DE2813416C2 - New antibiotics C-14919 E-1 and E-2 and processes for their preparation - Google Patents

Description

The invention relates to new antibiotics with the designations C-14919 E-I and E-2 and a method for their Manufacturing.

A large number of soil and other samples have been taken by the applicant, and those of Microorganisms isolated from these specimens were subjected to a screening test for that produced by them Subjected to antibiotics. The research led to the finding that certain microorganisms from these samples new antibiotics are able to form that these microorganisms belong to the genus Nocardia and that it is possible by cultivating these microorganisms in a suitable nutrient medium is to enrich the antibiotics in the culture medium. The new antibiotics were called Antibiotics C-14919 E-I and E-2

The invention accordingly

1) the antibiotic C-14919 E-I and there? Antibiotic C-14919 E-2 as well

2) the production of the antibiotics C-14919 E-I and / or E-2 by a process that is characterized is that Nocardia IFO 13723 is cultivated in a nutrient medium and the antibiotics C-14919 E-I and / or E-2 with the usual separation and cleaning methods for lipophilic and neutral substances isolated or the obtained antibiotic C-14919 E-2 by oxidation into the antibiotic C-14919 E-I converts

In the present specification, antibiotic C-14919 E-I and antibiotic C-14919 E-2 sometimes referred to as C-14919 E-I or C-14919 E-2 for short.

The microorganism used for the purposes of the invention which is the new antibiotic C-14919 E-I and / or E-2 is the actinomycete strain No. C14919 (hereinafter sometimes referred to as strain No. C-14919), which, as the following description shows, is a new type of microorganism of the genus * ■

Nocardia is.

25th

A) Microbiological characteristics of strain C-14919

The microbiological characteristics of the strain no. C-14919 were examined by methods which are analogous to the methods described by Schirling and Gottiieb (International Journal of Systematic Bacteriology 16 [1966] 313-340). The cultivation during a period of 21 days at 28 ⁰ C had the following results:

1) Morphological properties

The vegetative mycelium spreads widely and develops branches both on agar and in liquid Media. Many of the hyphae are 0.8 to 1.2 μm in diameter, and the hyphae are dependent fragmented by the cultivation conditions.

The strain shows good growth on various taxonomic media, with the aerial mycelium superimposing the vegetative mycelium, but forming coremia-like bodies (50 to 200 μm x 200 to 1500 am) on which the air growth occurs. Much of the air mycelium is tortuous or straight, with a few cases of loosely spiral shape. Examination of aged cultures under the microscope shows that the conidia-like shape occurs in chains only in a few cases, while examination of the cell suspensions obtained from the surfaces of these cultures under the microscope shows that they have elongated elliptical bodies (0.8-1.2 x 3.7-6.7 μτη) and elliptical bodies that resemble Konidisn (0.8-1.2 x 1.0-2.0 μπι) contain.

Examination under the electron microscope revealed that these bodies had smooth surfaces.

2) Components of cells

The strain was inoculated into modified ISP No. 1 medium and shaken for 66 to 90 hours cultured at 28 ° C. After this time the cells were separated and rinsed. They were then after the Method of B. Becker and coworkers (Applied Microbiology 12 [1964] 421) and according to the method of M. P. Lechevalier et al. (Journal of Laboratory and Clinical Medicine 71 [1968] 934) on diaminopimelic acid and sugar composition examined. It has been found that the former is in the meso form was present while localized sites corresponding to galactose and arabinose were clearly noted became.

3) Merkmaie on taxonomic media

The strain shows relatively good growth on various media, with the vegetative mycelium in the initial phases of the culture colorless to pale yellow and in later phases light yellowish brown to yellowish brown is. Furthermore, essentially no soluble pigments were used in those used for the present strain laxonomic media formed. The aerial mycelium is covered with flour-like dust and grows moderately. It is white to yellow or light yellowish brown.

The characteristics of the strain in various taxonomic media are given in Table 1 below

Table 1 _v

Cultural characteristics of the strain C-14919 on taxonomic media tß

s A) sucrose nitrate agar || i

Growth (W): bad, colorless to light yellow ff

Aerial mycelium (LM): not available | j

Soluble pigment (LP): not available |

ίο B) glycerol nitrate agar ^; > \

W: bad, colorless to light yellow; Formation of coremia-like bodies j

LM: sparse, light ivory, Lt Ivory (2ca) * f

LP: not available |;

C) Glucose asparagine agar J:

W: moderate, colorless to light yellow, Brite-Yellow (3na) *; Formation of coremia-like bodies r; l

LM: sparse, white to light ivory, Lt Ivory (2ca) * fr

LP: not available V-

D) Glycerin-asparagine agar ^ i W: moderate, white to light orange-yellow; Formation of coremia-like bodies ji LM: sparse, light ivory (Lt Ivory (2ca) * ' LP: not available "

E) starch agar

W: moderate, Lt Ivory (2ca) * to Lt Wheat (2ea) *; Formation of coremia-like bodies LM: abundant, Lt Ivory (2ca) *
LP: not available

