CH637137A5 - Antibiotic and process for its preparation - Google Patents

Description

The present invention thus relates to:

(1) the antibiotic C-15003, which corresponds to the following general formula:

40

3rd

(I)

60

where R

-CO-C

-CH,

Vh,

-CO-CH1-CH -.- CH, or

-CO-CHi-CH

/ CH,

N,

CH,

65

means

(2) a process for producing the antibiotic C-15003. which consists in taking the antibiotic C-15003

637 137

4th

producing strain of the genus Nocardia in a medium, and the said antibiotic C-15003 from said which obtains assimilable carbon sources and digestible stick broth, and contains substance sources, is grown to produce the strain in the culture broth (3) a process a connection of the following

Formula for the formation and enrichment of the antibiotic C-15003:

(II)

which consists in taking the antibiotic C-15003 of the following general formula:

Ci CH3 0

(I)

where R

45

XU,

-CO-CH.

CH,

-CO-CH, -CH, -CH, or

In the present specification, the term “antibiotic C-15003” generally means the three compounds of the above-mentioned general formula I as a group or a mixture of two or three of the said compounds or ultimately any of these compounds. With reference to general formula I, the compound in which R represents the 50 radical

/ CH,

.CH,

-CO-CH.

-CO-CH, -CH

\

XCH,

means manufactured according to the process defined above and then hydrolyzed reductively.

The antibiotic C-15003 is obtained by cultivating a microorganism belonging to the genus Nocardia, which is capable of producing antibiotic C-15003, in a culture medium containing assimilable carbon sources and digestible nitrogen sources until the antibiotic is concentrated therein, after which it is isolated .

As an example of a strain of a microorganism producing the antibiotic C-15003, there can be cited an Actinomyces strain No. C-15003 which has been isolated from the soil or other samples in the screening of antibiotic-producing microorganisms.

CH,

55, referred to in the present description as "antibiotic C-15003 P-3" or only "C-15003 P-3". The compound in which R represents the radical -CO-CHo-CH ^ -CH is referred to as "antibiotic C-15003 P-3 '", or only "C-15003 P-3'". The connection in which R the rest

60

-C0-CH, -CHC ^

CH, CH,

65, is referred to in the present description as "antibiotic C-15003 P-4" or only "C-15003 P-4" and the compound in which R is the hydrogen atom (with which this compound corresponds to general formula II), will be next

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637 137

referred to as «antibiotic C-15003 P-0» or only «C-15003 P-0».

As an example of a strain of a microorganism producing the antibiotic C-15003, there can be cited an Actinomyces strain No. C-15003 which has been isolated from the soil or other samples in the screening of antibiotic-producing microorganisms.

The microbiological properties of strain No. C-15003 were researched in a manner analogous to that proposed by Schirling & Gottlieb [International Journal of Systematic Bacteriology 16, 313-340 (1966)]. The results of these observations at 28 ° C over a period of 21 days can be found below.

1) Morphological properties The vegetative mycelium expands well and develops into branches, both on agar and in the liquid medium. Some of the hyphae are 0.8 to 1.2 µm in diameter and, in certain cases, can divide to form fragments that resemble rod-shaped bacteria or branched short hyphae. The strain shows good growth on various taxonomic media, with an aerial mycelium accumulating on the vegetative mycelium. Often, coremia-like bodies (50-200 x 200-1000 µm) also form on which further aerial mycelium grows. Some of the aerial mycelia are tortuous, with straight or wide spiral-like configurations occasionally occurring. The microscopic examination of older cultures shows that the conidia-like cells are only in chains in a few cases, while the cell suspensions obtained from the surfaces of such cultures, when viewed under the microscope, extended variously, ellipsoidal (0.8-1.2 [in x 4.8-6.8 (im) and ellipsoidal (0.8-1.2 | in x 1.0-2.0 µm) bodies that resemble arthrospores.

Observations under the electron microscope showed

that these bodies have smooth surfaces.

2) Cell composition The strain was in a modified ISP No. 1 medium was grown under shaking at 28 ° C for 66-90 hours, after which the cells were collected and rinsed. According to the method of B. Becker et al. [Applied Microbiology 12,421 (1964)] and the method according to M.P. Lechevalier [Journal of Laboratory and Clinical Medicine 71,934 (1968)], the whole cells thus obtained were examined for their content of diaminopimelic acid and sugar composition. The acid was in the meso form, while sites could be observed which indicated galactose and arabinose.

3) Properties on taxonomic media The strain showed relatively good growth on various media, the vegetative mycelium being colorless to pale yellow in the initial stages of cultivation and pale yellow-tan to yellowish tan in the latter phases. The strain produced soluble pigments that were yellow to yellowish tan in various taxonomic media. The aerial mycelium was powdery and generally showed moderate growth with a white to yellow or light yellowish tan color. The properties of the strain in various taxonomic media can be found in Table 1 below.

Table 1

Breeding properties of the strainNo. C-15003 on taxonomic media

(A) Sucrose Nitrate Agar:

Growth (G): massive bright melon yellow (3 ia) 4 to amber tan (3 lc) *. Formation of coremia-like structures aerial mycelium (AM): sparse, white soluble pigment none or pale yellowish-tan (SP):

(B) Glycerol Nitrate Agar:

G: moderate, light ivory (2 ca) *, formation of coremia-like structures AM: moderate, white SP: none

(C) glucose-asparagine agar:

G: moderate, light marigold (3 pa) * to bright yellow (2 pa) *

AM: sparse, white SP: bright yellow (2 pa) *

(D) Glycerin Asparagine Agar:

G: moderate, light ivory (2 ca) *, formation of coremia-like bodies AM: sparse, white SP: none

(E) Starch agar:

G: moderate, light ivory (2 ca) * to light wheat

(2 ea) *, formation of coremia-like bodies AM: abundant, ivory-colored (2 ca) *

SP: no

F) Nutrient agar:

G: moderate, light ivory (2 ca) * to khaki yellow (2ga) *, formation of coremia-like bodies AM: sparse, white SP: none

(G) calcium maleate agar:

G: moderate, light ivory (2 ca) * to light wheat

(2 ea) *, formation of coremia-like bodies AM: moderate, white to light ivory (2 ca) *

SP: no

(H) Yeast extract-malt extract agar:

G: moderate, amber (3 lc) * to bright yellow (31a) *, formation of coremia-like bodies AM: moderate, white to light ivory (2 ca) *

SP: no

(I) oatmeal agar:

G: moderate, light ivory (2 ca) * to khaki yellow

(2 ga) "\ Formation of coremia-like bodies AM: scanty, white to light yellow SP: none

(J) Peptone Yeast Extract Iron Agar:

G: moderate, khaki yellow (2 ga) *

AM: none

SP: khaki yellow (2 ga) *

(K) Tyrosine agar:

G: moderate, light ivory (2 ca) * to light melon yellow

(3 ea) *, formation of coremia-like bodies AM: moderate, white to light ivory (2 ca) * SP: camel (3 ie) *

* Color code according to "Color Harmony Manual", 4th edition (Container Corporation of America, 1958)

4) Physiological properties The physiological properties of the strain can be found in the following Table 2. The temperature range for the growth was 12 to 38 ° C. The temperature range in which there was good growth of air mcel on agar (ISP No. 2) was 20 to 35 ° C.

