CS214749B2 - Means for treating the diseased plants and method of preparation of active substance - Google Patents

Abstract

A novel Antibiotic C-15003 is produced by cultivating a microorganism of the genus Nocardia. The Antibiotic C-15003 is useful as an antifungal agent or an antiprotozoan agent.

Description

The invention relates to a composition having antimicrobial, antiprotozoal and antifungal activity suitable for the treatment of diseased plants and to a process for preparing the active ingredient of the composition.

According to the present invention, many soil and other samples were collected and microorganisms isolated from the samples were examined and sorted. These experiments have shown that some of the microorganisms of interest are capable of producing a novel antibiotic, said microorganisms belonging to the genus Nocardia, and that it is possible to collect said antibiotic in a culture medium by culturing the microorganisms in suitable nutrient media. It has also been found according to the invention that various derivatives of said antibiotic can be prepared.

The subject of the present invention is that it contains an antibiotic as an active ingredient. General formula C-15003:

wherein R is a group

CHs /

—CO — CH \

CH3 —CO — CH2 — CH2 —CH3,

CH3 / -CO-CH2-CH

CH 3 or a hydrogen atom.

The method of preparation of the active ingredient of the composition of the present invention is characterized by culturing the microorganism of Nocardia strain C-15003 IFO 13726 in a culture medium containing assimilable carbon sources and digestible nitrogen sources at a temperature in the range of 20 ° C to 35 ° C with an initial pH of about 7 under aerobic conditions until the C-15003 antibiotic accumulates in said nutrient medium, and then optionally the C-15003 antibiotic thus obtained is subjected to reductive hydrolysis to form a compound of formula :

In the advantageous. carrying out the process according to the above. According to the invention, the abovementioned reduction hydrolysis is carried out with the aid of a complex metal hydride. As a complex metal hydride, the use of lithium aluminum hydride is preferred.

The advantages. The compositions of the present invention possess excellent antimicrobial, antiprotozoal and antifungal properties useful in the treatment of plants.

In the description of the present invention, the term "antibiotic C-15003" generally refers to either a group of all three compounds having the aforementioned general formula I, or a mixture of two or three of said compounds, or individually any of the foregoing. compounds. A compound of formula I wherein R is a group of formula

CH3 /

—CO — CH .....

\

CHs

-15003 P-3 “; a compound of formula I wherein:. R represents a group of the formula —CO — CH2 — CH2 — CH3, referred to as "antibiotic C-15003 P-3‘ "or abbreviated as" C-15003 P-3 ‘"; a compound of formula I wherein R is a group. formulas

CH3 /

—CO — CH2 — CH \

CH 3 is referred to as "antibiotic C-15003 P-4" or, in short, "C-15003 P-4"; and a compound of formula I wherein R is an atom. hydrogen (i.e. the compound of formula II) is referred to as an "antibiotic. C-15003 P-O 'or' C-15003 P-O '.

An example of a strain of microorganisms producing the antibiotic C-15003 is antinomycetes strain number. C-15003, which the inventors isolated from the soil. also from other samples at. studying and sorting of antibiotic-producing microorganisms.

The microbiological features of strain number C-15003 were determined by analogous methods to those described by Schirling & Gottlieb (International Journal of Systematic Bacteriology 16, 313-340 (1966)). The results of observations made over 21 days at 28 ° C are shown below.

1. Morphological features

The vegetative mycelium grows well and develops in the form of branched fibers, both on agar and in a liquid culture medium. Numerous hyphae have a diameter of 0.8-1.2, dark. and in some cases they may disintegrate into rod-like bacteria particles or short branched hyphae. The strain grows well on various taxonomic. nutrient. soils, where ... airborne mycelium forms on the surface of vegetative mycelium,. which often form cromium-like formations. (bundles of conidiophors of dimensions 50, -200. X 200—1000 μΐη), per. which. is located. future aerial mycelium. Numerous aerial mycelia are wavy, straight or. loosely helical as the shape was determined on various occasions. Microscopic evaluation of mature cultures shows that only rarely do conidium-like cells appear on the fibers, while cell suspensions obtained from the surfaces of said cultures contain. as determined microscopically, numerous elongated ellipsoidal (0.8-1.2 µm XX 4.8-6.8 .. µϊίι) and ellipsoidal (0.8-1.2 µ XX 1.0—2.0 µΐη ) bodies similar to arthospores.

When viewed with an electron microscope was. . it has been found that these bodies have a smooth surface.

is referred to herein as "C-15003 P-3 antibiotic"; or. shorter 'C2. Cell components

The strain was cultivated for. shaking on Mode 221449 fed ISP broth # 1 at 28 ° C for 66-90 hours and after that time cells were harvested and rinsed. All obtained cells were then evaluated by the method described by B. Bocker et al. (Applied Microbiology 12, 421 (1964)) and by the method described Μ. P. Lechevalier (Journal of Laboratory and Medical Medicine 71, 934 (1968)) on the content of -diamtoopimelic acid and on the composition of sugars. The first substance was found to be present in the meso-form, and traces of substances corresponding to galactose and a.rabinose were detected,

3 .. Characteristics features on different taxonomic media

Strain. It exhibits relatively good growth in various nutrient media, with the vegetative mycelium being colorless to pale yellow in the early stages of cultivation and light yellow to brown in the later stages. In various taxonomic environments, the strain produces soluble dyes, yellow to yellow-brown. Airy, mycelium is dusty, usually medium-sized, white to yellow or light yellow-brown. The strain characteristics on the different taxonomic soils are shown in Table 1.

Table 1

Characteristic cultivation features - strain number C-15003 on various taxonomic soils

A. Sucrose nitrate agar:

Growth (G):

lush, melon-yellow color (3 iaj * - -to 'amber yellow (3 1c) *, formation of coremium-like formations

Air, mycelium (AM):

sparse, white

Soluble dyes (SP): no or light yellow-brown

B. - Nitrate agar with glycerol:

G: medium. color - light, - pitch bones (2 ca) *, formation of corium-like structures AM: medium, white

SP: none

C. - Asparagine glucose agar:

G; medium, color yellow - (3 pa) * to- bright- yellow (2 pa) *

AM.:, Sparse, white

SP: yellow (2 * a) *

D. Asparagine agar with glycerol:

G: medium, pale-color - (2 car, corium-like formation AM: -spore, white

SP: none.