F) nutrient agar

W: moderate. Colonial Yellow (2ga) * to Brite Maize (31a) *; Formation of coremia-like bodies LM: moderate, white to pearl pink (3ca) *
LP: not available
35

G) calcium maleate agar

W: moderate, light yellow to Colonial Yellow (2ga) *; Formation of coremia-like bodies LM: moderate, light yellow to yellow

LP: not available,

Another property: Calcium is made soluble

H) yeast extract-malt extract agar

W: moderate, amber amber (3nc) *; Formation of coremia-like bodies LM: moderate, white
LP: not available

I) Oatmeal Agar

W: moderate. Amber (3nc) * to Brite Yellow (3na) *; Formation of coremia-like bodies LM: moderate, light yellow
LP: not available

J) tyrosine agar

W: moderate, amber amber (31c) *; Formation of coremia-like bodies LM: sparse, light yellow
LP: not available

K) KartofTel stitch culture

W: moderate, Lt Amber (3ic) *; Formation of coremia-like bodies gg LM: not available

LP: the sting turns pale yellowish tan

L) Peptone Yeast Extract Iron Agar

W: moderate, light yellowish brown to light orange yellow
LM: not available

LP: not available;

* The color names correspond to the Color Harmony Manual, 4th edition (Container Corporation of America 1958).

4) Physiological properties

The physiological properties of the strain are given in Table 2. Temperature range for the
Growth: 12 to 38 ° C. The temperature range in which air mycelium will grow well on agar (ISP # 2)
takes place is 20 to 32 ° C.

Table 2

Physiological properties of strain C-14919

Growth temperature range: 12 to 38 ° C Temperature range for sporulation: 20 to 28 ° C Liquefaction of gelatin: positive Hydrolysis of starch: positive Reduction of nitrates: positive Milk peptonization: positive Coagulation of milk: doubtful positive Decomposition of casein: positive Formation of black pigments: negative (peptone yeast extract iron agar) negative (tyrosine agar) Decomposition of tyrosine: positive Decomposition of xanthine: negative Decomposition of hypoxanthine: negative Compatibility with lysozyme: positive Compatibility with sodium chloride: 2%

5) Utilization of various carbon sources

The recovery of various carbon sources was based on the method of Pridham and Gottlieb
(Journal of Bacteriology 56 [1948] 107) examined. The spectrum obtained is shown in Table 3. 35

Table 3

Utilization of carbon sources by the strain C-14919

+++ = lush growth.
++ = good growth.
+ = Growth.
± = poor growth.
- = no growth. ⁶⁵

Source of coal growth Source of coal growth D-xylose + Raffinose ± L-arabinose - Melibiosis + D-glucose ++ i-inositol - D-galactose ++ D-sorbitol - D-fructose +++ D-mannitol + L-rhamnose ± Glycerin + D-mannose + Soluble starch + Sucrose ++ control - Lactose ± Maltose ± Trehalose +

6) Other features

The cells were isolated in the manner described under (2). Following the procedure of J. Murmar et al (Journal of Molecular Biology 3 [1961] 208) DNA was prepared. The G-C content of the DNA was found to be about 71 mol%.

Gram staining of the vegetative mycelium of this strain gave a positive reaction.
The above characteristics of the strain C-14919 were described in "The Actinomycetes", Volume 2 by SA Waksman (The Williams and Wilkins Co. 1961), RE Buchanan and NE Gibbons in "Bergey's Manual of Determinative Bacteriology", 8th edition 1974 and other references. It was

ίο assumed that this strain belongs to group III of the genus Nocardia. However, there is no species with the characteristics described so far could be found among the known species, the applicant moved the conclusion that it is a new species of microorganisms.

The strain C-14919 used according to the invention was obtained from the Institute for Fermentation, Osaka (IFO), Japan, deposited under accession number IFO 13723.

The nutrient medium used for the purposes of the invention can be any liquid or solid medium provided that it contains the nutrients usable by the strain, however, it will be a liquid medium preferred for large-scale production. The medium can contain carbon and nitrogen sources, which the strain C-14919 May assimilate and digest, contain inorganic substances, trace nutrients, etc. As carbon sources are for example glucose, lactose, sucrose, maltose, dextrin, starch, glycerin,

Mannitol, sorbitol and fats and oils (ζ. B. soybean oil, lard oil, chicken oil, etc.). Suitable sources of nitrogen are, for example, meat extract, yeast extract, dry yeast, soybean meal, corn steep liquor, pcptone, cottonseed meal, spent molasses, urea, ammonium salts (ζ, Β. Ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium acetate), etc. The medium can also contain salts of sodium, potassium salts. Calcium, magnesium, etc., salts of iron, manganese, zinc, cobalt, nickel, etc. and salts with organic acids, e.g. B. Acetate and Propionate contain. Furthermore, various amino acids (e.g. glutamic acid, aspartic acid, alanine, lysine, methionine and proline), peptides (e.g. dipeptides and tripeptides), vitamins e.g. B. B ,, B ₂ , nicotinic acid, B ₁₂ and vitamin C), nucleic acids (e.g. purine, pyrimidine and the corresponding derivatives) etc. can be added. Inorganic or organic acids, alkali, buffers or the like can be added to adjust the pH of the medium. Oils and fats, surfactants, etc. can also be added in suitable amounts for anti-foaming purposes.