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40

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50

55

60

65

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Table 2

The physiological properties of strain No. C-15003 were the following:

Temperature range for growth Temperature range for growth of aerial mycelium Liquefaction of gelatin Hydrolysis of starch Reduction of nitrates Peptonization of milk Coagulation of milk Decomposition of casein Production of melanoid pigments

Decomposition of tyrosine Decomposition of xanthine Decomposition of hypoxanthine Tolerance in relation to lysozyme Tolerance in relation to sodium chloride

12 to 38 ° C

20 to 35 ° C

positive positive positive positive negative positive negative (peptone--

Yeast extract iron

Agar),

positive (tyrosine

Agar)

positive negative negative positive

2%

5) Use of Different Carbon Sources The use of different carbon sources was examined using a medium as described in Pridham and Gottlieb [Journal of Bacteriology 56,107 (1948)] and a basic medium of the same composition plus 0.1% yeast extract. The values shown in Table 3 below were determined.

Table 3 Use of carbon sources by strain no. C-15003

25th

carbon

growth

carbon

Growth source

source

D-xylose

+ ++:

Raffinose

± ± *

L-arabinose

+ +

Melibiosis

-1- +

D-glucose

+ + + +

i-inositol

- -

D-galactose

+ +

D-sorbitol

- -

D-fructose

+ + + + +

D-mannitol

+ + + +

L-rhamnose

+ +

Glycerin

- +

D-mannose

+ + + + +

soluble starch

+ +

Sucrose

+ + + +

Try blindly

- -

Lactose

- - *

Maltose

± +

Trehalose

+ + +

Base medium when adding 0.1% yeast extract Note: + + +: abundant growth + +: good growth +: growth ±: poor growth no growth

6) Other properties

The cells were collected by the method given further above 2) and DNA by an analogous method to that of J. Marmur et al. [Journal of Molecular Biology, 3,208,1961)]. The G-C (guanine cytosine) content of the DNA was approximately 71 mol%.

The gram staining of the vegetative mycelium of this strain was positive.

The above characteristics of strain No. C-15003 have been described in S.A. Waksman's "The Actinomycetes" Vol. 2 [The Williamsand Wilkins Co., 1961]; R. E. Buchanan and N. E. Gibbons, "Bergey's Manual of Determinative Bacteriology, 8th Edition, 1974"; and other references.

It was believed that this strain belonged to Group III of the

Genus would belong to Nocardia. However, no species with the described properties could be found among the known strains. It can be concluded from this that this strain represents a new type of microorganism.

5 The said strain No. C-15003 is at the Fermentation Research Institute, Agency of Industrial Science and Technology (FERM) under number 3992, on March 22, 1977 at the Institute for Fermentation. Osaka, (IFO) »under number IFO 13726 and on March 31, 1977 at« The American Type 10 Culture Collection (ATCC), Maryland, USA. » registered under number 31281.

While strain No. As just mentioned, C-15003 is a new species in the genus Nocardia, as microorganisms generally do, it can enter into variations and mutations, either spontaneously or under the influence of a mutagen. For example, the various variants of the strain, which can be obtained by exposure to X-rays, y-rays, ultraviolet light, etc., by monocell isolation, by cultivation on various chemicals, or by any other mutagenic treatment , as well as the mutants which are derived spontaneously from the strain are essentially not to be regarded as representatives of another specific species. Any variants and mutants capable of forming the antibiotics C-15003 P-3, P-3 'and / or P-4 can be used for the purposes of the present invention.

For example, strain No. C-15003 by various mutagenic treatments mutants which do not produce soluble pigments, so to speak, mutants with substrate mycelia which are colorless, yellowish green, reddish tan or orange-red, mutants whose hyphae can easily be broken down into bacillus-like elements or branched, short hyphae, and Mutants with abundant, white aerial mycelia or essentially without aerial mycelia.

The medium used to grow a strain producing an antibiotic C-15003 can be any liquid or solid medium, provided that it contains nutrients useful by the strain. However, a liquid medium is preferably used to achieve high production. The medium can contain carbon and nitrogen sources, which are caused by strain no. C-15003 are assimilated and digested, contain inorganic substances, trace nutrients etc. Examples of carbon sources in question include glucose, lactose, sucrose, maltose, 45 dextrin, starch, glycerin, mannitol, sorbitol etc., fats and oils, e.g. B. soybean oil, bacon oil, chicken oil, etc., and the like. Examples of nitrogen sources include meat extract, yeast extract, dried yeast, soybean meal, corn steep liquor, peptone, casein, cottonseed meal, treated molasses, urea, ammonium salts, e.g. As ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc., and the like can be used. The medium may also include sodium, potassium, calcium, magnesium salts etc., iron, magnesium, zinc, cobalt, nickel salts etc., salts of Phos-55 phosphoric acid, boric acid etc. and organic salts e.g. B. acetates and propionates. Furthermore, the medium can also contain various amino acids, such as. B. glutamic acid, aspartic acid, alanine, glycine, lysine, methionine, proline etc., peptides. B. dipeptides, tripeptides etc., vitamins, e.g. B. Vitamins B |, B2, nicotinic acid, B | 2, C, E etc., nucleic acids, e.g. B. purine, pyrimidine and derivatives thereof. An inorganic or organic acid, an alkali, a buffering agent or the like can also be used to adjust the pH of the medium. Appropriate amounts of oils, fats, surfactants, etc. can also be added as an anti-foaming agent.

The cultivation can under any stationary conditions, with shaking, under submerged aerobic conditions

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40

50

60

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637 137

or under other breeding conditions. In order to obtain a high yield, submerged aerobic cultivation is preferred. The breeding conditions depend of course on the condition and the composition of the medium, the strain, the breeding method and other factors. Usually it is done at 20 to 35 ° C with an initial pH of around 7.0. Preferably one will use a temperature of 23 to 30 ° C at an initial pH of 6.5 to 7.5 as the cultivation temperature. The incubation period also varies depending on the factors mentioned above, the incubation being continued until the titer of the desired antibiotic product has reached a maximum value. In the case of shake cultures or aerobic submerged cultures in a liquid medium, the time normally required is approximately 48-144 hours.

The effectiveness of the antibiotic was tested with Tetrahy-mena pyriformis W as the test organism. The microorganism mentioned above was tested on a test medium [20 g proteose peptone (Difco), 1 g yeast extract (Difco), 2 g glucose, 1000 ml distilled water and 10 ml 1-molar phosphate buffer (pH 7.0)] at 28 ° C grown for 44-48 h,

whereupon the effectiveness of the antibiotic is determined by the serial dilution method while observing the turbidity due to growth and by thin-layer chromatography, abbreviated TLC, according to the information described below.

The new antibiotics C-15003 P-3, P-3 'and / or P-4 were obtained both extracellularly and intracellularly in the culture broth obtained and enriched.

These substances have also been detected by TLC. The fermentation broth was thus divided into cells and filtrate by filtration or centrifugation and the filtrate was extracted with the same volume of ethyl acetate. The same amount of 70% aqueous acetone as the amount of the filtrate was then added to the cells, after which the suspension was filtered after stirring at 20 ° C. for one hour. The acetone was removed from the filtrate and the aqueous filtrate obtained was extracted with ethyl acetate. Each of these extracts was concentrated in a volume ratio of 1: 100 and over a silica gel glass plate (Merck, German Federal Republic, silica gel 60 F254.0.25 mm, 20x20) (solvent system: chloroform and methanol in a mixing ratio of 9: 1) Subjected to thin layer chromatography. Efficacy was determined based on the intensity of the spots observed when 2537 Â was irradiated with ultraviolet light.

The C-15003 P-3, P-3 'and / or P-4 produced in this way in the culture broth are lipophilic and neutral substances,

which can be isolated by separation and purification methods which are normally customary in the production of such microbial metabolites. For example, you can work by taking advantage of the differences in solubility between the antibiotic and the contaminants. But you can also use the fractional adsorption affinity of various adsorbents such. B. activated carbon, macroporous, non-ionic resins, silica gel, aluminum oxide, etc. apply. The impurities can also be removed with the aid of ion exchange resins. However, other methods or the aforementioned methods can also be used in a suitable combination, or else repeatedly.