E. Starch agar:

G: medium, light ivory color (2 ca) ´ to light corn color (2nd & a) * formation of corium-like formations AM: abundant, light ivory color (2 ca) *

SP: none

F. Nutrient agar:

G: medium, pale color (2 ca) * to ancient yellow (2. ga) *, formation of corium-like forms

AM: sparse, white

SP: none

G. Calcium malate agar:

G: medium color - light ivory (2 -ca) * to light corn (2 ea) *, formation of corium-like formations

AM: Medium, white to light ivory (2 ca) *

SP: none

H. Agar with yeast extract and malt extract:

G: medium, amber (3 1c) * to bright yellow (3 lap, corium-like formation)

AM: Medium, white to light, ivory (2 'ca.) *.

SP: none

I. Agar with oat flour:

G: - medium, light ivory color (2 -ca.) * -To ancient yellow (2 * a) * - formation of corium-like formations

AM: -sparing, white to pale yellow

SP: none .....

J. Pepton agar with yeast extract and iron:

G: - medium, antique yellow color (2 ga) *

AM: None

SP: ancient yellow (2 -ga) *

K. Tyrosine Agar:

G: medium, light ivory color - bones (2 ca) * to - light melon yellow (3 ea) *, formation of -coremic-like formations.

AM: Medium, white to light ivory (2-c.) *

SP; brownish pink (3 ie) *

(i) - Color designation according to Color Hormony Manual, 4 edition (Container Corporatlon -of America, 1958).

4. Physiological properties

Physiological. strain properties are listed in Table 2. The strain grows over a temperature range

12—38. Deň: 32 ° C. The temperature range at which good airborne growth on the agar (ISP # 2) occurs is 20-35 ° C.

Table 2

Physiological properties of strain number C — 15003

Temperature range suitable for growth: 12 to 38 ° C

Temperature range suitable for air growth: 20 to 35 ° C

Liquidation of gelatin: positive

Starch hydrolysis: positive

Nitrate reduction: positive

Peptonization of milk: positive

Milk coagulation: negative

Casein decomposition: positive

Melanin dye formation: negative (peptone agar with yeast extract and iron) positive (tyrosine agar) Tyrosine decomposition: positive

Xanthine decomposition: negative

Hypoxanthine breakdown: negative Lysozyme tolerance: positive

Tolerance to sodium chloride: 2%

5. Use of various carbon sources

The utilization of various carbon sources by the C-15003 strain was monitored using the medium described by Pridham and Gottlleb (Journal of Bacteriology 56, 107 (1948)) and using a basic nutrient medium of the same composition to which 0.1% yeast extract was added. The spectrum obtained is shown in Table 3 below

Table 3

Utilization of various carbon sources by strain 015003

Carbon source Grow D-xylose + 4-4- * L-arabinose + + D-glucose ++ —1_ D-galactose 4- + D-fructose 4-4 * 4- 4-4- L-rhamnosa 4- + D-mannose 4—1 — H ++ sáčharosa 4-4- 4-4- lactose - - maltose 4- 4- trehalosa X 4-4-

Carbon source Grow rafinosa + 4- * melibiosa 4- 4- i-inositol - D-sorbitol -. D-mannitol 4-4- -4- glycerol - soluble starch + control -

*) Basic medium with addition

Paznámka:

4 — F + lush growth

- | —j- good growth + growth + weak growth - no growth

6. Other characteristic features

Cells were cultured as described above under 2. and DNA was prepared as described by J. Marmur et al. (Journal of Molecular Biology, 3, 208 (1961)). The content of G-C (guanine-cytosine) in the DNA was found to be about 71 mol%.

Gram staining of the vegetative mycelium of this strain was positive.

The above characteristics of strain number C-15003 were compared with the descriptions in SA Waksman's monograph "The Actinomycetes", Volume 2 (edited by Williams and Wilkins Co., 1961), with the descriptions in RE Buchana and NE Gibbons "Bergey * s Manual of Determinative Bacteriology ', 8th Edition, 1974, and provides descriptions

1.1% yeast extract.

in other literature.

Although the inventors believed that this strain belongs to the group III of the genus Nocardia, they failed to find any species among the known strains having the characteristics described above and therefore conclude that this strain represents a new species of microorganism.

The strain No. C-15003 is deposited with the Fermentation Research Institute (FERM) under accession number 3992, and the Institute for Fermentation in Osaka (IFO / J). under the accession number IFO 13726, and in the American Type Culture Collection (ATCC), Maryland, USA under the accession number 31281.

Although strain C-15003, as mentioned above, is a new species of the genus Nocardia, it is able, like all microorganisms, to undergo various variations and mutations, either spontaneously or by the action of mutagens. For example, numerous variants of the strain can be obtained by X-rays, gamma-rays, ultraviolet light, etc., isolation of unicellular cells, culture of the strain on nutrient media containing various chemicals, or any other mutagenic action, as well as mutants arising spontaneously from the original. the strain may not be considered essentially another distinct species, but rather any such variant or mutant of the original strain capable of producing the antibiotic C-15003 P-3, P-3 'and / or P-4 may be used without modification for the purposes of this invention as well as strain number-C-15003. When the strain number C-15003 is treated with various mutangens, for example, mutants that are substantially lacking in the ability to produce soluble dyes, or mutants whose basic myoelia are colorless, yellowish green, reddish or orange red, or mutants whose hyphae are capable of disintegrating into rod-like particles or short branched fragments of hyphae, or mutants with abundant white air-mycelium or almost no air mycelium at all.