The cultivation can be carried out in stationary culture, shaking culture, aerobic submerged culture and other cultivation methods. For large-scale production, of course, aerobic submerged culture is preferred. The cultivation conditions naturally depend on the condition and composition of the medium, the strain used, the cultivation method and other factors, but it is preferred to work at 20 to 32 ° C. with an initial pH value close to the neutral point. A temperature of 23 to 26 ^° C. in an intermediate stage of the cultivation with an initial pH of 6.0 to 7.0 is particularly expedient. The duration of the cultivation is also different depending on the factors mentioned, but it is appropriate to carry out the cultivation until the titer of the desired antibiotic product is at its maximum. In the case of shake culture or submerged aerobic culture in the liquid medium, the time required is usually in the range of about 72 to 192 hours.

A pH value close to the neutral point is suitable for the production of antibiotics; a range from 6.5 to 7.0 is optimal. The yield can be increased by regulating the mean pH value during cultivation by adding an acid or an alkali or by choosing the appropriate composition of the medium. For example, the yield of antibiotics increases significantly if components of the medium such as inorganic phosphates are added in an amount that is greater than required for the growth of the strain, ie 1000 to 7000 ppm, or certain assimilable carbohydrates which are capable of forming acids, e.g. . B. glucose and mannitol, can be used as carbon sources along with appropriately selected nitrogen sources.
The antibiotics C-14919 EI and E-2 formed in this way by the strain used according to the invention are normally extracellular, but there are cases in which the cells contain about 10 to 30% of the total yield of the antibiotics. The antibiotics can be tested for effectiveness using the cylinder method or the paper disk method using Candida albicans IFO 0583 as the test microorganism and using TSA (Trypticase Soy Agar, BBL) as the test medium.
The antibiotics are present both in the filtrate and in the cells isolated from the culture medium.

Therefore, the culture medium is filtered or centrifuged to collect the cells and the filtrate or supernatant separately. The effectiveness of the filtrate is tested immediately. The effectiveness of the cells is tested after they ^{have been stirred for one hour at 20 °} C. with the same volume of 70% acetone-water as the filtrate. One of the above test methods is carried out using Candida albicans as the test microorganism.

Since C-14919 E-I and E-2 formed in the culture medium are lipophilic and neutral substances, can them in a simple manner by separation and purification processes that are normally used to isolate such Metabolites applied by microorganisms can be isolated. For example, a method is suitable taking advantage of the difference in solubility between the antibiotic and the impurities is, a process whereby the difference in adsorption affinity between antibiotic and contaminant to various adsorbents such as activated carbon, macroporous nonionic resins, Silica gel, aluminum oxide, etc. is exploited, and a process in which the impurities with the help of removed by ion exchange resins. These methods can be used alone, in suitable combination, or be applied repeatedly. Since, as already mentioned, C-14919 E-I and E-2 both in the filtrate and in the

If cells are present, the following methods can be used at will: 1) The culture medium is extracted as such with a solvent prior to the separation of the cells. 2) The cells and the filtrate or the supernatant obtained by filtration or centrifugation are separated with solvents extracted.

When the cells and the filtrate are extracted separately, the following procedure can be used to advantage are: As a solvent for the extraction of the filtrate, for example, organic solvents that are immiscible with water, e.g. B. fatty acid esters such as ethyl acetate and amyl acetate, alcohols, e.g. B. butanol, halogenated hydrocarbons, e.g. Chloroform, and ketones, e.g. B. methyl isobutyl ketone is used. the Extraction is carried out near the neutral point, preferably with ethyl acetate, the culture medium adjusted to pH 7 beforehand. The extract is washed with water and concentrated, using C-14919 E-2 is obtained in the form of raw crystals. That which remains after the separation of these crude crystals Filtrate is rich in E-I.

Mixtures of water with a water-miscible organic are suitable for extraction from the cells Solvents, e.g. B. a mixture of water with a lower alcohol (e.g. methanol and ethanol) or a mixture of water with a ketone (e.g. acetone and methyl ethyl ketone) and with water not miscible organic solvents, e.g. B. halogenated hydrocarbons (e.g. methylene dichloride). It is however, it is advisable to use 70% acetone-water. For example, the cells are given 70% acetone water in the the same volume as the corresponding filtrate was added, whereupon the mixture was 3 hours at room temperature is stirred. Here, the C-14919 E-I and E-2 present in the cells are completely extracted.