Since, as mentioned above, C-15003 P-3, P-3 'and P-4 are present both in the filtrate and in the cells, the antibiotics, if one is used, become either direct with the aid of such an adsorbent or after extraction with a solvent in the case of a filtrate or after extraction with a solvent in the case of microbial cells, separate and clean. The extraction with a solvent can be carried out by any of the following methods or by another method, for example (1) by solvent extraction from the culture broth before separating the cells and (2) by solvent extraction of the cells and the filtrate obtained by filtration. Centrifugation or otherwise. In order to extract the filtrate and the cells independently of one another, one will advantageously use the following method.

Suitable solvents for the extraction of the filtrate are water-immiscible, organic solvents, such as. B. fatty acid esters such as ethyl acetate or amyl acetate, alcohols such as butanol, halogenated hydrocarbons such as chloroform, and ketones, e.g. B. methyl isobutyl ketone. The extraction takes place at a pH value close to the neutral pH value and preferably the culture liquid previously adjusted to a pH value of 7 is extracted by means of ethyl acetate. The extract is washed with water and concentrated under reduced pressure. The concentrate is then washed with a non-polar solvent, e.g. B. petroleum ether or hexane, and the crude product I, which contains the active compound, is obtained. Since in addition to the antibiotic C-15003 a certain number of spots are found in thin layer chromatography, the product I is then subjected to the following cleaning processes. This shows that a routine cleaning method, in particular adsorption chromatography, is valuable, for which purpose one of the usual adsorbents, such as silica gel, aluminum oxide, a macroporous, nonionic adsorption resin, etc., can be used. Silica gel is particularly valuable for cleaning the raw product I. Development can be carried out, for example, starting from petroleum ether and hexane, while the elution of the antibiotic C-15003 is carried out by adding a polar solvent, such as ethyl acetate, acetone, ethanol or methanol. According to a typical method, silica gel (Merck German Federal Republic, 0.05 to 0.2 mm) is used as the carrier and the column chromatography is carried out with a mixture of hexane and ethyl acetate, the proportion of hexane increasing successively. The eluate is collected and checked by TLC, collecting the fractions containing the antibiotic C-15003 and concentrating under reduced pressure. Then the concentrate is mixed with petroleum ether or hexane, whereby the crude product II is obtained. Because this product still contains contaminants, it will be further cleaned as described below. For example, product II can be cleaned with the help of a second silica gel column using a different solvent system. The development system used for this purpose can consist of a halogenated hydrocarbon, e.g. B. dichloromethane or chloroform, with the addition of a polar solvent such as an alcohol, e.g. B. ethanol or methanol, a ketone, e.g. B. acetone or methyl ethyl, or the like. The antibiotic C-15003 can be isolated in this way. The order of the solution systems for these first and second silica gel columns can also be reversed, it also being possible, if necessary, to use conventional organic solvents in conjunction with the above systems.

If a macroporous adsorption resin is used to clean the crude product II, the antibiotic C-15003 is eluted with a mixture of water with a lower alcohol, a lower ketone or an ester. As a lower alcohol, for example, methanol, ethanol, propano! or butanol and use, for example, acetone or methyl ethyl ketone as the lower ketone. The ester can be, for example, ethyl acetate. According to a typical procedure, the crude product II is dissolved in 60% aqueous methanol and adsorbed on a column of Diaion HP-10 (Mitsubishi Kasei K.K.). This column will then be 70rf iges

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40

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50

55

60

65

637 137 8

Washed aqueous methanol and eluted with 90% aqueous phie the methanol corresponding to C-15003-P-4, P-3 'and P-3. In this way, the antibiotic C fractions are concentrated under reduced pressure and ethyllace-15003 is eluted from the column. did admit the concentrates. In this way, the compounds corresponding to the antibio are obtained in the form of crystals in each of the aforementioned methods. Fractions containing Usenikum C-15003 collected and concentrated under 5 a macroporous, non-ionic adsorption resin, so reduced pressure. The dried product is then subjected to a gradual elution with fluctuating Ver then with 5 to 8 times the volume of ethyl acetate and the mixture is left in alcohol, ketone or ester with respect to the water, whereupon crystals of the antibiotic are applied . Eliminate elution medium C-15003 when using the stages. These crystals contain C-15003 P method using 60% aqueous methanol and 3, P-3 'and P-4. These compounds are then separated with the aid of a 10 95% strength aqueous methanol with the addition of 5% adsorbent, for example of the type mentioned above, sodium chloride is obtained in the above order C. So you use silica gel or a 15003 P-3, P-3 'and P-4. After treatment with thin-layer micro-macroporous, non-ionic adsorption resin and the above matography, each group of the active Frak-mentioned solvents is so that the desired compounds can be concentrated under reduced pressure and fertilized from ethyl acetate fractionally eluted. For example, use is made to crystallize. The isolated crystals contain silica gel, so development takes place using hexane, ethyl acetate as the crystallization solvent and show ethyl acetate or a mixture of chloroform and methanol, after drying over phosphorus pentoxide for 8 hours, whereby C-15003-P-4, P- 3 'and P-3 in the series 70 ° C mentioned, the following physical and chemical sequence can be obtained. After thin-layer chromatography.

Table 4

Antibiotic C-15003

P-3

P-3 '

P-4

C, 2H43C1N20 "= 635.169

C ,, H43C1N, 0, = 635.169

C "H45C1N: 0.) = 649.196

Melting point (° C)

190-192 ° C

182-185 ° C

-J

1

) - 1 00

0

0

0

Specific

-136 ° ± 10 °

-134 ° ± 10 °

0

O

rH

+1

O

s?

r-H

1

Rotation [a] 2o

(c = 0.375 CHCI3)

(c = 0, ll CHCI3)

(c = 0.522 CHClj)

Elemental analysis

C 60.06

60.09

60.65

Found: (%)

H 7.04

7.02

7.05

N 4.33

4.34

4.25

Cl 5.37

5.99

5.23

Elemental analysis

C 60.51

60.51

61.05

Range: (%)

H 6.82

6.82

6.99

N 4.41

4.41

4.32

Cl 5.58

5.58

5.46

Ultraviolet absorption spec

233 (30250) 240 (sh 28450)

233 (30155) 240 (sh 28250)

233 (29900) 240 (sh 28240)

strand (e) (in methanol

252 (27640) 280 (5750)

252 (27600) 280 (5750)

252 (27590) 280 (5712) 288 (5680)

288 (5700)

288 (5700)

Infrared absorption spectrum

1740.1730.1670.1580.1445,

1740.1730.1670.1580.1445,

1740,1730,1670.1580.1445.

(cm "') KBr

1385, 1340, 1255, 1180, 1150,

1385, 1340, 1255, 1180, 1150,

1385, 1340, 1255, 1180, 1150.