Any liquid or solid culture medium can be used as the culture medium for the strain-producing antibiotic C-15003 only if it contains nutrients that the strain can utilize; the liquid medium is advantageous in large batch production. The medium must contain sources of carbon and nitrogen, which strain C-15003 is capable of assimilating and digesting, inorganic substances, micro-nutrients and other substances. Examples of said carbon sources include glucose, lactose, sucrose, maltose, dextrin, starch, glycerol, mannitol, sorbitol and the like, fats and oils (e.g., soybean oil, lard oil, chicken oil and the like) -atd. Examples of nitrogen sources which may be used are meat liquor, yeast liquor, dried yeast, soybean meal, corn liquor, peptone, cottonseed meal, fermented molasses, urea, ammonium salts (e.g. ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate) etc.) and similar raw materials. The broth may further comprise salts of sodium, potassium, calcium, magnesium, etc., salts of iron, manganese, zinc, cobalt, nickel, etc., salts of phosphoric acid, boric acid, etc., and salts of organic acids such as acetates and propionates. The culture medium may further comprise, as addition, various amino acids (e.g. glutamic acid, aspartic acid, alanine, glycine, lysine, methionine, proline, etc.), peptides (e.g. dipeptides, tripeptides, etc.), vitamins (e.g. -vitamin Bi and Bz, nicotinic acid, vitamins B12, C, E and others), nucleic acids (e.g., purine, pyrimidine and derivatives thereof) and other substances. Inorganic or organic acid, alkali, buffer and the like can be added to adjust the pH of the nutrient medium. Further, suitable amounts of antifoam agents such as oils, fats, surfactants and the like can be added to the environment.

The cultivation of the strain can be carried out under stationary conditions, with shaking, under submerged aerobic conditions and under other cultivation conditions. For large-scale batches, of course, submersible aerobic cultivation is advantageous. The conditions of cultivation depend, of course, on the form and composition of the culture medium, the type of strain, the method of cultivation -and other factors; normally, incubation at 20-35 ° C, at an initial pH of about 7.0 or the like is preferred. A particularly preferred medium temperature of cultivation is 23 to 30 ° C, at an initial pH of 6.5 to 7.5. The cultivation time is also variable and depends on the same factors as mentioned above; it is advisable to continue the cultivation until the titer of the resulting antibiotic has reached its maximum. In the case of shaking cultivation or in the case of aerobic submersible cultivation in a liquid culture medium, the required cultivation time is normally within the range of 48 to 144 hours.

The amount of antibiotic formed is determined using Tetrahymena -pyriformis W as a test organism. The microorganism is grown on a test broth (20 g - proteasepepton (Difco), 1 g yeast extract (Difco brand), 2 g glucose, 1000 ml distilled water and 10 · - ml fosf-phosphate buffer / pH 7.0) at 28 [deg.] C. for 44 to 48 hours and the amount of antibiotic is determined by a serial dilution method which monitors the turbidity caused by the microorganism cells; at the same time, the amount of antibiotic is determined based on its inhibitory effect on -ascitic tumor cells, and by thin layer chromatography (TLC) as described below.

The novel antibiotics C-15003 P-3, P-3 ‘- and / or P-4 are produced by the microorganism and accumulated in the culture broth both extracellularly and intracellularly.

Said antibiotics are also detected by thin-layer chromatography. The fermentation liquid is separated by filtration or centrifugation into a cell-containing fraction and the filtrate and the filtrate is shaken with an equal volume of ethyl acetate. -Add the cells with the same quantity of 70 ^o / o ornti aqueous acetone as -k filtrate -míchá suspension for one hour at 20 ° C -then filtered. Acetone was removed under reduced pressure from the filtrate and the remaining aqueous portion was shaken with ethyl acetate. Each individual ethyl acetate extract is concentrated to 1/100 of the original volume and the concentrate is subjected to thin layer chromatography on a silica gel layer on a glass plate (Merek, Federal Republic of Germany, Kieselgel 60 F254, 0.25 mm, -20 XX 20 cm) for use of solvent system -chloroform-methanol 9: 1. The amount of substances is determined based on the fluorescence intensity of the spots after detection by ultraviolet light irradiation at 2537 A.

The C-15003 P-3, P-3 'and / or P-4 antibiotics produced by the culture medium in this manner are lipophilic and neutral substances which can be isolated and purified by conventional separation and purification procedures, which are normally used in the isolation of similar microbial metaholites. For example, methods that utilize differences in solubility between the antibiotic and the impurity can be used, or methods that utilize the fractional adsorption affinity of various adsorbents such as activated carbon, macroporous non-ionic resins, silica gel, alumina, and the like, or processes using ion exchange resins to remove impurities, or similar processes; said processes may be applied singly or in suitable combinations or repeatedly.

Since, as mentioned above, the C-15003 P-3, P-3 ‘and P-4 antibiotics occur both in the culture medium filtrate and in the cells, the antibiotics mentioned. isolated and purified by means of said adsorbents either directly or after solvent extraction of the filtrate or solvent extraction of microbial cells. The solvent extraction of the antibiotics may be carried out by one of the methods described below or by other methods, for example:

1. by solvent extraction from culture broth - without cell separation, and 2. solvent extraction from cells and filtrate after separation by filtration, centrifugation or other means. The following procedure may be advantageously used to extract the filtrate and cells, each fraction separately.