The solvent extracts are poured together, whereupon the solvent, e.g. B. acetone, under is removed under reduced pressure. As in the case of the filtrate, the aqueous solution is treated with ethyl acetate extracted and the solvent layer washed with water and concentrated under reduced pressure. To the Hexane or the like is added to concentrate to precipitate the active components. A mixture can be used here from C-14919 E-I and E-2 by centrifugation or filtration. The separation of the mixture in its components can be determined by adsorption chromatography on various adsorbents. to for this purpose the various carriers that are usually used for the adsorption of antibiotic are suitable Substances are used. Examples are adsorptive resins, silica gel and aluminum oxide too to name. A mixture of water is used to elute the antibiotics from these adsorptive resins a water-miscible organic solvent, e.g. B. a lower alcohol or a lower one Ketone, used. Suitable lower alcohols are, for example, methanol, ethanol, propanol and butanol. Acetone and methyl ethyl ketone, for example, are suitable as lower ketones. Esters, e.g. B. ethyl acetate, can also be used. The following procedure is typical: The raw product is 40% Dissolved methanol-water and adsorbed it in a column of the resin "Diaion HP-10". The column is first with 40% Washed methanol-water and then eluted with 60% methanol-water to obtain C-14919 E-2 fraction will. Elution with 90% methanol-water gives the C-14919 E-1 fraction. Each faction will concentrated under reduced pressure and left to stand after adding methanol. Here are crystals of C-14919 E-I and crystals of C-14919 E-2 obtained from the respective fractions. Using Silica gel as an adsorbent can be developed using a non-polar solvent be started using a polar solvent, e.g. B. methanol, is added in small portions. This elutes C-14919 E-I and E-2. The crystals of C-14919 E-I are made from ethyl acetate, Recrystallized from methanol, aqueous methanol or the like. The C-14919 E-2 can also consist of the same or similar Solvents are recrystallized.

The antibiotics C-14919 EI and E-2 obtainable in this way can be converted into one another by reversible oxidation-reduction. For example, reducing C-14919 EI with a reducing agent (e.g. hydrosulfite, ascorbic acid, NaBH ₄ and zinc with acetic acid) produces the antibiotic C-14919 E-2, while oxidizing C-14919 E-2 with an oxidizing agent (e.g. ferric chloride, nitric acid, AgO ₂ , MnO. and air) C-14919 EI is obtained.

The physical and chemical properties of the new crystalline obtained in the above manner Antibiotics C-14919 E-I and E-2 are listed in Table 4 below.

50 Table 4

C-14919 E-I

C-14919 E-2

1) melting point

2) Appearance

3) solubility

187 ° C (dec.)

yellow crystals
(Needles or prisms)

insoluble in petroleum ether, hexane, water

sparingly soluble in diethyl ether, benzene

soluble in ethyl acetate, chloroform, butanol, methyl isobutyl ketone, ethanol, acetane, methanol

Easily soluble in dimethyl sulfoxide

148 ° C (dec.)

pale yellow crystals
(Needles or prisms)

insoluble in petroleum ether, hexane

sparingly soluble in diethyl ether, benzene, water, chloroform

soluble in ethyl acetate, butanol, methyl isobutyl ketone, ethanol, Acetone, methanol

Easily soluble in dimethyl sulfoxide

continuation

C-14919 E-I

C-14919 E-2

4) acidic, neutral or basic

5) Elemental analysis,%

10

6) Gross formula

7) UV adsorption spectrum

¹⁵ £? "· N-HCl (9: 1) (nm) (£} *")

'• buk l ⁿⁿ U ( ^£ lcm)

λ ™.? ^μ N-NaOH (9: 1) (nm) (E \ ^% _m . ²⁰ 8) Infrared absorption spectrum (KBr)

25th

9) Specific rotation

30 10) Color reactions 35 40

60 65

11) Nuclear magnetic resonance spectrum (100 MHz) in dimethyl sulfoxide neutral substance
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

274 (455), 240 (sh), 397 (43)
274 (455), 240 (sh), 397 (43)
236 (585), 265 (500), 550 (56)

Fig. 1

Predominant peaks (cm " ¹ )

3430, 3340, 2950, 2910, 1740,
1692, 1660, 1645, 1605, 1500,
1375, 1315, 1120, 1100, 10U5,
1060, 1025

[a) ff + 350 ° ± 10 °
(C = 0.5, methanol)
negative: ninhydrin reaction

Honest reaction
Peptide reaction,
Magnesium Acetate Reagent
and 1% ferric chloride-1%
Ferricyanide reagent (1: 1)

neutral substance

C 62.32; 62.07 H 8.58; 8.43 N 4.82; 4.78 O 20.81; 20.81

-, - JfH ₂ O

238 (560), 255 (295), 308 (sh) 238 (550), 255 (290), 308 (sh) 236 (505, \ 265 (420), 550 (50)

Fig. 2

Predominant peaks (cm " ¹ )

3480, 3250, 2980, 1685, 1625, 1598, 1472, 1390, 1370, 1315, 1207, 1090, 1065, 1042, 1030