1100, 1080, 1038

1100, 1080, 1038

1100, 1080, 1038

nuclear magnetic resonance l, 27 (d) (3H)

l.06 (t) (3H)

1.03 (d) (6H)

spectra (ppm) 100 MHz in l, 28 (d) (3H)

CDClj

Mass spectra (m / e)

573, 485, 470, 450

573, 485, 470, 450

587, 485, 470, 450

Solubility in petroleum ether, hexane and insoluble in petroleum ether, hexane insoluble in petroleum ether, hexane

Water insoluble in benzene and water, sparingly soluble in and water, sparingly soluble in

and ether sparingly soluble, in

Benzene and ether, soluble in

Benzene and ether, soluble in

Chloroform, ethyl acetate, ace

Chloroform, ethyl acetate, ace

Chloroform, ethyl acetate, ace

clay, ethanol, methanol, pyritone, ethanol, methanol, pyritone, ethanol, methanol, pyritone

din, tetrahydrofuran and di-

din, tetrahydrofuran and di-

din, tetrahydrofuran and dime

methylsulfoxyd soluble methylsulfoxcyd thylsulfoxyd

Color reactions

Dragendorff: positive

Dragendorff: positive

Dragendorff: positive

Beilstein: positive

Beilstein: positive

Beilstein: positive

Based on the molecular formula above and the following could deliver and vegetable or natural, organic

reproduced antimicrobial and tumor-preventing origin would lead to the Maytanacingruppen.

The new antibiotic was compared with the efficacy values in particular due to the molecularly known groups of antibiotics in question. In literature 65 mein it was suspected that the antibiotic maytanacin

no group of compounds containing 2 nitrogen atoms similar to the antibiotic C-15003

determine. Research on substances that are similar. Maytanacin was obtained as a plant component

ultraviolet absorption values such as the present antibiotics. This is described in the Journal of American Chemical Society 97,

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637 137

5294 (1975). The mass spectrum for Maytanacin is the following:

M "- (a) M" - (a + b) 485-CH, 485-C1 545 485 470 450

(a) = H, 9 + HNCO

(b) = R-C-OH O!

The values m / e 485,470 and 450 for C-15003 P-3, P-3 'and P-4 lead to the belief that these compounds have an identical skeletal structure with that of Maytanacin with the difference in the acyl group present in the 3-position . It is clear from this that the antibiotic C-15003 is a new compound. When C-15003 P-3, P-3 'and P-4 5 are treated with an alkali and analyzed by gas chromatography for the released carboxylic acids, it is found that isobutyric acid, butyric acid and isovaleric acid from C-15003 P-3, C-15003 P-3 'and C-15003 P-4 are available. 1 shows the structures based on the above data for C-15003 P-3, P-3 'and P-4.

Fig. 1

R

Cl? H3 0

p-3 -CO-CH

/ CH3

\

ch

3rd

p-3 '-co-ch2-ch2-ch3

p 4 -co-ch2-ch:

CH-

■ ch-

Biological effects:

A) Antimicrobial activity: With trypticase soy agar (BBL) as the test medium, the inhibitory concentrations against the microorganisms mentioned below were determined using the paper disk method. The filter paper discs (Toyo Seisakusho) with a diameter of 8 mm were each impregnated with 0.02 ml of a 300 (xmg / ml solution of C-15003 P-3, P-3 'or P-4, after which these discs were placed on plates ordered which were inoculated with the microorganisms mentioned below to determine the minimum inhibitory concentrations, and the results obtained show that the antibiotics were ineffective with respect to the following microorganisms:

Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus substilis, Bacillus cereus, Klebsiella pneumoniae, Serratia marcescens, Mycobacterium avium.

On the other hand, agar plates containing 3.5 g of disodium hydrogen phosphate, 0.5 g of monopotassium dihydrogen phosphate, 5 g of yeast extract (Difco), 10 g of glucose, 15 g of agar and 1000 ml of distilled water at a pH of 7 were used as the test medium. 0

contained that tested growth inhibition against Talaromyces avellaneus. In this experiment the minimum inhibitory concentrations were 3 µg / ml for C-15003 P-3 and P-3 'and 1.5 µg / ml for C-15003 P-4. Furthermore, the wild strain of Tetrahymena pyriformis W was used as a test organism on a test medium consisting of 20 g proteose peptone (Difco), 1 g yeast extract, 2 g glucose, 1000 ml distilled water and 10 ml 1-molar phosphate buffer with a pH value of 7.0, grown at 28 ° C for 44-48 h and then the inhibition of the growth of this particular microorganism by the antibiotic compounds was determined by the serial dilution method. The growth inhibition occurred at 1 µg / ml for C-15003 P-3 and P-3 'and at 0.5 (µg / ml for C-15003 P-4.

The fungicidal activity can be found in Table 5. As can be seen from Table 5, the antibiotic C-15003 has an inhibitory effect on the growth of microorganisms which cause plant diseases. The filter paper disks impregnated with 0.02 ml of a 50 1000 [ig / ml solution of C-15003 were placed on plates inoculated with the microorganisms mentioned in Table 5.

40

45

Table 5 Antimicrobial spectrum

Test organisms

IFO number medium

Hours

Inhibition diameter

Alternariakikuchiana

7515

PSA *

48

38

Fusicladium levieri

6477

PSA *

90

68

Helminthosporium sigmoideum var. Irregular

5273

PSA *

48

55

Pyricularia oryzae

-

PSA *

48

53

Elsino

8417

PSA *

90

55

Fusarium oxysporum f. cucumerinum

-

PSA *

48

20th

Guignardia laricina

7888

PSA *

48

12

Cochlioborus miyabeanus

5277

PSA *

48

60

Diaporthe citri

9170

PSA *

48

55

637 137

10th

\ v. r_- if'iMÜLii

IFO number

medium

Hours

In h never »

Gibberella / eae

8850

PSA *

48

37

Sclerotinia sclerotiorum

9395

PSA *

90

65

Venturia pirina

6189

PSA *

48

50

Pellicularia sasakii

9253

PSA *

48

50

Pythium aphanidermatum

7030

PSA *

48

58

Botrytis cinerea

-

PSA *

48

48

Aspergillus niger

4066

PSA *

48

0

Pénicillium chrysogenum

4626

PSA *

48

35

Rhizopus nigricans

6188

PSA *

48

25th

Saccharomyces cerevisiae

0209

PSA *

48

0

Rhodotorula rubra

0907

PSA *

48

28

Trichophyton rubrum

5467

GB **

48

38

Trichophyton mantagrophytes

7522

GB **

48

38

Candida albicans

0583

GB **

48

0

Candida utilis

0619

GB **

48

0

Cryptococcus neoformans

0410

GB **

48

43

PSA: Potato-sucrose agar medium. "GB: glucose nutrient agar medium

B) Cytostatic effect:

The therapeutic effect of C-15003 P-3, P-3 'and P-4 on mouse P 388 leukemia (1x10 ° cells / animal) was determined after nine days of intraperitoneal treatment. The results have shown that the cytostatic effect of these compounds causes an extension of life which corresponds to a value of 200% for a dose of 0.00625 mg / kg / day.

C) Toxicity:

In an acute toxicity test using mice as test animals, which were injected intraperitoneally with C-15003 P-3, P-3 'and P-4, these antibiotics showed an LDjn value of more than 0.313 mg / kg.

As already mentioned, the antibiotic C-15003 has a strong inhibitory action against fungi and protozoa and is therefore very valuable as a fungicidal agent or as an antiprotozoal agent. Furthermore, since the antibiotic C-15003 in mammals suffering from tumors, e.g. B. mice, has a life-prolonging effect, one can also assume that this compound will be valuable as a cytostatic.

The antibiotic C-15003 can be used as a fungicidal agent and as an antiprotozoal agent with advantage also in connection with the bacterial ecology in the soil, in activated sludge, in the animal body fluid or the like. If you want to isolate valuable bacteria from a soil sample, or if you want to assess the effects of bacteria independently of those in front of fungi and protozoa in the processing and analysis of activated sludge, such as those used in the treatment of waste water, you can do the new one Use an antibiotic to promote selective growth of the bacterial flora without promoting simultaneous growth of the fungi and protozoa. According to a particular example, a sample can be added to a liquid or solid medium and, in addition, 0.1 ml of a 10 to 100 µg / ml solution of the antibiotic in a 1% aqueous methanol solution per ml of the medium, after which an incubation is initiated.