Suitable solvents for extracting the filtrate include water-immiscible organic solvents such as fatty acid esters, for example ethyl acetate or amyl acetate; alcohols such as butanol; halogenated hydrocarbons such as chloroform; and ketones such as methyl isobutyl ketones. The extraction is carried out at a pH close to neutral, and preferably the culture liquid pretreated to pH 7 is extracted with ethyl acetate. The extract was washed with water and concentrated under reduced pressure. A non-polar solvent such as petroleum ether or hexane is then added to the concentrate to give a crude product I containing the active ingredient. Since product I contains a number of stains other than antibiotic C-15003 when evaluated by TLC, the crude product is subjected to further purification operations. Particularly suitable as a routine purification procedure is adsorption chromatography to which one of the following conventional adsorbents can be used: silica gel, alumina, macroporous non-ionic adsorption resins and the like. Silica gel is most preferred for purification of the crude product. The elution can be carried out, for example, first with petroleum ether and hexane, and then with mixtures thereof with polar solvents, for example ethyl acetate, acetone, ethanol or methanol. In a typical procedure, silica gel column chromatography (Merck, Federal Republic of Germany, 0.05-0.2 mm] is used as the support and the elution of the substances is first carried out with hexane, then with a mixture of hexane with increasing amounts of ethyl acetate, and finally The eluate was collected by TLC fractions and the fractions containing the -antibiotic C-15003 were combined and concentrated under reduced pressure, and petroleum ether or hexane was added to the concentrate to give crude product II. For example, the crude product II is repeatedly chromatographed on a silica gel column in a different solvent system, for example an elution system consisting of a halogenated hydrocarbon such as dichloromethane or chloroform is suitable. with the addition of a polar solvent such as an alcohol, for example methanol or ethanol, keto of acetone or methyl ethyl ketone, or the like, to give pure C-15003 antibiotic. The order of the solvent systems used for the first and second column chromatography on silica gel can be reversed, and can also be used in combination with the above systems and optionally other conventional organic solvents.

If a macroporous adsorption resin is used to purify crude product II, elution of the antibiotic - C-15003 with a mixture of water - is carried out with a lower alcohol, a lower ketone or an ester. As the lower alcohol, for example, methanol, ethanol, propanol or butanol can be used, as a lower ketone, for example acetone or methyl ketone, and as an ester, for example ethyl acetate. In a typical procedure, the crude product II is dissolved in 60 µl ^. % aqueous methanol, the solution is adsorbed onto a column of Dianion HP-10 resin (Mitsubishi Kasei KK product column is washed with 70% aqueous methanol and then eluted with 90% aqueous methanol) to remove the solvent. The column elutes antibiotic C-15003.

In any of the above purification methods, fractions containing the antibiotic C-15003 are combined and the solvents distilled off under reduced pressure. 5 to 8 parts by volume of ethyl acetate are added to the dry residue, and the mixture is left to stand, thereby eliminating the crystals of the C-15003 antibiotic; these crystals contain antibiotic C-15003 P-3, P-3 ‘and P-4. Said compounds are separated one from the other by chromatography on a suitable adsorbent, for example on one of the abovementioned adsorbents. For example, by using silica gel or macroporous non-ionic adsorption resin and the above solvent systems, the desired compounds can be eluted fractionately. It is used, for example, to separate said silica gel mixture, eluting with hexane, ethyl acetate or a mixture of chloroform and methanol; fractions containing the antibiotic C-15003 P-4, then fractions containing C-15003 P-3 ‘, and finally fractions containing C-15003 P-3, flow out of the column. After TLC evaluation, the fractions containing C-15003 uniforms P-4, P-3 ‘and P-3 were combined, the solvents were distilled off under reduced pressure, and ethyl acetate was added to the residue. In this way, pure compounds can be obtained in crystalline form. If a macroporous non-ionic adsorption resin is used to separate the macroporous adsorption resin, a gradient elution is applied in the ehromatography using an increasing amount of alcohol, ketone or ester in water. For example, in a gradient elution using 60% aqueous methanol to 95% aqueous methanol, with the addition of · 5% sodium chloride, the fractions containing C-15003 P-3, then the 'fractions' containing C-15003 P-3 'and finally the fraction containing C-15003 P-4. Fractions were collected, evaluated by TLC, solvents from each group of fractions containing the same active ingredient were distilled off under reduced pressure, and the residue was recrystallized from ethyl acetate.

The obtained crystalline substances contain ethyl acetate as a crystal solvent; after drying over phosphorus pentoxide at 70 ° C (for 8 hours), the C-15003 P-3, P-3 ‘and P-4 antibiotics exhibit the following physical and chemical properties (see Table 4).

Table · 4

Physical and chemical properties of antibiotic C-15003

Antibiotic C-15003 P — 3 P — 3 ‘P — 4

C32H43CIN2O9 = 635.169 C32H43CIN2O9 = 635.169 C33H45CIN2O9 = 649.196

Melting point

Specific rotation

14 ²²

Elementary analysis

Found (%)

Elementary analysis

Calculated (%)

Ultraviolet absorption spectrum nm (ε) (in methanol) Infrared absorption absorption spectra (cm ^-1 ) KBr

Nuclear Magnetic Resonance Spectrum (PPm)

100 MHz in CDCl3

Mass Spectrum (m / e)

Solubility

190-192 ° C -136 ° + 10 ° (c = 0.375, CHCl 3)

C60.06

H7.04

N4.33

Cl5.37

C60.51

H6.82

N4.41

Cl5.58

233 (30250) 240 (sh 28450) 252 (27640) 280 (5750) 288 (5700)

1740, 1730

1580, 1445, 1385,

1340, 1255, 1180,

1150, 1100, 1080, 1038 L, 27 (d) (3H) 1,28 (d) (3H)

573, 485, 470, 450 non-res. in petroleum ether, insol. petroleum ether, hexane and water 'sparingly soluble in benzene and ether soluble in chloroform, ethyl acetate, acetone, ethanol, methanol, pyridine, tetra-toluene, pyridine, tetra-toluene, pyridine, tetrahydrofuran, dimethyl-hydrofuran, dimethyl-hydrofuran, dimethyl sulfoxide sulfoxide. sulfoxide

182-185 ° C - 134 ° + 10 ° (c = 0.11, CHCl 3)

60.09

7.02

4.34

5.99

60.51

6.82

4.41

5.58

233 (30155) 240 (sh 28250) 252 (27600)

280 (5750) 288

1740, 1730

1580, 1445, 1385,

1340, 1255, 1180,

1150, 1100, 1080, 1038 L, 06 (t) (3H)

573, 485, 470, 450 hexane and water sparingly soluble in benzene and ether soluble in chloroform, ethyl acetate, acetone, ethanol, me177 to 180 ° C -142 ° + 10 ° (c = · 0.522, CHCl 3)