[a \ f + 62 ° ± 4 ° (C = 0.5, methanol) negative: ninhydrin reaction

Ehrlich reaction peptide reaction and magnesium acetate reagent

positive: 1% ferric chloride-1% ferricyanide reagent (1: 1, blue)

Potassium permanganate number Potassium permanganate δ d m discolored number Reagent: discolored the
Per Reagent: (ppm) d m J the
Per δ J tone d S. (Hz) tone (ppm) (Hz) 3H 0.63 S. S. 3 H 3H 0.82 m m 7th 3H 0.63 d 6.5 3H 0.89 S. S. 7th 3H 0.94 d 6.5 3H 1.16 2.1-2.2 m d 7th 3H 0.96 d 6.5 3H 1.55 2.65 d 1.4 S. 1.83 2.86 d 3 H 1.88 S. 3.13 d 1.4-1.8 m 3.24 2.2-2.4 m 3H 3.2 3.0-3.2 m 3H 3.39 3H 3.23 S. 3H 4.54 3 H 3.27 S. 4.64 3.43 S. IH 5.02 311 3.3 m IH 5.21 IH 4.5 S. IH 5 IH 5.21 d 10 IH 8th IH 5.28 d IH 8th St. 5.59 dd 8.10 10 6.22 t 11

continuation

11) Nuclear magnetic reso δ J number δ J number nance spectrum (100 MHz) (ppm) (Hz) the
Per (ppm) (Hz) the
Per in dimethyl sulfoxide tone tone 6.32 s (disappear 5.86 t 10 IH det at Zu 5.98 s-like IH sentence of
D ₂ O) 6.23 s (disappear
det through IH Addition of D ₂ O) 6.41 d 2 IH 6.81 d 2 IH 6.95 d 11 IH 6.35 d 2.5 IH 9.3 s (disappear 6.45 d 2.5 IH det at Zu 7.51 s (disappear IH sentence of det through D ₂ O) Addition of D ₃ O) 8.64 s (dto.) IH 8.85 s - IH C-14919 E-I C-14919 E-2

12) Persistence

Room temperature:

resistant in neutral and

weakly acidic solutions;

inconsistent in basic

solution

8O ⁰ C, 1 hour: inconsistent

Room temperature:

consistent under neutral and

weakly acidic conditions;

inconsistent under basic

conditions

80 ⁰ C, 1 hour: inconsistent

13) Thin layer chromatography adsorbent: silica gel, spot film (Tokyo Kasei)

Chloroform-methanol (9: 1) chloroform-methanol (19: 1) ethyl acetate-acetone (9: 1) Ethyl acetate-methanol (9: 1)

0.50
0.22
0.50
0.78

Antimicrobial activity

Test microorganisms

* Escherichia coli K-12 Proteus vulgaris Pseudomonas aeruginosa Salmonella typhimurium Alcaligenes faecal I is

IFO No.

C-14919 EI
MIC (µg / ml)

C-14919 E-2
MICUg / ml) ⁺ )

3301 > 100 > 100 3045 > 100 > 100 3080 > 100 > 100 12529 > 100 > 100 13111 > 100 > 100

The antimicrobial activity of antibiotics C-14919 E-I and E-2 was determined by the serial dilution method using Trypticase Soy Agar (BBL), Glucose Nutrient Agar and Glycerin Nutrient Agar as test media determined (Table 5). In addition, the activities against Tetrahymena pyriformis W after the Serial dilution method using a test medium consisting of 20 g proteose peptone, 1 g yeast extract, 2 g glucose, 1000 ml distilled water and 10 ml 1 molar phosphate buffer (pH 7.0) are determined. 55

Table 5

28 13 416 C-14919 E-I C-14919 E-2 continuation MIC (µg / ml) MIC (iig / ml) ⁺ ) Test microorganisms IFO No. > 100 > 100 50 100 Serratia marcescens 3046 50 100 Bacillus pumilus 3813 100 > 100 Bacillus subtilis 6633 3134 > 100 > 100 Bacillus subtilis PCI219 3513 50 50 Bacillus cereus 3514 50 50 Bacillus megaterium 12108 > 100 > 100 Bacillus brevis 3331 50 50 Staphylococcus aureus 209P 12732 50 50 Sarcina lutea 3232 25th 6.25 Micrococcus flavus 3242 > 100 > 100 Candida albicans 0583 100 100 ** Candida tropicalis 1400 > 100 > 100 Candida pseudotropicalis 0617 50 25th Candida utilis 0619 > 100 > 100 Candida parapsilosis 1396 50 25th Candida krusei 1395 > 100 > 100 Candida albicans 0583 100 100 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 50 25th Hormodendrum pedrosoi 6071 > 100 > 100 Penicillium chrysogenum 4626 > 100 > 100 *** Mycobacterium avium Mvcobacterium Dhlei

**** Tetrahymena pyriformis W

10

⁺ ) MIC = minimum inhibitory concentration. * Trypticase Soy Agar. ** Glucose nutrient agar. *** Glycerin nutrient agar. **** Proteose peptone solution.

toxicity

The assumed LD ₅₀ values, determined by intraperitoneal administration of C-14919 EI and E-2 in a test to determine the acute toxicity using mice as test animals, are given below _:

C-14919 E-I C-14919 E-2

50-100 mg / kg 25-100 mg / kg.

