CS277547B6 - Process for producing macrotetrolide insecticides - Google Patents

Abstract

Macrotetrolides of the general formula I where R1.R2.R3 and R4 are methyl or ethyl groups, are natural pesticides with insecticidal, acaricidal, coccidiostatic and anthelmintic activity, produced by various species of streptomycetes. Their activity increases with the number of ethyl groups in the molecule. Streptomyces griseus, used for the development of the production process according to the present invention, produces a mixture of nonactin, monactin, dynactin and tetranactin in the ratio 8 : 27 : 57 when cultivated in submerged culture on medium with starch, distillery dregs and dried yeast; 8. When cultivated on a medium of the same composition, to which sodium propionate and potassium hydrogen phosphate were added, dinactin, trinactin and tetranactin are produced in the ratio 2 : 26 : 72. By chemical isolation of this complex from the mycelium of production cultures, a mixture of trinactin and tetranactin in the ratio 1 : 3 was obtained, containing only a trace amount of dinactin.

Description

The present invention relates to a process for the production of the macrotetrolide insecticides trinactin and tetranactin by a biosynthetic route.

Losses caused by harmful insects and mites in the cultivation and storage of agricultural crops account for up to 30% of world production each year. Effective plant protection against pests is therefore one of the main factors determining the amount and quality of the harvest, and thus one of the most readily available reserves in agriculture. However, long-term application of synthetic agrochemicals has negative ecological consequences (accumulation of non-degradable toxic substances in soil or crops, development of resistant forms of pests, death of harmless organisms, etc.), and therefore increased attention is paid to research and development of natural insecticides, especially microorganism products. A number of microbial metabolites with pesticidal effects have been discovered in recent years, but only a few have properties suitable for practical application (Misato et al., Adv. Appl. Microbiol. 21, 53-88, 1977; Mistato, J. Pesticide Sci. 7, 301-305, 1982; Deshpande et al., Enzyme Microb. Technol. 10, 455-473, 1988). These include macrotetrolide-type substances (Dominguez et al., Helv. Chim. Acta 45, 129-138, 1962; Beck et al., Helv. Chim. Acta 45, 620-630, 1962; Kellar-Schierlein et al., Antimicrob Agents Chemother (1966, 644-650, 1967; Ando et al., J. Antibiot. 24, 418-422, 1971) of formula I

(I) wherein R _HR R _2, R ₃ and R ₄ are methyl or ethyl groups:

^R 1 ^R 2 ^R 3 ^R 4 la Nonactin ch ₃ ch ₃ ch ₃ ch ₃ Ib Monactin ch ₃ ch ₃ ch ₃ ch ₂ ch ₃ Ic Dinactin ch ₃ ch ₃ CH ₂ ^CH 3 ch ₂ ch ₃ Id Trinactin ch ₃ ^CH 2 ^CH 3 CH ₂ ^ch ₃ ^CH 2 ^CH 3 le Tetranactin ^c H2 ^ch 3 ch ₂ ch ₃ ^CH 2 ^CH 3 ch ₂ ch ₃

CS 277547 B6 2

Macrotetrolides are active against insects and mites (Oishi et al., J. Antibiot. 23, 105-106, 1970; Ando et al., J. Antibiot., 24, 347-352, 1971; Sagawa et al., J. Econ. Entomol., 65, 372-375, 1972) and also have coccidiostatic effects (Sakamoto et al., Deutsche Patentschrift DE 2802455 C2, 1978) and anthelmintic effects (Nippon Kayaku Co. Ltd., and Chugai Pharm. Co., Ltd., Japan Kokai Tokkyo Koho 8157, 714, 1981). They are harmless to warm-blooded animals (Oishi et al., J. Antibiot. 23, 105-106, 1970; Sagawa et al., J. Econ. Entomol. 65, 372-375, 1972) and to plants (Hirano et al. , J. Econ. Entomol. 66, 349-352, 1973) and readily degradable by soil microflora (Sasaki et al., Appl. Environ. Microbiol., 40, 264-268, 1980). Their activity increases with the number of ethyl groups in the molecule; the most effective are trinactin (formula Id) and tetranactin (formula le).