60.65

7.05

4.25

5.23

61.05

6.99

4.32

5.46

233 (29900) 240 (sh 28240) 252 (27590) 280 (5712) 288 (5680)

1740, 1730, 1670, 1580, 1445, 1385, 1340, 1255, 1180, 1150, 1100, 1080, 1038 L, 03 (d) (6H)

587, 485, 470, 450 non-res. in petroleum ether, hexane and water sparingly soluble in benzene and ether soluble in chloroform, ethyl acetate, acetone, ethanol, MeColor reactions

Dragendorff: Positive Dragendorff: Positive Dragendorff: Positive Beilstein: Positive Beilstein: Positive Beilstein: Positive

1S

The above-described antibiotic C-15003 was compared on the basis of molecular weights and the antimicrobial and antineoplastic activity shown below with known classes of antibiotics. The literature search did not find any obvious class of antibiotics similar to the antibiotic C-15003. However, when searched among known plant ingredients and other naturally occurring organic compounds for substances with a similar ultraviolet absorption spectrum as the antibiotics prepared by the method of the present invention, it was found that similar spectra of the maytanacin group antibiotic, and comparing the respective molecular weights and summary of the formulas, it was concluded that antibiotic C-15003 belongs to the group of maytacin antibiotics with two nitrogen atoms. Maytanacin was previously isolated from the bark of some plants and described in Journal of the American Chemical Society 97, 5294 (1975).

Maytanacin has the mass spectrum shown below.

M ⁺ - (а) M + - 485 - С 485 - Cl (a- | -b)

545 485 470 450 (a) = H10 + HNCO (b) = R-C-OH

O

The presence of m / e fragments 485, 470 and 450 in the C-15003 P-3, P-3 * and P-4 antibiotics convinced the inventors that these compounds have a skeleton structure identical to maytanacin, and that they differ from maytanacin by the acyl group Thus, it is clear that the C-15003 antibiotic is a novel compound. When the individual antibiotics C-15003 P-3, P-3 'and P-4 were alkaline degraded and the released carboxylic acids were analyzed by gas chromatography, it was found that isobutyric acid was obtained from C-15003 P-3, from C-15003 P-3 'butyric acid and C-15003 P-4 isovaleric acid. The above data shows the structure of the antibiotics C-15003 P-3, P-3 ‘and P-4 shown in Figure 1.

Figure 1

P — 3, R = —CO — CH \

СНз

P-3 ‘, R = -CO-CH 2 -CH 2 -CH 3

СНз

P — 4, R = —СО — CH2 — CH \

СНз

Biological efficacy:

A. Antimicrobial activity

The inhibitory concentration of antibiotic C-15003 was tested by the paper disc method using tryptocase-soy agar (BBL) as a test broth. Filter paper rolls (manufactured by Toyo Seisakusho, thin type, 8 mm diameter) impregnated with 0.02 ml of C-15003 P-3, P-3 'or P-4 antibiotic solution at a concentration of 300 µg / ml were placed on agar The plates were inoculated with the microorganisms below and the minimum inhibitory concentrations of antibiotics were monitored. The tests showed that the antibiotics in question were not effective against the following microorganisms: Escherichia coli, Proteus vulgaris, Proteus mirabelis, Pseudomonas aeruginosa, Staphylococcus aureus,

Bacillus subtilis, Bacillus cereus, Klebsiella pneumoniae, Serratia marcescens, Mycobacterium avium.

On the other hand, inhibitory activity against growth of Talaromyces avellaneus grown on agar plates (composition of nutrient medium: 3.5 g sodium hydrogen phosphate, 0.5 g potassium dihydrogen phosphate, 5 g yeast extract (Difcoj, 10 g glucose, 15 g agar) 1000 ml of distilled water, pH 7.0) In these tests, a minimum inhibitory concentration of 3 [mu] g / ml and 1.5 [mu] g were observed for C-15003 P-3 and P-3 ' Next, a wild strain of Tetrahymena pyriformis W as a test organism was cultivated in a nutrient medium (composition: 20 g proteaseopepton (Difco), 1 g corn extract, 2 g glucose, 1000 ml distilled water and 10 ml 1 M phosphate buffer pH 7). The inhibitory activity of the antibiotic C-15O0.3 against the growth of this specific microorganism was determined at 28 ° C for 44-48 hours and by a serial dilution method at a concentration of 1 µg / ml. for C-15003 P-3 and P-3 ‘and at a concentration of 0.5 µg / ml for C-15003 P-4.

The antifungal activity of antibiotic C-15003 is shown in Table 5. As can be seen from this table, antibiotic C-1-5003 has inhibitory activity against the growth of microorganisms that cause various plant diseases. Inhibitory potency was determined by a disk method in which filter paper rolls impregnated with 0.02 ml of 1000 µg / ml C-15003 antibiotic solution were placed on agar plates inoculated with the following microorganisms (see Table 5).

Table 5

Antimicrobial spectrum of antibiotic C-15003

Tested microorganism IFO number Environment Time (throw.) Inhibition average Alternaria kikuchiana 7515 PSA * 48 38 Fusicladium levieri 6477 PSA * 90 68 Helminthosporium sigmo- ideum var. irregulare 5273 PSA * 48 55 Pyricularia oryzae - PSA * 48 53 Elsinoe fawcetti 8417 PSA * 90 55 Fusarium -oxysporum f. cucumerinum - PSA * 48 20 May Guignardia laricina 7888 PSA * 48 12 Cochlioborus miyabeanus 5277 PSA * 48 60 Di-aporthe citri 9170 PSA * 48 55 Gibberella zeae 8850 PSA * 48 37 Sclerotinia sclerotiorum 9395 PSA * 90 65 Venturia pirina 6189 PSA * 48 50 Pellicul-aria sasakii 9253 PSA * 48 50 Pythium aphanidermatum 7030 PSA * 48 58 Botrytis cinerea - PSA * 48 48 Aspergillus niger 4066 PSA * 48 0 Pe ^; nicillium chrysogenum 4626 PSA * 48 35 Rhizopus nigricans 6188 PSA * 48 25 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 utllis 0619 GB ** 48 0 Cryptococcus neoformans 0410 GB ** 48 43

) PSA: Potato agar with sucrose *) GB: Nutrient agar with glucose

B. Anti-tumor efficacy

Antibiotics - C-15003 P-3, P-3 'and P-4 were administered intraperitoneally for nine consecutive days to P 388 leukemia mice (1 X 10® cells per animal, transplanted intraperitoneally) and the therapeutic effect of the administered substances. The compounds showed an antitumor activity, manifested in an increase in the lifespan of the test animals by more than 200% at a dose of 0.00625 mg / kg / day.