The antibiotic C-15003 according to the invention can also be used as an antimicrobial agent for the treatment of plant diseases which are caused by the microorganisms mentioned in Table 5 above.

According to a typical application method, the antibiotic C-15003 is used in the form of a 1% methanolic, aqueous solution which contains 0.5 µg / ml to 5 [xg / ml of the antibiotic. For example, you can use the antibiotic C-15003 to combat reddish-brown leaf sheath rot, Brusone disease, leaf spot disease caused by Helminthosporium and leaf sheath burn

25th

use of rice plants.

It follows that C-15003 P-3, P-3 'and P-4 are new compounds which have the same basic structure. They can also be used as intermediates for the production of other pharmaceutically valuable compounds

30 can be used. For example, the antibiotic according to the invention can be obtained by deacylation reaction C-15003 P-0 (Mayt-ansinol) with a hydroxyl group in the 3-position. Since the acyl group is in the β position to the carbonyl group, reductive hydrolysis is used. It is therefore possible to use a complex metal hydride, e.g. As lithium aluminum hydride (Li A1H4), at low temperature, for example -2 ° C to 0 ° C, hydrolytically cleave the O-ester bond in the 3-position, without other functional groups, for. B. the carbonyl, epoxy, carbon-carbon double bonds, etc., too

40 in order to obtain maytansinol in this way. The physical and chemical data obtained with a maytansinol sample thus obtained are in good agreement with the data obtained according to Kupchan et al, The Journal of American Chemical Society 97.5294-5295

45 (1975).

The antibiotic C-15003 can also extend the survival of warm-blooded animals, i.e. H. from animals and humans who suffer, for example, from leukemia, sarcomas, mastocytomas, melanomas or carcinomas.

50 The antibiotic C-15003 also has a regressive effect on solid tumors which are injected into other organs by subcutaneous or intramuscular administration.

Examples of warm-blooded animals are mammals and humans.

55 In an acute toxicity test using mice as test animals, intraperitoneal injections of the antibiotics C-15003 P-3, P-3 'and P-4 showed that an LDjn value of more than 313 mcg / kg was consistently achieved could.

61 The injection form is generally used to administer the antibiotic C-15003 to warm-blooded animals. The injection can be intramuscular, intrathoracic, intra-abdominal, intrauterine, intrathecal, intra-artery, subcutaneous or intravenous.

6: Such an injection means can be produced in a manner known per se. For example, you can use the antibiotic C-15003 in an aqueous solution. liquid medium, which is a common solubilizing agent, e.g. B. an alcohol such as

11

637 137

Ethanol, a polyalcohol. like propylene glycol. Polväthvlen-glycol 200 to 300, a nonionic surfactant, a physiological saline and / or an isotonic solution, dissolve or suspend.

Typical examples of a liquid medium are 0.85% physiological saline containing 1% ethanol or 2.5% methanol or 0.85% physiological saline containing 1% Tween 80.

The concentration of antibiotic C-15003 in the liquid is advantageously between 0.5 mcg / ml and 500 mcg / ml.

The injection solutions or suspensions can be sterilized and / or auxiliary substances, e.g. Preservative. Stabilizers, wetting agents and emulsifiers included.

The antibiotic C-15003 can be administered to warm-blooded animals in a dose between 0.8 and 50 mcg / kg / day.

As already mentioned, the antibiotic C-15003 contains the antibiotics C-15003 P-3, P-3 'and P-4. In pharmaceutical preparations, a single component of said antibiotic, namely antibiotic C-15003 P-3, P-3 'or P-4, can be used. Mixtures of one or more of these components can also be used, i.e. a mixture of the antibiotics C-15003 P-3 and P-4, a mixture of the antibiotics C-15003 P-3 'and P-4, a mixture of the antibiotics C-15003 P-3 and P-3' and a mixture of Antibiotics C-15003 P-3, P-3 'and P-4. In practice, a mixture of the antibiotics C-15003 P-3 is preferred. P-3 'and P-4.

The individual components or a mixture thereof do not necessarily have to be cleaned.

In the present description, the terms tumor, neoplasm, leukemia, sarcoma or carcinoma are more or less equivalent to one another.

All subsequent experiments regarding the cytostatic activity of the antibiotic C-15003 were carried out according to the information in the National Cancer Institute, National Institute of Health, USA [forg. Cancer Chemotherapy Reports (Part 3) Vol. 3, No. 2, 1972, pp. 1 -103] with regard to the cultivation and evaluation of the cytostatics.

Melanomas B 16 and leukaemias L1210 obtained from the National Cancer Institute, USA in 1971 were measured in C57BL / 6 and DBA / 2 mice [Cancer Chemotherapy Reports (Part 1) Vol. 50, No. 5.1975, pp. 919-928]. P388 leukemia obtained from the Japan Cancer Chemotherapy Center in 1975 was maintained in DB A / 2 mice. P815 mastocytoma in DBA / 2 mice and Ehrlich carcinoma and Sarcom 180 in JCR-JCL mice were also used. In each of these examples, a suitable strain of male or female mice, which were 5 to 7 weeks old, was used for each tumor.

The following examples illustrate the present invention without restricting it. The parts are parts by weight, unless otherwise stated. The ratio between parts and parts by volume corresponds to that between g and ml. The expression “%” refers to “weight / volume” unless otherwise stated.

example 1

NocardiaNo. C-15003 (IFO 13726: FERM3992: ATCC 31281). which had been grown on a medium (yeast extract-malt extract agar) was used to make a 200 volume part fermentation container containing 40 volume parts of a seed culture medium (2% glucose, 3% soluble starch, t% raw soybean meal , 1% corn steep liquor, 0.5% polypeptone, 0.3% NaCl, 0.5% CaCO- ,, pH7.0). The inoculated medium was incubated at 28 ° C for 48 hours to obtain an inoculum. 0.5 part by volume of the inoculum thus obtained was placed in a fermentation container holding 200 parts by volume, which contained 40 parts by volume of a fermentation medium which consisted of 5% dextrin, 3% corn steep liquor, 0.1% polypeptone and 0 , 5% calcium carbonate (pH 7.0), contained, introduced and grown for 90 hours at 28 ° C. in order to obtain an inoculum (seed culture) in this way.

As determined by the serial dilution method using Tetrahymena pyriformis W as the test organism and C-15003 P-3 as the standard sample, the above culture had a titer of 25 xg / ml.

10th

Example 2

10 parts by volume of the inoculum obtained according to Example 1 (inoculum) were added to a fermentation container holding 2000 parts by volume, which contained 500 parts by volume of a seed-15 culture medium of the same composition as above, followed by 48 hours at 28 ° C brooded. 500 parts by volume of the culture thus obtained was put in a 50,000 part by volume stainless steel tank containing 30,000 parts by volume of seed culture medium, followed by cultivation under 20 aeration (30,000 parts by volume / min) Bred 28 ° C and stirred at an internal pressure of 1 kg / cm2 at 280 revolutions per minute (Vi DT), in order to obtain a seed culture in this way. This culture was used to inoculate a 200,000 parts by volume stainless steel tank containing 100,000 parts by volume of a fermentation medium similar in composition to Example 1 above while maintaining a 10% inoculation rate. The inoculated medium was incubated with aeration (100,000 parts by volume / min) at 28 ° C. and shaken at an internal pressure of 1 kg / cm 2 for 90 h at 200 revolutions per minute (/: DT). When determining the titer in the same manner as in Example 1, it was found that the culture obtained in this way had a titer of 25 xg / ml.