As the antibiotic spectrum in Table 5 shows, C-14919 E-I and E-2 are against gram-positive bacteria, Mushrooms and yeasts effective. Hence, these antibiotics are valuable as germicides or disinfectants against pathogenic bacteria or fungi of the same species as the test microorganisms.

10

Anti-tumor effect

The therapeutic effect (intraperitoneal administration on consecutive days) of C-14919 EI and E-2 against P388 leukemia in mice (1 × 10 ⁶ cells / mouse, transpired intraperitoneally) was examined. The anti-tumor effect, expressed as life extension, was 144% at a dose of 5 mg / kg per day.

Since the antibiotics C-14919 E-I and E-2 have a life-prolonging effect on mammals (e.g. mice), those affected by tumors are expected to be valuable as anti-tumor agents.

By formulating antibiotics C-14919 E-I or E-2 as an aqueous solution containing 10 to 100 µg / ml of aqueous Contains ethanol (with, for example, 5% ethanol), the antibiotics used to disinfect bird cages, Laboratory equipment, hands, etc. are used.

The invention is further illustrated by the following examples. In these examples, the Parts are parts by weight unless otherwise specified. "Parts" relate to "parts of space" like grams Cubic centimeter. The percentages are based on "weight / volume", unless otherwise stated.

15 Example 1

The strain C-14919 (IFO13723) was inoculated on the tyrosine agar medium and then incubated for 240 hours at 28 ° C. The well-grown mycelium was suspended in a 0.6% strength sodium glutamate solution and stored in the refrigerator. A part of 1 part by volume of the mycelium suspension was used for inoculating a fermenter which had a capacity of 2000 parts by volume and contained 500 parts by volume of an inoculation culture medium (20 parts glucose, 30 parts soluble starch, 10 parts corn steep liquor, 10 parts soybean meal, 5 parts peptone, 3 Parts of sodium chloride and 5 parts of calcium carbonate / 1000 parts by volume of water, pH 7.0). The inoculated medium was incubated on a reciprocating shaker for 48 hours at 28 ° C. This culture (500 parts by volume) was used to inoculate a stainless steel tank having a capacity of 50,000 parts and containing 30,000 parts by volume of an inoculating medium. This seed culture was carried out under the following conditions: 28 ° C., aeration 30,000 parts / minute, 280 rpm, internal pressure 0.98 bar, 48 hours. 10,000 parts by volume of the preculture obtained were used to inoculate a 200,000 parts by volume tank made of stainless steel, the 100,000 parts by volume of a fermentation medium (5% glycerine, 2% corn steep liquor, 2% yeast extract, 2% KH ₂ PO ₄ , 0.5% MgCl ₂ and 0.1% CaCO ₃ , pH 6.5) and was sterilized. In the initial phase of the fermentation, the following conditions were used: temperature 28 ° C., ventilation at 100,000 volume parts / minute, 180 rpm, internal pressure 0.98 bar, 24 hours. Here, the microorganism grew well. Cultivation was then carried out for a further 114 hours under the same conditions, except that the cultivation temperature was 24 ° C. and stirring was carried out at 200 rpm. After this time, the mycelium was filtered off from the filtrate. The filirate contained 380 μg / ml activity, while the titer in the mycelium was 90 ag / cells in 1 part of fermentation medium. Efficacy testing was performed by the cylinder method or the paper disk method using Candida albicans IFO 0583 as the test microorganism and using C-14919 EI as the standard test compound.

Example 2

The experiment described in Example 1 was repeated up to the seed culture stage. The cultivation was then carried out in a 200,000 volume tank made of stainless steel containing 100,000 volume parts of a nutrient medium (5% mannitol, 3% dry yeast, 0.5% MgCl ₂ and 0.1% CaCO, pH 6.5) contained and was sterilized. In the initial phase of cultivation, work was carried out at 26 ° C., with aeration of 100,000 parts / minute, 180 rpm and an internal pressure of 0.98 bar for 18 hours. The cultivation was then carried out for a further 106 hours at 24 ° C. and 200 rpm under otherwise identical conditions. After the cultivation time mentioned, the activity of the filtrate was 310 μg / ml and the activity of the cells was 100 μg / cells in 1 ml of culture medium.