C. Toxicity

The acute toxicity of C-15003 P-3, P-3 ‘, and P-4 was determined in mice following intraperitoneal injection. All antibiotics administered were found to have a LD50 of greater than 0.313 mg / kg.

As mentioned above, the C-15003 antibiotic prepared by the method of the invention has high inhibitory activity against fungi and protozoa and is therefore a valuable antifungal and antiprotral agent. Furthermore, it can be expected that the antibiotic C-15003 extends the survival time of the experimental animals. (e.g., mice) with some tumors that the compound will find use in the treatment of cancer.

The C-15003 antibiotic can be used as a substance with antifungal and antiprotozoal efficacy for the evaluation of bacterial ecology in soil, in active sludge, in animal body fluids and the like. For example, if valuable bacteria are to be isolated from soil samples, or if the activity of single bacteria is to be evaluated independently of the activity of different fungi and protozoa, for example in connection with the operations and analyzes of the active sludge system used in treatment, The use of antibiotics C-15003 can be used to allow the selective growth of the bacterial flora and not to be allowed. growth of the accompanying fungi and protozoa in the sample. In a typical example of this use, the test sample is added to a liquid or solid nutrient medium, then 0.1 ml of a C-15003 antibiotic solution in ¹⁰ % aqueous methanol is added to each 10 ml of culture medium. 100 µg / ml, and incubated.

Antibiotics C-15003 can also be used as an antimicrobial agent for the treatment of plant diseases caused by the microorganisms listed in Table 5 above.

In a typical application, the C-15003 antibiotic used in the form of a 1% aqueous-methanolic solution containing 0.5 to 5 µg / ml of active ingredient. The solution can be used, for example, to protect against reddish-brown rot of leaf scabbard, against gangrene, against helmiinthospore stripiness of leaves and against gangrene of rice plants.

From the above description, it is evident that the C-15003 P-3, P-3'-and P-4 antibiotics are novel compounds having the same structural skeleton, and can also be used as intermediates to produce other antibiotics. of pharmaceutically important compounds. For example, by the deacylation reaction, antibiotic c-15003 P-O (maytansinol) -with a free hydroxyl-group at position 3 of the molecule can be obtained from said antibiotics. Since the acyl group is located in the β-position to the carbonyl, conventional reductive hydrolysis can be advantageously used for deacylation. For example, by treatment with a complex metal hydride (e.g. lithium aluminum hydride, LiAlHa) at low temperatures (e.g. -20 ° C to 0 ° C), the O-ester at the 3-position can be hydrolytically cleaved to give maytansinol without affecting other functional groups such as carbonyl, epoxy, C = C double bonds, etc. The physical and chemical properties of a sample of the maytansinol thus obtained are in good agreement with the data described by Kupchan and co-workers in Journal of the American Chemical Society 97, 5294-5295 (1975).

The process according to the invention is further illustrated by the following non-limiting examples. Unless otherwise indicated, "parts" means weight parts, and the relationship between "parts" and "parts by volume" corresponds to the relationship between "grams" and "milliliters"; and "%" -relate, unless otherwise stated, to the weight / volume ratio.

Examples

Example 1

Kmen -č. C-15003 (IFO 13726; FERM 3992; ATCC 31281) of Nocardia cultivated on nutrient broth (yeast and malt extract agar) was used to inoculate 40 vol volumes of seed culture medium. (2% glucose, 3 ° / o soluble starch, 1 ° / o raw soy meal, 1% corn steep liquor, 0.5% polypepton, 0.3% sodium chloride, 0.5% calcium carbonate, pH 7.0] The inoculum medium is incubated for 48 hours at 28 ° C to obtain an inoculum, 0.5 parts by volume of the inoculum thus prepared is transferred to a fermenter of 200 parts by volume. in which 40 parts by volume of fermentation broth is composed of 5% dextrin, 3% corn steep liquor, 0.1% -polypepton and 0.5% calcium carbonate [pH 7.0], and cultured for -90 hours at 28 ° C to obtain an inoculum (seed culture).

Using the serial dilution method, using Tetrahymena pyriformis W as the test organism and the antibiotic C-15003 P-3 as standard, the above culture was found to have a titer of 25 µg / ml.

Example 2

A batch of 10 parts by volume of the inoculum (seed) obtained by the procedure described in Example 214749 du 1 is transferred to a fermenter of contents. 2000 parts by volume containing 500 parts by volume of seed culture medium (same composition as above in Example 1) and incubating the culture at 28 ° C for 48 hours. A batch of 500 parts by volume of the culture thus obtained is transferred to a 50,000 volume by volume stainless steel fermentation tank containing 30,000 parts by volume of seed culture medium and the microorganism is cultured at 28 ° C under aeration (30,000 parts per minute). ], with stirring [280 rpm (1 / 2DT)] and an internal pressure of 1 kg / cm ^{2 to} obtain a seed culture, and the seed culture thus obtained is used to inoculate a 200 000 volume by volume stainless steel fermentation tank, containing 10,000 parts by volume of fermentation broth of a similar composition to that used in Example 1, at an inoculation ratio of 10% The inoculated soil is incubated at 28 ° C, under aeration (100,000 parts per minute), with stirring [200 revolutions per minute (1/2 DT)] and at an internal pressure of 0.1 MPa for a period of 90 hours. 1, it was found that the culture obtained as above had a titer of 25 µg / ml.