35 Example 3

95,000 parts by volume of the culture obtained according to the information in Example 2 were mixed with 2000 parts of Hyflo-supercel® (Johnes and Manville Products, USA) and, after thorough mixing, the mixture was filtered through a pressure filter, 85,000 parts by volume . Parts of filtrate and 32000 parts of moist cells were obtained. The filtrate (85,000 parts by volume) was stirred and extracted with 30,000 parts by volume of ethyl acetate. This measure was repeated one more time. The ethyl acetate layers were collected 45, washed twice with 30000 parts by volume of water, dried by adding 500 parts of anhydrous sodium sulfate, and concentrated to 200 parts by volume under reduced pressure. Then the concentrate was mixed with petroleum ether and the resulting precipitate was collected by filtration (53 50 parts). This crude product I was stirred with 100 parts by volume of ethyl acetate, whereupon the insoluble constituents were filtered off. The filtrate was stirred with 10 parts of silica gel (Merck, German Federal Republic, 0.05 to 0.2 mm) and the ethyl acetate was removed under reduced pressure. The residue 55 was added on top of a silica gel column (400 parts by volume). Then with 500 parts by volume of hexane, 500 parts by volume of a mixture of hexane and ethyl acetate (mixing ratio 3: 1), 500 parts by volume of a mixture of hexane and ethyl acetate (mixing ratio 1: 1), 500 parts by volume Share a mixture of 6f) hexane and ethyl acetate (mixing ratio 1: 3). 500 parts by volume of ethyl acetate and 1000 parts by volume of a mixture of ethyl acetate and methanol (mixing ratio 50: 1) eluted. the eluate was collected in fractions of 100 parts by volume.

5 1 part by volume of each fraction was evaporated to dryness, after which 0.1 part by volume of ethyl acetate was added to the concentrate. The mixture was then placed on a location 2.5 cm above the lower edge of a silica gel glass plate (Merck, Federal Republic of Germany. 60 F34.0.25 mm, 20x20)

637 137

12

applied and developed in a length of about 17 cm in a solvent system of ethyl acetate and methanol (mixing ratio 19: 1). After development, the desired product was determined using ultraviolet light (2537 Â).

The active fractions No. 23-28 with Rf 0.6-0.65 were collected and each reduced to about 20 parts by volume under reduced pressure. These concentrates were each mixed with 150 parts by volume of petroleum ether, giving 15 parts of a crude product II.

Example 4

32,000 parts of the moist cells obtained according to Example 3 were extracted with 50,000 parts by volume of 70% aqueous acetone with stirring for 3 hours and then filtered on a pressure filter. The extraction using 50,000 parts by volume of 70% aqueous acetone and the subsequent filtration were repeated once more. The filtrates were collected and the acetone was removed by concentration under reduced pressure. The resulting aqueous system was passed through a column of 5000 parts by volume of Diaion HP-10 (Mitsubishi Kasei K.K.). The column was washed with 20,000 parts by volume of water and 50% aqueous methanol and then eluted with 15,000 parts by volume of 90% aqueous methanol. The eluate was concentrated to 3000 parts by volume under reduced pressure and shaken with 3000 parts by volume of water and 3000 parts by volume of ethyl acetate. This procedure was repeated once more. The ethyl acetate layers were combined, washed with water, dried by adding anhydrous sodium sulfate, and concentrated to a volume of 200 parts under reduced pressure. Then petroleum ether was added and the resulting precipitate was collected by filtration to give 28 parts of crude product I. This product was purified on a silica gel column in a similar manner to that in Example 1 to obtain 8.0 parts of crude Product II.

Example 5

0.5 part of the crude product II obtained according to Example 3 above was dissolved in 10 parts by volume of ethyl acetate and the solution was stirred thoroughly with 4 parts of silica gel (Merck, Federal Republic of Germany, 0.05 to 0.2 mm). The ethyl acetate was removed under reduced pressure. The residue was introduced on top of a column of 300 parts by volume of silica gel and the column was first washed with 500 parts by volume of chloroform and then with 500 parts by volume of a mixture of chloroform and methanol (mixing ratio 50: 1), 500 parts by volume . Parts of a mixture of chloroform and methanol (mixing ratio 20: 1) and 500 parts by volume of a mixture of chloroform and methanol (10: 1) eluted. These eluates were collected in fractions of 25 parts by volume.

0.5 part by volume of each fraction was concentrated under reduced pressure. The concentrate was mixed with 0.05 parts by volume of ethyl acetate and the mixture was subjected to thin layer chromatography (development system: mixture of chloroform and methanol in a mixing ratio of 9: 1).

Fractions # 39 and # 40 absorbent at 2537, which were in the zone of RF 0.50-0.60, were collected and concentrated to dryness under reduced pressure. The residue was then mixed with 2 parts by volume of ethyl acetate and the mixture was allowed to stand, whereupon 0.150 part of antibiotic C-15003 was obtained in the form of crystals.

The above crystals of the antibiotic C-15003 (0.150 parts) were dissolved in 15 parts by volume of methanol, and 0.300 parts of sodium chloride and 15 parts by volume of water were then added. A column charged with 200 parts by volume of Diaion HP-10 (Mitsubishi Kasei K.K.) was filled with 600 parts by volume of 50% aqueous

Methanol containing 5% sodium chloride treated. The solution thus prepared was passed through a column, and gradient elution was carried out using 1500 parts by volume of 60% aqueous methanol containing 5% sodium chloride and 1500 parts by volume of 95% aqueous methanol. The eluate was collected in different fractions of 15 parts by volume each and each fraction was checked by means of silica gel by thin layer chromatography. Fractions 145 to 153 contained C-15003 P-3, fractions 167 to 180 contained C-15003 P-3 'and P-4 and fractions 185 to 190 contained C-15003 P-4.

Each group of the fractions was concentrated and dissolved by adding 50 parts by volume of water and 100 parts by volume of ethyl acetate. The solution was shaken in a separatory funnel and the aqueous layer separated. After washing twice with 50 parts by volume of water each time, the ethyl acetate layer was dried over anhydrous sodium sulfate, concentrated and left to stand. In this way, crystals were obtained in each group of the fractions. These crystals were collected by filtration and dried.

C-15003 P-3 0.070 parts

C-15003 P-3 ', P-4 0.018 parts

C-15003 P-4 0.015 parts

The mixed crystals of C-15003 P-3 'and P-4 (0.018 parts) were dissolved in 0.3 parts by volume of ethyl acetate and on a line at a distance of 2.5 cm above the lower edge of a silica gel glass plate (Merck, German Federal Republic, silica gel 60 F254 0.25 mm, 20 x 20) applied, whereupon it was developed with a mixture of ethyl acetate and methanol (mixing ratio 19: 1). After this development to approximately 18 cm, the absorption band at Rf 0.68 (P-4) and Rf 0.65 (P-3 ') was scraped off and each band independently extracted twice with ethyl acetate containing little water. The resulting ethyl acetate extract was washed with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure and left to stand.

0.010 part of the crystals of C-15003 P-4 and 0.003 part of the crystals C-15003 P-3 'were obtained from the fractions with Rf 0.68 and Rf 0.65, respectively.

Example 6

1000 parts by volume of the culture according to Example 2 were inoculated in a stainless steel tank, which had a capacity of 200,000 parts by volume and contained 100,000 parts by volume of a seed culture medium, and the inoculated medium with aeration (100,000 parts by volume). Parts / min) and incubated with stirring at 200 revolutions per min for 48 h at 28 ° C. in order to obtain a seed culture. This seed culture was put in a stainless steel tank with a capacity of 2,000,000 parts by volume, which contained 1,000,000 parts by volume of a fermentation medium of the same composition as in Example 1, at a transplantation rate of 10%. The cultivation was carried out with aeration (1,000,000 parts by volume / min) at 28 ° C. for 90 hours with stirring at 120 revolutions per minute ('/ s DT) and at an internal pressure of 1 kg / cm 2. The resulting culture had a titer of 20 (xg / ml) when tested according to the method of Example 1.