Example 3

The culture medium obtained according to Example 1 (95,000 parts by volume) was mixed with 2000 parts of "Hyflo-Supercel" well mixed. The mixture was filtered with a filter press, leaving 85,000 parts by volume of filtrate and 31,000 Parts of wet cells were obtained. The filtrate (85,000 parts by volume) was ethyl acetate with 30,000 parts by volume extracted while stirring. This extraction was repeated twice. The ethyl acetate layers were poured together, washed twice with 30,000 parts by volume of water, by adding 500 parts dried anhydrous sodium sulfate and concentrated under reduced pressure to 200 parts by volume. The concentrate was left to stand in the cold room and the crude crystals of C-14919 E-2 formed were filtered off (8.2 parts). The filtrate was further concentrated to 50 parts by volume and then with 300 parts by volume of petroleum ether offset. The precipitate formed in this way was filtered off. The obtained crude crystals (38 parts) were in Dissolved 50 parts by volume of methanol. After adding 5 parts of silica gel (0.05-0.2 mm), the methanol became distilled off under reduced pressure. The residue was on top of a column of 500 parts by volume of silica gel (same product as above) abandoned. The column was washed with 500 parts by volume of hexane and with 1000 parts by volume of hexari-ethyl acetate (1: 1), 1000 parts by volume of ethyl acetate and 1000 parts by volume of ethyl acetate-methanol (30: 1) eluted. The eluate was collected in fractions of 100 parts by volume each. C-14919 E-I was eluted from the column in fractions No. 13 to No. 16, while C-14919 E-2 in fractions

No. 21 to No. 23 was eluted. The fractions containing C-14919E-1 (400 parts by volume) were combined- $

poured and concentrated and, after the addition of 80% aqueous methanol, left to stand in the cold room. The ?,

crystals formed thereby were filtered off. As a result, 10 parts of crystals were obtained. Si

The fractions that contained C-14919 E-2 (300 parts of volume) were also poured together on | 4

Restricted 100 parts of the room and left to stand in the refrigerator. The crystals formed in this way were isolated and if

dried. This gave 6.8 parts of C-14919 E-2 in the form of crystals. I.

Separately from this, 31,000 parts of the moist cells were washed twice with 40,000 parts by volume of 70% acetone each time

ser extracted. The extracts obtained were poured together and poured under to remove the acetone

reduced! Pressure restricted. The aqueous extract was diluted to 30,000 parts by volume with water and replaced by f.

ίο the prepared column of 1000 parts of the adsorbent »Diaion HP-10« passed, with the active Component has been adsorbed. The column was filled with 2000 parts water and 2000 parts 40% ' Washed methanol-water in the order mentioned and then with 2500 parts by volume of 60% methanol-water and 2500 parts by volume of 90% methanol-water are eluted in that order. The eluate was divided into fractions, "ι collected by 250 room parts each. Fractions Nos. 3 and 4 were combined under reduced pressure

concentrated and left to stand in the refrigerator. The resulting crude crystals of C-14919 E-2 were t ^;

filtered off and dried (1.8 parts). Fractions 13, 14 and 15 were merged, under ί

reduced! The pressure was concentrated and, after the addition of 80% methanol-water, left to stand in the cold room. The one here- ':'. when crude crystals of C-14919 EI formed, this was filtered off and dried (5.8 parts).

Example 4!

The filtrate obtained according to Example 3 (85,000 parts by volume) was ethyl acetate twice with 30,000 parts by volume extracted as in Example 3. The ethyl acetate layers were poured together, 30,000 parts by volume Washed water and concentrated under reduced pressure to 500 parts by volume. The concentrate were;

4000 parts by volume of methanol and then 4000 parts by volume of water and 3000 parts by volume of hexane were added. That The mixture was stirred and the bottom layer then separated. The bottom layer became a solution of 20 parts of iron (III) chloride in 8000 parts by volume of water were added. The mixture was under occasional Stirring left to stand for 5 hours at room temperature and cooled after this time. The here formed crude crystals of C-14919 E-I were filtered off and dried (18 parts). The filtrate was continued with

5000 parts by volume of ethyl acetate extracted. The ethyl acetate solution was washed with water and concentrated. f After adding petroleum ether to the concentrate, 13 parts of a crude powder of C-14919 E-I was obtained. As in Example 3, the crude powder was applied to a column (200 parts by volume) of silica gel (0.05-0.2 mm) using chromatographed of the solvents mentioned. This gave 7.2 parts of crude C-14919 E-I in the form of Get crystals.

Example 5

The crystalline C-14919 E-I (0.400 parts) obtained according to Examples 3 and 4 was in 40 parts by volume Dissolved ethyl acetate. In a separating funnel with a capacity of 100 parts were

30 parts by volume of water added. The solution was then shaken vigorously with 0.500 parts of sodium hydrosulfite (Na ₃ S ₂ O _4). The mixture was allowed to stand and the lower layer discarded. The upper layer was shaken well with 30 parts by volume of water and 0.500 parts of sodium hydrosulfite (Na ₂ S ₂ O _4). The lower layer was discarded, and the ethyl acetate layer was washed well with water and dried by adding anhydrous sodium sulfate. After drying, the ethyl acetate layer was evaporated to dryness under reduced pressure at low temperature. The residue was dissolved by adding 10 parts by volume of methanol and filtered. After adding 40 parts by volume of water, the filtrate was left to stand in the cold room. The obtained crystals of C-14919 E-2 were filtered off and dried. Melting point 147-148 ° C (dec.).