Example 3

To 90,000 parts by volume of the culture liquid obtained as described in Example 2, 2000 parts of Hyflo-supercel (g) (manufactured by Johnes and Manville Products, USA) were added and after thorough mixing the mixture was filtered through a pressure filter; 85,000 parts by volume of filtrate and 32,000 parts by weight of wet cells are obtained. The filtrate (85,000 parts by volume) was extracted with stirring with 30,090 parts by volume of ethyl acetate, and the operation was repeated once more. The ethyl acetate portions were combined, washed twice with 30,000 parts by volume of water, dried by adding 500 parts of anhydrous sodium sulfate and concentrated under reduced pressure to 200 parts by volume. To the concentrated solution was added petroleum ether and the precipitate formed was filtered off (yield 53 parts). The crude product I obtained is stirred with 100 parts by volume of ethyl acetate and the insoluble material is filtered off. The filtrate was stirred with 10 parts silica gel (Merck, Federal Republic of Germany, 0.05-0.2 mm) and the ethyl acetate was removed under reduced pressure. The residual silica gel with the adsorbed product is applied to the upper 2 ends of a silica gel column (400 parts by volume of silica gel) and the products are eluted successively with 500 parts by volume of hexane, 500 parts by volume of a mixture of hexane and ethyl acetate. ^. (3: 1), 500 parts by volume of hexane / ethyl acetate (1: 1), 500 parts by volume of hexane / ethyl acetate (1: 3), 4,500 parts by volume of ethyl acetate, 1000 parts by volume. 50 parts by volume of ethyl acetate / methanol (50: 1), and the eluate was collected in 100 volume fractions.

One volume of solution was taken from each fraction, the solvent was distilled off to dryness and 0.1 volume of ethyl acetate was added to the residue. The mixture is deposited at a distance of 2.5 cm from the bottom edge on a glass plate with a layer of silica gel (Merck, Federal Republic of Germany, 60 F54, 0.25 mm, 20 X 20 cm) and developed to a distance of about 17 cm in an ethyl acetate solvent system. methanol (19: 1). After development, ultraviolet light (2537 A) is detected.

Fractions 23-28 containing the active substance having an R _{f of} 0.60 to 0.65 are combined and concentrated under reduced pressure to about 20 parts by volume. To the concentrated solution was added 150 parts by volume of petroleum ether to give 15 parts of crude product II.

Example 4

000 parts of the filtered wet cells obtained by the method described in Example 3 was extracted by stirring 50,000 parts by volume of ^{70 0} / o »aqueous acetone for 3 hours. The mixture is filtered using a pressure filter and the extraction is repeated once more with 50,000 volumes of 70% aqueous acetone followed by filtration. The filtrates were combined, the acetone was distilled off under reduced pressure, and the remaining aqueous fraction was adsorbed onto a column of 5,000 volumes of Diaion HP-10 ion exchange resin (manufactured by Mitsubishi Kasei К, K.). The column was washed with 20,000 volumes of water and 50% aqueous methanol and eluted with 15,000 volumes of 90% aqueous methanol. The eluate is concentrated under reduced pressure to 3000 parts by volume, 3000 parts by volume of water are added and the mixture is shaken with 3000 parts by volume of ethyl acetate. This operation is repeated once more, the ethyl acetate portions are combined, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure to 200 parts by volume. To the concentrated solution was added petroleum ether and the precipitate formed was filtered off. 28 parts of crude product are obtained which, after purification by silica gel column chromatography, yields 8.0 parts of crude product II.

Example 5

Dissolve 1.5 parts of crude product II obtained as described in Example 3 in 10 parts by volume of ethyl acetate and mix well with 4 parts of silica gel (Merck, Federal Republic of Germany, 0.05-0.2 mm). The ethyl acetate was evaporated under reduced pressure and silica gel with the adsorbed product was applied to the upper end of a column made up of 300 parts by volume of silica gel; the column is washed first

214743

500 parts by volume of chloroform and then eluting successively with 500 parts by volume of a mixture of chloroform and methanol (50: 1), 500 parts by volume of a mixture of chloroform and methanol (20: 1) and 500 parts by volume of a mixture of chloroform and methanol (10: 1); The eluate was collected in 25 volume fractions.

0.5 part by volume of the solution was removed from each fraction, the solvent was distilled off under reduced pressure, 0.05 part by volume of ethyl acetate was added to the residue, and the mixture was subjected to thin layer chromatography on silica gel (solvent system: chloroform-methanol 9: 1).

Fractions number 39 and 40, absorbs at 2537 and zone of _Rf from 0.50 to 0.60 were combined and evaporated under reduced pressure. 2 volumes of ethyl acetate were added to the residue and the mixture was allowed to stand; 0.150 parts of the crystalline antibiotic C-15003 are precipitated.

The above-obtained C-15003 antibiotic crystals (0.150 parts) are dissolved in 15 parts by volume of methanol, and 0.300 parts of sodium chloride and 15 parts by volume of water are added to the solution. The chromatography tube was filled with 200 volumes of Diaion HP-10 ion exchange resin (manufactured by Mitsubishi Kasei К. К.) and the column was washed with 600 volumes of 50% aqueous methanol containing 5% sodium chloride. The antibiotic solution prepared above was adsorbed onto an ion exchange column and the substances eluted by gradient elution 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 15 parts by volume fractions and each fraction was evaluated by thin layer chromatography on silica gel. Fractions 145-153 contain C-15003 P-3, fractions 167-180 contain C-15003 P-3 ‘and P-4, and fractions 185-190 contain C-15003 P-4.