900,000 parts by volume of the culture liquid thus obtained were mixed with 900,000 parts by volume of acetone and, after stirring for one hour, 20,000 parts of Hyflo-Supercel (Johnes & Manville, USA) were added. This mixture was further stirred and filtered in a pressure filter machine.

1700000 parts by volume of the filtrate thus obtained was mixed with 500000 parts by volume of water and the mixture was extracted in a Podbielniak device (Podbielniak, Inc., USA) with 1000000 parts by volume of ethyl acetate. The ethyl acetate layer

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

13

637 137

was washed with water, dried by adding anhydrous sodium sulfate, and concentrated under reduced pressure. Petroleum ether was added to the concentrate and the resulting precipitate was collected by filtration and dried. In this way, 68 parts of the crude product I were obtained. This crude product was then purified as described in Examples 3, 4 and 5. In this way, 9.5 parts of C-15003 P-3, 0.300 parts of C-15003 P-3 'and 2.5 parts of C-15003 P-4 were obtained.

Example 7

0.015 part of the antibiotic C-15003 in the form of the crystals obtained in Example 5 was dissolved in 1 part by volume of tetrahydrofuran, followed by cooling to -5 ° C. and adding 0.012 part of lithium aluminum hydride. After the addition of 0.5 part by volume of a 1% aqueous sulfuric acid solution, the reaction mixture was extracted with 2 parts by volume of ethyl acetate. The ethyl acetate layer was washed with water, dried by adding anhydrous sodium sulfate, and concentrated under reduced pressure. This was followed by preparative thin layer chromatography with silica gel, whereupon the zone was scraped off with Rf 0.25 to 0.3. Then, ethyl acetate containing little water was extracted. The extract was washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure, whereupon crystals separated out. These crystals were collected by filtration and dried. In this way, 0.010 part of C-15003 P-0 with a melting point of 174 ° C. was obtained.

Elementary analysis for C-> 8I I ^ CIN ^ O ^:

calc .: C = 59.52: H = 6.60: N = ~ 4.96; Cl = 6.27;

Found: C = 59.65; H = 6.58; N = 5.02; Cl = 6.51.

IR: 1715, 1670, 1580 (cm'1).

UV (nm): 232 (32750). 244 (sh, 30850), 252 (31650), 281 (5750), 288 (5700).

In terms of properties, this product was identical to Maytansi-nol.

Effect of C-15003 (a mixture of C-15003 P-3, C-15003 P-3 'and C-15003 P-4) on survival in mice inoculated with P388 leukemia

(C57BL / 6 x DBA / 2) F! (BDFi) mice (in a group of 3 or 5 mice) weighing 18-22 g were inoculated intraperitoneally with 1 x 10 ° C leukemia P388 on day 0.

The antibiotic C-15003 [a mixture of C-15003 P-3 (60 to 65% by weight), C-15003 P-4 (30 to 35% by weight) and C-15003 P-3 '] was suspended in 0.85% physiological saline containing 1 '7 Tween 80 and the suspension was vaccinated intraperitoneally with tumor-containing animals (0.2 ml / mouse) once a day for 9 days, the treatment being started 24 hours after the tumor vaccination.

To evaluate the cytostatic effect of the antibiotic C-15003, the T / C9c, i.e. H. calculated the ratio times 100 of the average survival of the P388-containing mice treated with the antibiotic C-15003 (T) to that of the untreated mice (C). A value of more than 125 for T / C <? R was considered positive in terms of cytostatic activity according to the NC1 standard.

Table 6

Dose mcg / kg / day

Number of mice average survival in days

T / C%

25th

3rd

24.5

227

12.5

3rd

20.0

185

6.25

3rd

21.5

199

(Blind test)

11

10.8

50

5

9.5

82

25th

5

22.5

194

12.5

5

22.5

194

6.25

5

22.5

194

3.13

5

19.5

168

1.6

5

15.0

129

0.8

5

12.5

108

0.4

5

12.5

108

(Blind test)

20th

11.6

As can be seen from the results in Table 6, intraperitoneal administration of daily doses of 1.56 to 25 mcg / kg of the antibiotic for 9 days can result in an extended survival in the treated mice.

The most significant lifespan extension of the treated mice was found at a dose of 25 mcg / kg (optimal dose) (T / C% of 227).

Effect of C-15003 P-3 and C-15003 P-4 on survival in mice inoculated with P388 leukemia

BDFr mice (5 mice per test group) weighing 18 to 22 g were inoculated intraperitoneally with 1X10 ° cells from leukemia P388 on day 0. Each antibiotic was dissolved in a 0.85% physiological saline solution containing 2.5% methanol and the solution was administered intraperitoneally once a day for 9 days to mice (0.2 ml / mouse), the administration being 24 hours after the tumor had occurred -culation took place.

As described in Example 8, more than 125 in T / C% according to the NCI method was considered positive in terms of cytostatic activity. As can be seen from Table 6, iritraperitoneal administration of C-15003 P-3 prolonged at daily doses of 1.6 to 25 mcg / kg or C-15003 P-4 at daily doses of 0.8 to 50 mcg / kg the survival of the treated mice in a significant way. The maximum effect was observed with a daily dose of 25 mcg / kg with each of the above test agents.

Table 7

Dose mcg / kg / day

T / C%

C-15003 P-3

C-15003 P-4

50

118

125

25th

223

195

12.5

177

177

6.25

168

180

3.13

159

145

1.6

144

141

0.8

123

132

0.4

105

105

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

637 137

14

Effect of antibiotic C-15003 (a mixture of C-15003 P-3. C-15003 P-3 'and C-15003 P-4) on the survival of mice inoculated with B 16 melanoma

1 g of the tumor was homogenized with 10 ml of a cold 0.85% physiological saline solution, whereupon 0.5 ml of this homogenized material containing the tumor was vaccinated intraperitoneally in BDFr mice (5 animals per test group) according to the information in NCl directives.

Antibiotic C-15003 [a mixture of C-15003 P-3 (60-65% by weight). C-15003 P-4 (30-35 wt%) and C-15003 P-3 '] was suspended in 0.85% physiological saline containing 1% Tween 80 and the suspension was intraperitoneally mice once daily for 9 days for 9 days The treatment was started 24 hours after tumor inoculation (5 mice in each group and 15 mice in the blind test).

T / C% in the mean survival was calculated according to the information in Example 8. T / C% above 125 was considered a positive cytostatic effect.

As can be seen from Table 8, the survival of the mice treated with the antibiotic C-15003 was significantly extended, in contrast to the untreated mice.

Table 8

Dose mcg / kg / day

Number of mice average survival in days

T / C

24th

5

29.5

159

12

5

31.5

170

6

5

35.5

192

3rd

5

34.5

186

1.5

5

29.0

157

(Blind test)

15

18.5

24th

5

31.5

213

12

5

28.5

193

6

■ 5

22.5

152

3rd

5

16.2

109

(Blind test)

15

14.8

Effect of C-15003 P-3 and C-15003 P-4 on the survival of mice inoculated with B 16 melanoma

BDF, mice (5 animals per test group) were inoculated intraperitoneally with a suspension of B 16 melanoma in the same manner as in Example 3.