Nonactin, monactin, dinactin and trinactin are produced by a number of species of streptomycetes, for example by various strains of Streptomyces griseus, S. chrysomallus, S. flaveolus, S. Tsusimaensis neo S. werraensis (Smith, J. Antibiot. 28, 1000-1003, 1975). Tetranactin in a mixture with dinactin and trinactin was isolated only from S. aureus cultures (Ando et al., J. Antibiot. 24, 347-352, 1971), whereas S. roseochromogenes forms nonactin, monactin, dinactin, trinactin and tetranactin in quantitative 22: 33: 33: 9: 3 (Smith. J. Antibiot. 28, 1000-1003, 1975). A complex of macrotetrolides (so-called polynactin) containing dinactin, trinactin and tetranactin in a ratio of 10:40:50 (Shichi et al., U.S. Pat. 4,843,092, 1989) is manufactured by the Japanese company Chugai Pharm. Co., Ltd., by fermentation using S. aureus as a production microorganism and successfully applied in practice either alone or in a mixture with chemical pesticides, for example for large-scale protection of tea or orange plantations (Sagawa et al., Japan Kokai Tokkyo Koho 7406656, 1974 Chugai Pharm. Co., Ltd., and Hokko Chern. Industry Co., Ltd., Japan Kokai Tokkyo Koho 5846006 and 5846009, 1983).

Neither a patented strain of S. aureus nor a commercial preparation of polynactin is available in the Czechoslovak Socialist Republic. Although tetranactin can be prepared by chemical synthesis (Schmidt and Werner, J. Chem. Soc. Chem: Commun. 1986 (13), 996-997), this procedure is very laborious and expensive and gives only small yields of the desired substance. These disadvantages have been overcome by the development of a new process according to the invention, the essence of which is that sodium propionate and potassium hydrogen phosphate are added to a submerged culture of Streptomyces griseus at the beginning of the culture and the finished product is isolated after 96 hours of fermentation. The addition of propionate inhibits the biosynthesis of nonactin, monactin and dinactin while stimulating the production of trinactin and tetranactin. The addition of hydrogen phosphate eliminates the inhibitory effect of propionate on the growth of the production culture, leading to a significant increase in both desired macrotetrolides. The biosynthetic production process according to the invention is simpler than the chemical production process because it proceeds spontaneously from start to finish in the fermentation vessel without human intervention until the finished product is isolated.

The production method of the present invention is apparent from the following examples:

Example 1

a) Inoculum preparation.

Streptomyces griseus strain J-249 (derived from the soil isolate S. griseus LKS after repeated application of UV light) was cultured for 10 days at 28 ° C on slant agar with sporulating soil with yeast and malt extract (Shirling and Gottlieb, Int. J. Syst. Bacteriol., 14, 313-340, 1966). Well sporulated cultures were stored at 4 ° C and used to prepare a vegetative inoculum for up to three months.

The vegetative inoculum was grown on a rotary shaker (2.7 Hz) at 28 ° C in 500 ml boiling flasks filled with 60 ml of fermentation broth containing 40 g of soluble starch, 7 g of distillery effluent and 5 g of dried yeast in tap water. . The initial pH of the soil after sterilization was 6.8 (adjusted to 6.9 to 7.0 before sterilization). The total content of inorganic phosphate in the soil (determined by the method of Weil-Malherbe and Green, Biochem. J. 49, 286-292, 1951) was 2.5 mM. The medium was inoculated with an agar culture inoculum containing approximately 6 x 10 ° spores. The vegetative inoculum was cultured for 24 hours.

