0.400 part) obtained in Example 3 were dissolved in 60 parts by volume of methanol, followed by the addition of 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 resulting C-14919 E-1 crystals were collected by filtration, washed with 20% methanol-water and dried. m.p. 186-187"C (decomp.) Example 7 The crude crystals of C-14919 E-1 (4 parts) obtained according to Example 3 were dissolved in 600 parts by volume of methanol and, after filtering, 200 parts by volume of water were added. The mixture was allowed to stand in the cold room and the resulting purified crystals of C-14919 E-1 were collected by filtration and dried (2.8 parts). m.p.

187-188"C (decomp.) Similarly, 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 and, after filtering 500 parts by volume of water were added. The mixture was cooled and the resulting purified crystals of C-14919 E-2 were collected by filtration (2.1 parts) m.p. 148"C (decomp.) WHAT WE CLAIM IS: 1. Antibiotics C-14919 E-1 and/or E-2, which have the following properties: (a) Antibiotic C-14919 E-1 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: dimethylsulphoxide

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (7)

1. **WARNING** start of CLMS field may overlap end of DESC **.

   concentrated under reduced pressure and, after the addition of 80% methanol-water, allowed to stand in the cold room. The resulting crude crystals of C-14919 E-1 were collected by filtration and dried (5.8 parts).

   Example 4 The filtrate obtained in Example 3 (85,000 parts by volume) was extracted twice with 30,000 parts by volume portions of ethyl acetate as in Example 3 and the ethyl acetate layers were pooled, washed with 30,000 parts by volume of water and concentrated under reduced pressure down to 500 parts by volume. 4,000 parts by volume of methanol were added to the concentrate, followed by the addition of 4,000 parts by volume of water and 3,000 parts by volume of hexane. After stirring, the bottom layer was separated. A solution of 20 parts ferric chloride in 8,000 parts by volume of water was added to the bottom layer, and, with occasional stirring, the mixture was allowed to stand at room temperature for 5 hours, at the end of which time it was cooled. The resulting crude crystals of C-14919 E-1 were collected by filtration and dried (18 parts). The filtrate was further extracted with 5,000 parts by volume of ethyl acetate. The ethyl acetate solution was washed with water and concentrated. Upon the addition of petroleum ether to the concentrate, 13 parts of crude powders of C-14919 E-1 were obtained. As in Example 3, the crude powders were chromatographed on a colum (200 parts by volume) of silica gel (Merck, West Germany, 0.05 to 0.
2. 2 mm) using the solvents mentioned. 7.2 parts of crude C-14919 E-1 crystals were obtained by the above procedure.

   Example 5 The C-14919 E-1 crystals (0.400 part) obtained in Examples 3 and 4 were dissolved in 40 parts by volume of ethyl acetate and, in a separatory funnel of 100 parts by volume capacity, 30 parts by volume of water were added. Then, with 0.500 part of sodium hydrosulphite (Na2S204), the solution was shaken intensively. The mixture was allowed to stand and the bottom layer was discarded. The upper layer was then thoroughly shaken with 30 parts by volume of water and 0.500 part of sodium hydrosulphite (Na2S204). The bottom layer was discarded and the ethyl acetate layer was thoroughly washed with water and dried by the addition of anhydrous sodium sulphate. After drying, the ethyl acetate layer was concentrated to dryness under reduced pressure at a 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 filtrate was allowed to stand in the cold room.

   The resulting C-14919 E-2 crystals were collected by filtration and dried. m.p. 147-148"C (decomp.) Example 6 The C-14919 E-2 crystals (0.400 part) obtained in Example 3 were dissolved in 60 parts by volume of methanol, followed by the addition of 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 resulting C-14919 E-1 crystals were collected by filtration, washed with 20% methanol-water and dried. m.p. 186-187"C (decomp.) Example 7 The crude crystals of C-14919 E-1 (4 parts) obtained according to Example 3 were dissolved in 600 parts by volume of methanol and, after filtering, 200 parts by volume of water were added. The mixture was allowed to stand in the cold room and the resulting purified crystals of C-14919 E-1 were collected by filtration and dried (2.8 parts). m.p.