Each of the above fractions was combined, the solvents were distilled off and the residue was dissolved by adding 50 ml of water and 100 ml of ethyl acetate. The mixture was shaken in a separatory funnel, the aqueous layer was separated, the ethyl acetate portion was washed twice with 50 ml of water, dried over anhydrous sodium sulfate, concentrated to a small volume and the concentrated solution was discarded for crystallization. In this way a crystalline substance is obtained from each of the above fractions; the crystals formed are filtered off and dried. It is obtained

0.070 parts of antibiotic C-15003 P-3,

0.018 parts of a mixture of antibiotics C-15003 P-3 ‘and P-4, a

0.015 parts of antibiotic C-15003 P-4.

Dissolve the mixed crystals of C-15003 P-3 'and P-4 (0.018 parts) in 0.3 parts by volume of ethyl acetate, apply the solution in a narrow strip at a distance of 2.5 cm from the bottom edge to a silica gel glass plate. (Merck, Federal Republic of Germany, Kieselgel 60 F254, 0.25 mm, 20 x X 20 cm) and the compounds are developed in an ethyl acetate-methanol (19: 1) solvent system to a distance of about 18 cm. Zones of substances having R _f 0.68 (antibiotic P-4) and R _F 0.65 (antibiotic P-3 ') were scraped, and each separately extracted twice with ethyl acetate containing a small amount of water. The resulting ethyl acetate extracts were washed with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrated solutions were discarded for crystallization. From fractions of _Rf 0.68 was obtained 0,010 parts of crystalline antibiotic C-15003 P-4, and the fractions of _Rf 0.003, 0.65 parts of crystalline antibiotic C-15003 P-3 '.

Example 6

100,000 volumes of seed culture medium contained in a 200,000 volume stainless steel fermentation tank are inoculated with 1000 volumes of culture obtained as described in Example 2, and the seeded soil is incubated at 28 ° C, under aeration ( 100,000 parts per minute) and with stirring (200 rpm) for 48 hours. The seed culture obtained is transferred to a stainless steel fermentation tank containing 2,000,000 parts by volume containing 1,000,000 parts by volume of a fermentation broth of the same composition as described above in Example 1 at a transplantation rate of 10%. Cultivation is carried out at 26 ° C, under aeration (1000,000 parts per minute), with stirring [120 rpm (1/3 DT)] and at an internal pressure of 1 kg / cm ² , for 90 hours. The culture obtained had a titer of 20 µg / ml as determined by the test method described in Example 1.

To 900,000 parts by volume of the culture liquid obtained as above, 900,000 parts by volume of acetone are added, the mixture is stirred for one hour, and then 20,000 parts by weight of diatomaceous earth (manufactured by Johnes & Manville, USA) are added. The mixture was further stirred and then filtered using a pressure filter apparatus. To 1 700 000 parts by volume of the filtrate obtained, 500 000 parts by weight of water are added and the mixture is extracted on a Podbielniak column (manufactured by Podbielniak, Inc) by 1000 000 parts by volume of ethyl acetate. The ethyl acetate extract was washed with water, dried by adding anhydrous sodium sulfate and concentrated under reduced pressure. Petroleum ether is added to the concentrated solution and the precipitate formed is filtered off and dried. 68 parts of crude product I are obtained, which is then purified according to the methods described in Examples 3, 4 and 5. 9.5 parts of C-15003 P-3, 0.300 parts of C-15003 P-3 'and 2 are obtained. , 5 parts antibiotic C-15003 P-4.

Example 7

0.015 parts of crystalline antibiotic C-15003 obtained as described in Example 5 was dissolved in 1 part by volume of tetrahydrofuran, cooled to -5 ° C, and then 0.012 parts of lithium aluminum hydride was added thereto. The reaction mixture is allowed to stand at room temperature for 2 hours, then the excess reducing agent is quenched by the careful addition of 0.5 parts by volume of a 1% aqueous sulfuric acid solution and the mixture is shaken with 2 parts by volume of ethyl acetate. The ethyl acetate extracts were washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The once-filtered solution was subjected to preparative thin layer chromatography on silica gel; R _F zone of 0.25 to 0.3 was scraped off and the product is extracted with ethyl acetate · containing a small amount of water. The ethyl acetate extract was washed with water, dried over anhydrous sodium sulfate and concentrated to a small volume under reduced pressure. The precipitated crystals are filtered off and dried; 0.010 parts of antibiotic C-15003 P-0 having a melting point of 174 ° C are obtained.

Elementary analysis:

For C28H37ClN2O8

calculated: 59,52% C, 6.60% H, 4.96% N, 6.27% Cl; found: 59,65% C, 6.58% H, 5.02% N, 6, ^ 1% Cl.

Infrared spectrum: 1715, 1670, 1580 cm @ -1

Ultraviolet Spectrum (nm): 232 (32750),

244 (sh, 30850); 252 (31650); 281 (5750); 288 (5700);

The substance obtained corresponds to the physical and chemical properties of maytansinol.

Claims (4)

OBJECT OF THE INVENTION What is claimed is: 1. A composition for the treatment of diseased plants comprising as active ingredient an antibiotic C-15003 of the general formula 2. A process for the preparation of an active ingredient of a composition according to claim 1 of the formula wherein R represents a group in which R represents a group CH3 / —CO — CH \ CH3 —CO — CH2 — CH2 — CH3, CH3 from —CO — CH2 — CH CH 3 or a hydrogen atom. CH3 / —CO — CH \ CH3 —CO — CH2 — CH2 — CH3, CHs / —CO — CH2 — CH \ CH3 or a hydrogen atom, characterized in that the microorganism of the strain -Nocardium-nr. C-15003 IFO 13726 in a culture medium comprising assimilable carbon sources and digestible nitrogen sources at a temperature ranging from 20 ° C to · 35 ° C with an initial pH of about 7 under aerobic conditions as long as said antibiotic C- 15003 does not accumulate in said nutrient medium, and then the optionally obtained antibiotic C-15003 is subjected to reductive hydrolysis to give a compound of the formula: 3. The process of claim 2 wherein the reduction hydrolysis is carried out using a complex metal hydride. 4. The method of claim 3, wherein the complex metal hydride is lithium aluminum hydride.