b) Preparation of the second vegetative generation

The actual submerged cultivation according to the invention took place in the second vegetative generation on the same fermentation broth and under the same conditions as in the preparation of the inoculum for 96 hours. Cultures were inoculated with 3 ml of vegetative inoculum.

c) Analytical determination of macrotetrolides

At the end of the culture, the mycelium from two parallel cultures of the second vegetative generation (120 ml of fermentation broth) was separated by centrifugation, washed with 50 ml of distilled water and extracted with 2 x 50 ml of methanol and 1 x 50 ml of a mixture of methanol and chloroform (1: 1). The combined extracts (150 ml) were evaporated to dryness after the addition of 1 g of diatomaceous earth (Celite No. 545). The residue was extracted with 40 ml of a mixture of chloroform and methanol (95: 5), and the extract was filtered. The remaining sediment on the filter was extracted with 40 ml of the same mixture and the extract was filtered. The two filtrates were combined and evaporated to dryness. The residue was dissolved in 10 ml of dichloromethane and 1 ml of the solution was used for quantitative spectrophotometric determination (at 380 nm) of the total macrotetrolide content by the method of Suzuki et al. (J. Antibiot. 24, 675-679, 1971), based on the complexation of these substances with sodium picrate. The remaining 9 ml of solution was evaporated to dryness and the residue was dissolved in 1 ml of a mixture of methanol and chloroform (3: 2). The solution (1 to 5 μΐ, depending on the concentration of macrotetrolides in the sample) was used for thin layer chromatography on Kieselgel ₆₀ plates (0.25 mm, 200 x 200 mm) in ethyl acetate-chloroform-dichloromethane (7: 3: 1) . After developing twice, the chromatogram was sprayed with concentrated sulfuric acid and heated at 150 ° C for 10 minutes. The quantitative ratio between the components of the macrotetrolide complex was determined densitometrically on a Shimadzu CS-930 instrument at 590 nm.

Under the indicated culture conditions, the S. griseus J-249 strain achieved an average production of 626 mg of macrotetrolide complex in 1 L of fermentation broth. This complex contained nonactin, monactin, dinactin and trinactin in a ratio of 8: 27: 57: 8.

d) Determination of dry matter, mycelia -

To determine the absolute values of the growth of the cultures, the dry matter, mycelia, washed with distilled water and freed from the interfering presence of macrotetrolides by repeated extraction according to the procedure described in paragraph c) was determined. The mycelium was dried on pre-weighed paper filters for two days at room temperature (until constant weight).

The average dry mycelial mass from the culture grown under the conditions mentioned in paragraph b) was 6.73 mg / 1 ml.

Example 2

The same procedure as in Example 1 was used, except that a sterile aqueous solution of potassium hydrogen phosphate (1 ml) at a concentration of 46 mg / 60 ml of soil was added to the cultures of the second vegetative generation at the beginning of the cultivation. The total inorganic phosphate content was thus increased (compared to the data in Example 1) from 2.5 nM to 7.0 mM.

Under these culture conditions, the S. griseus J-249 strain achieved an average production of 732 mg of macrotetrolide complex of fermentation fluid villas, i.e. 16.9% more than under the conditions described in Example 1. The complex contained nonactin, monactin, dinactin and trinactin in the ratio 6: 26: 58: 10. The average dry mycelial mass was 7.58 mg / 1 ml, i.e. 12.6% higher than the cultures grown according to the procedure in Example 1.

Example 3

The same procedure as in Example 1 was used, except that a sterile aqueous solution of sodium propionate (2 ml) at a concentration of 500 mg per 60 ml of soil was added to the second vegetative generation cultures at the beginning of the cultivation. Compared to the data in Example 1, under these conditions the total yield of macrotetrolides decreased to 353 mg per 1 l of fermentation broth (i.e. by 43.7%), at the same time the composition of the complex containing monactin, dinactin, trinactin and tetranactin in a ratio of 3: 9: 32: 56 (along with a trace amount of nonactin).