Example 6

The crystalline C-14919 E-2 (0.400 parts) prepared according to Example 3 was methanol in 60 parts by volume solved. 32 parts by volume of a 1% strength aqueous solution of ferrous chloride were added to the solution. The mixture was left to stand at room temperature for 1 hour. The resulting crystals of C-14919 E-I were filtered off, washed with 20% methanol-water and dried. Melting point 186 to 187 ° C (decomp.). :

Example 7

The crude crystals of C-14919 E-I (4 parts) obtained in Example 3 were divided into 600 parts by volume Dissolved methanol. After filtration, 200 parts by volume of water were added. The mixture was in the cold room; ditched. The purified crystals of C-14919 E-I thereby formed were filtered off and dried (2.8 parts). Melting point 187-188 ° C (dec.).

Likewise, 3.5 parts of the crude crystals of the crude antibiotic C-14919 E-2 prepared according to Example 3 were dissolved in 100 parts by volume of methanol. After filtration, 500 parts by volume of water were added. The mixture =,. was cooled and the purified crystals of C-14919 E-2 thus obtained were filtered off (2.1 parts). : Melting point 148 ° C decomp.). ^

For this purpose 2 sheets of drawings S-

Claims (2)

Claims: 1. Antibiotics C-14919 E-I and E-2 with the following features: a) Antibiotic C-14919 E-I A) Melting point 187 ° C (decomp.) B) Appearance: yellow crystals (needles or prisms) C) solubility: insoluble in petroleum ether, hexane and water sparingly soluble in diethyl ether and benzene soluble in ethyl acetate, chloroform, butanol, methyl isobutyl ketone, ethanol, acetone and Methanol Easily soluble in dimethyl sulfoxide Ü) acidic, neutral or basic: neutral substance E) 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 F) Gross formula C ₃₀ -S ₂ H ₄₂ -I ₈ ^ O ₈ -? G) UV absorption spectrum -C. ⁰ ": N-HCl (9: 1) (nm) (£ j *,): 274 (455), 240 (sh.), 397 (43) ^ ⁰ """"'(£ | cm): 274 (455), 240 (sh.), 397 (43) / C ⁰ ": N-NaOH (9: 1) (nm) (£ | I _n ): 236 (585), 265 (500), 550 (56 ) H) Infrared absorption spectrum (KBr): Major peaks (cm " ¹ ) at 3430, 3340, 2950, 2910, 1740, 1692, 1660, 1645, 1605, 1500, 1375, 1315, 1120, 1100, 1085, 1060, 1025 I) Specific rotation: [a] f + 350 ° ± 10 ° (c = 0-5, methanol) J) Color reactions: negative in ninhydrin reaction, Ehrlich reaction, peptide reaction and reaction with 1% iron chloride-1% Ferricyanide (1: 1) b) Antibiotic C-14919 E-2 A) Melting point 148 ° C (dec.) B) Appearance: pale yellow crystals (needles or prisms) C) solubility: insoluble in petroleum ether, hexane Slightly soluble in diethyl ether, benzene, chloroform, water soluble in ethyl acetate, butanol, methyl isobutyl ketone, ethanol, acetone, methanol Easily soluble in dimethyl sulfoxide D) acidic, neutral or basic: neutral substance E) Elemental analysis: C, 62.32; 62.07% H 8.58; 8.43 N 4.82; 4.78 O 20.81; 20.81 F) Gross formula: Cjo-3 ₂ H ₄ 4-5oN ₂ 0 ₈ -9 · xH ₂ O G) UV absorption spectrum / M ' ⁰¹¹ : N-HCl (9: 1) (nm) (£ j * _m ): 238 (560), 255 (295), 308 (see) ; McO! I _lnm , _(£ j% j. _{238 (55O))} 255 ₍₂ 90), 308 (sh.) ; l ^cO ": N-NaOH (nm) (£! *",): 236 (505), 265 (420), 550 (50) H) Infrared absorption spectrum (KBr) Major peaks (cnT ¹ ) at 3480, 3250, 2980, 1685, 1625, 1598, 1472, 1390, 1370, 1315, 1207, 1090, 1065, 1042, 1030 I) Specific rotation: [äff ' + 62 ° ± 4 ° (c = 0.5, methanol) J) Color reactions: negative in ninhydrin reaction, Ehrlich reaction and peptide reaction; positive reaction with 1% iron chloride-1% ferricyanide (1: 1) (blue). 2. A method for obtaining the antibiotics C-14919 E-I and / or E-2 of claim 1, characterized in that that Nocardia IFO 13723 is cultivated in a nutrient medium and the antibiotics C-14919 E-I (.5 and / or E-2 isolated using separation and purification methods customary for lipophilic and neutral substances or converting the obtained antibiotic C-14919 E-2 into the antibiotic C-14919 E-I by oxidation.