   187-188"C (decomp.) Similarly, 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 and, after filtering 500 parts by volume of water were added. The mixture was cooled and the resulting purified crystals of C-14919 E-2 were collected by filtration (2.1 parts) m.p. 148"C (decomp.) WHAT WE CLAIM IS: 1. Antibiotics C-14919 E-1 and/or E-2, which have the following properties: (a) Antibiotic C-14919 E-1

   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: dimethylsulphoxide

   iv) Acid, neutral or basic: a neutral substance v) Elemental analysis: C, 65.31; 65.05; 64.85 (%) H, 7.71; 7.58; 7.62 N, 5.01; 4.95; 5.01 0, 21.37; 22.83; 22.45 vi) Empirical formula: C30-32H42-48N2O8-9 vii) Ultraviolet absorption spectrum: #maxMeOH: N-HCI(9: 1(nm) (El1c%n) :274(455), 240(sh.), 397(43) #maxMeOH(nm)(E1cm1%): 274(455), 240(sh.), 397(43) #maxMeOH N-NaOH(9:1)(nm)(E1cm1%): 236(585), 265(500), 550(56) 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: [alss5 + 350 + 10 (c=0.5, methanol) x) Colour reactions Negative ninhydrin, Erhlich, peptide and 1% iron chloride-1% ferricyanide (1:1) reactions (b) Antibiotic 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: diethyl ether, benzene, chloroform, water Soluble in: ethyl acetate, butanol, methyl isobutyl ketone, ethanol, acetone, methanol Readily soluble: dimethylsulphoxide iv) Acid, neutral or basic: a neutral substance v) Elemental analysis: C, 62.32; 62.07 (%) H, 8.58; 8.43 N, 4.82; 4.78 0, 20.81; 20.81 vi) Empirical formula: C30-32H44-50N2O8-9#xH2O (x=0-1) vii) Ultraviolet absorption spectrum: MeOH: N-HCl(9:1)(nm)(E1cm1%): 255(295), 308(sh.) ) H(nm)(El ): 255(290), 308(sh.) #maxMeOH:N-NaOH(nm)(E1cm2%): 236(505), 265(420), 550(50) viii) Infrared absorption spectrum (KBr): Dominant peaks (cm-l) at: 3480, 3250, 2980, 1685, 1625, 1598, 1472, 1390, 1370, 1315, 1207, 1090, 1065, 1042, 1030 ix) Specific rotation: [a]25 + 62 + 4 (c=0.5, methanol)

   x) Colour reactions Negative ninhydrin, Erhlich and peptide reactions; Positive 1% iron chloride-1% ferricyanide (1:1) reaction (blue) 2. A method of producing Antibiotic C-14919 E-1 and/or E-2 characterized in that said method comprises cultivating an Antibiotic C-14919 E-1 and/or E-2-producing strain of the genus Nocardia in a culture medium to cause said strain to elaborate and to accumulate Antibiotic C-14919 E-1 and/or E-2 in the resulting broth and harvesting said Antibiotic C-14919 E-1 and/or E-2 from the cultured broth.
3. 3. A method as claimed in Claim 2, wherein the resulting Antibiotic C-14919 E-2 is converted to Antibiotic C-14919 E-1 by subjecting Antibiotic C-14919 E-2 to oxidation.
4. 4. A method as claimed in Claim 2 or 3, wherein the microorganism is Nocardia No.

   C-14919 (ATCC 31280; IFO 13723; FERM-P No. 3991).
5. 5. A method according to claim 2 substantially as herein described with reference to any of the specific examples.
6. 6. Antibiotics C-14919 E-1 and E-2 when produced by a method as claimed in any of claims 1 to 5.
7. 7. A pharmaceutical composition comprising Antibiotic C 14919 E-1 and/or Antibiotic C 14919 E-2 together with a non-toxic, pharmaceutically acceptable carrier and/or diluent therefor.