Each antibiotic was dissolved in a 0.85% physiological saline containing 2.5% methanol, and the solution was intraperitoneally administered to the mice (0.2 ml / mouse) once a day for 9 days, with the administration after 24 hours after tumor inoculation was started.

As can be seen from Table 9, administration of C-15003 P-3 or C-15003 P-4 at daily dose levels of 1.6 to 50 mcg / kg caused a significant prolonged survival in the treated mice.

Table 9

dose

T / C

mcg / kg / day

C-15003 P-3

C-15003 P-4

50

> 219

> 219

25th

> 219

> 219

12.5

207

199

6.25

189

182

3.13

156

159

1.6

128

122

0.8

116

204

0.4

105

104

Effect of the antibiotic C-15003 (a mixture of C-15003 P-3, C-15003 P-3 'and C-15003 P-4) and C-15003 P-3 on the survival in mice inoculated with L1210 leukemia BDFp mice ( 5 animals per test group) were vaccinated intraperitoneally with lxlO3 cells from leukemia L1210 on day 0.

The antibiotic C-15003 [a mixture of C-15003 P-3 (60-65% by weight), C-15003 P-4 (30-35% by weight) and C-15003 P-3 '] and C-15003 P-3 were each dissolved in 0.85% saline containing 2.5% methanol and each solution intraperitoneally once a day for 9 days in tumor vaccinated mice (0.2 ml / mouse) administered starting 24 h after tumor inoculation.

In order to evaluate the cytostatic effect of the test agents, the T / C% was recorded according to the information in the NCl methodology. As already mentioned, this is the ratio times 100 of the mean survival time of mice suffering from L1210, which had been treated with the agents, and this compared to untreated mice (a group of 25 mice). T / C% of 120 was considered to be the minimum in relation to the cytostatic activity of the agents.

As can be seen from Table 10, the mixture of C-15003 P-3, C-15003 P-3 'and C-15003 P-4 and C-15003 P-3 was effective in extending the survival time of L1210-infected mice .

Table 10

medium

Dose mcg / kg /

Day

Number of mice average survival in days

TIC%

the mixture of

40

5

12.0

128

C-15003 P-3,

30th

5

12.2

130

C-15003 P-3

20th

5

11.0

117

and C-15003

10th

5

10.2

109

P-4

C-15003 P-3

40

5

11.0

117

30th

5

12.2

130

20th

5

11.2

119

10th

5

10.4

111

(Blind test)

25th

9.4

Effect of C-15003 P-3 and C-15003 P-4 on the survival of mice inoculated with sarcoma 180

ICR-JCL mice (supplied by Japan Clea, Tokyo; 5 animals per test group) were vaccinated intraperitoneally with 4x 106 sarcoma 180 cells on day 0.

Each antibiotic was dissolved in 0.85% physiological saline containing 2.5% methanol, and the solution was intraperitoneally administered to the tumor vaccinated mice (0.2 ml / mouse) once a day for 7 days, the administration 24 hours after tumor inoculation was started.

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

15

637 137

Table 12

The T / C% value for the mean survival time was calculated as described in Example 8.

As can be seen from Table 11, both agents were significantly effective in extending the survival of animals suffering from sarcoma 180.

Table 11

medium

Dose number mcg / kg / mice day average T / C% survival in days

medium

Dose mcg / kg /

Day

Number of mice average survival in days

TIC'Sr

C-15003 P-3

50

5

> 21.5

> 171

25th

5

> 21.5

> 171

12.5

5

> 21.5

> 171

6.25

5

> 21.5

> 171

C-15003 P-4

25th

5

> 21.5

> 171

(Blind test)

10th

12.6

C-15003 P-3 C-15003 P-4 (blind test)

25 25

5 5 10

24.5 33.5 13.0

188 258

C-15003 P-3

(Blind test)

50 25 12.5 6.25

5 5 5 5 10

33.5 30.5 15.5 17.8 11.8

284 258 131 150

20th

25th

Effect of C-15003 P-3 and C-15003 P-4 on the survival of mice vaccinated with Ehrlich carcinoma. ICR-JCL mice (5 animals per test group) were vaccinated intraperitoneally with 4x 10 "cells of Ehrlich carcinoma on day 0 .

Each antibiotic was dissolved in a 0.85% physiological saline solution containing 2.5% methanol and the solution was administered intraperitoneally once a day for 7 days to mice (0.2 ml / mouse), with the administration being continued for 24 hours tumor inoculation started.

The T / C% value for the mean survival was calculated as in Example 8.

As can be seen from Table 12, both agents were significantly effective in extending survival in Ehrlich carcinoma-bearing mice.

Effect of C-15003 P-3 and C-15003 P-4 on the survival of mice inoculated with P815 mastocytoma. BDFp mice (5 animals per test group) were vaccinated intraperitoneally with 1 x 10fl cells of P815 mastocytoma on day 0.

Each antibiotic was dissolved in a 0.85% physiological saline solution containing 2.5% methanol and the solution was administered intraperitoneally daily to tumor-vaccinated mice (0.2 ml / animal) for 7 days, the administration being carried out after the expiration started 24 hours after tumor inoculation.

The value T / C% for the mean survival was calculated as in Example 8.

As can be seen from Table 13, both agents were significantly effective in extending the survival of P815 mastocytoma-infected mice at the dose levels used in this experiment.

30th

Table 13

medium

Dose mcg / kg /

Day

Number of mice average survival in days

T / C%

35 C-15003 P-3

50

5

10.3

61

25th

5

> 28.5

> 170

12.5

5

> 28.5

> 170

6.25

5

> 28.5

> 170

C-15003 P-4

25th

5

> 28.5

> 170

40 (blind test)

10th

16.8

m

Claims (9)

637 137 PATENT CLAIMS 1. Antibiotic C-15003 of the following general formula: ci <rH3 p (i) where R is the rest / CH, -CO-CH XCH, -CO-CH2-CH2-CH3 or CH, -CO-CH-r-CH CH, represents. " 2. Antibiotic according to claim 1, wherein R represents the rest / CH, -CO-CH, x: h, represents. 3. Antibiotic according to claim 1, wherein R is -CO-CH2-CH2-CH. 4. Antibiotic according to claim 1, characterized in that R represents the rest / CH, -CO-CH, -CH 30 CH, means. 5. A process for the preparation of the antibiotic C-15003, characterized in that one of the genus Nocardia 35 belonging microorganism, which is able to produce the antibiotic C-15003, in a culture medium which contains assimilable carbon sources and digestible nitrogen sources, until the antibiotic is substantially enriched therein, after which the antibiotic C-40 15003 is isolated. 6. The method according to claim 5, characterized in that as a microorganism Nocardia No. C-15003 (IFO13726) is used. 7. A process for producing a compound of the following 45 formula: characterized in that the antibiotic C-15003 of the following general formula: 637 137 (I) where R is the rest -CO-C -CH, "CH, -CO-CH2-CH2-CH., Or -CO-CH, -CH / CH., VCH, 20th 25th 30th means, produces by the method according to claim 5 and then subjected to reductive hydrolysis. 8. The method according to claim 7, characterized in that the reductive hydrolysis takes place by using a complex metal hydride. 9. The method according to claim 8, characterized in that lithium aluminum hydride is used as the complex metal hydride. 35 For years, research has been carried out into substances which, after administration to warm-blooded animals, including people suffering from cancer, guarantee them an extended lifespan. For this purpose, various soil samples and other samples were collected and the microorganisms isolated from them were sighted. It was found that some of these microorganisms are able to produce a new antibiotic, that such microorganisms belong to the genus Nocardia and that the antibiotic C-15003 can be enriched in the culture broth by cultivating such microorganisms. It has also been found that derivatives can be produced from this antibiotic. These findings have led to the present invention.