The average dry matter of mycelia was 5.78 mg / 1 ml, i.e. 14.2% lower than the cultures grown according to the procedure in Example 1.

Example 4

The same procedure as in Example 1 was used, except that sterile aqueous solutions of sodium propionate (2 ml) at a concentration of 500 mg and potassium hydrogen phosphate (1 ml) at a concentration of 46 mg / 60 were added to the second vegetative generation cultures at the beginning of the culture. ml of fermentation broth.

Under these culture conditions, the S. griseus J-249 strain achieved an average production of 577 mg of macrotetrolide complex of fermentation fluid villas, i.e. only 7.3% less than in the procedure described in Example 1, and 35.7% more than under the conditions reported in Example 3. The complex contained dinactin, trinactin and tetranactin in a ratio of 2: 26: 72.

The average dry mycelial mass was 6.82 mg / 1 ml, i.e. 1.3% higher than the cultures grown according to Example 1, and 17.9% higher than the cultures obtained under the conditions described in Example 3.

Example 5

The cultivation was carried out as described in Example 4, except that the fermentation broth was filled into 130 100 ml flasks, and 833 mg of sodium propionate and 76.7 mg of hydrogen phosphate were added to the second vegetative cultures inoculated with 5 ml of inoculum. potassium / 100 ml soil.

After completion of the culture, the fermentation broth (13 L) was centrifuged, and the separated mycelium was washed with distilled water (3 L) and extracted with methanol (4 x 2 L) and a mixture of chloroform and methanol (1: 1) (4x21). The extracts were combined and after the addition of Celite (30 g). evaporated to dryness. The residue was extracted with a mixture of chloroform and methanol (95: 5) (3 x 300 mL). The filtered extracts were combined and evaporated to dryness. The residue (11.6 g) was crystallized from a mixture of hexane and acetone at -15 ° C. 1.6 g of a crystalline mixture of macrotetrolides were obtained. The mother liquor was evaporated to dryness. Methanol (15 ml) and water (200 ml) were added to the residue. The emulsion mixture was basified with sodium bicarbonate and extracted with chloroform (4 x 10 mL). The extract was evaporated to dryness and the residue was recrystallized from methanol. 1.8 g of a crystalline mixture were obtained.

The total macrotetrolide content, determined according to Example 1 in the crude mycelial extract, was 4.5 g. The yield of crystalline product after isolation was 3.4 g, i.e. 76%.

As in Example 4, nonactin and monactin were not found in the isolated product. Dinactin was present only in trace amounts. The main components were trinactin and tetranactin in a ratio of 1: 3. Their identity was confirmed by mass and NMR spectrometry in comparison with the literature data.

Although Streptomyces griseus is not a tetranactin producer according to published data, the process of the present invention has succeeded in obtaining tetranactin in high yield (while suppressing the undesired less active components of the macrotetrolide complex) in this microorganism as well. The composition of the final product (trinactin and tetranactin in a ratio of 1: 3, with a trace amount of dinactin) is even more advantageous than with a commercially produced Japanese preparation of polynactin, which contains dinactin, trinactin and tetranactin in a ratio of 1: 4: 5.

The general efficacy and utility of the procedures described in Examples 3 and 4 was also confirmed in eight other macrotetrolide-producing S. griseus strains kept at the Institute of Microbiology of the Czechoslovak Academy of Sciences. .

Claims (1)

Process for the preparation of the macrotetrolide insecticides trinactin and tetranactin of the general formula I where R 1 = CH ₃ or CH ₂ CH ₃ and R ₂ = = R ₄ = CH ₂ CH ₃ , by a biosynthetic path characterized in that the microorganism streptomyces griseus is grown under submerged conditions in fermentation broth with starch, distillery liquors and dried yeast , to which 8.33 mg of sodium propionate and 0.77 mg of potassium hydrogen phosphate per ml of soil were added at the zero hour of cultivation.