KR800001240B1 - Process for producing the polycyclic ether antibiotics - Google Patents

Abstract

A process is described for the manufacture of a polycyclic ether antibiotic with anticoccidial, insecticidal, and antimicrobial activity. Thus, Streptomyces hygroscoficus ATCC 31050 was grown in a liq. medium with aeration for 90-120 hr at 28-36≰C. Antibiotic 38295 was extd. from the fermentation broth by liq.-liq. extn. In the form of the free acid the compd. had an optical rotation of [α 25D + 36≰ at 1 % MeOH; av. compn. by wt. was C 60.58%, H 8.56%.

Description

Method for preparing polycyclic ether antibiotics

1 is the infrared spectrum of sodium / potassium salt

2 is an infrared spectrum of the free acid of compound 38295

3 is an infrared spectrum of the sodium salt of compound 38295

4 is an infrared spectrum of the potassium salt of compound 38295

The present invention relates to a process for the preparation of novel acidic polycyclic ether family antibiotics having biochemical activity against cationic transition in mitochondria. Antibiotics of this kind include monensin, nigericin, glyricin, dianemycin, salinomycin, X-537A, X-206 and A204A.

The polycyclic ether family antibiotics specified above were active against Gram-positive bacteria, fungi and crudes and exhibit potent anticoccidial activity. Specifically, the present invention relates to a method for preparing compound 38295, that is, an antibiotic represented by the following general formula.

Antibiotic of the present invention is prepared by aerobic growth of Streptomyces hygroscopic picus ATCC 31050 in a water-soluble nutrient medium.

Antibiotics of the invention and their cationic salts are potent antimicrobial agents and animal growth promoters. Microorganisms useful for the production of antibiotics of the present invention are isolated from samples of Japanese soil. The fungus was grown in different media to identify genus bacteria and was concluded to be similar to the characteristics of Streptomyces hygroscopyus.

The results of comparing the cultures of Streptomyces hygroscopicus obtained from cultures (Pfizer F. D 23604) and the German public culture collection center, Centra al Bureuer-Summel Culture (CBS) on several special media As follows. Comparisons and decisions were based on the concept of species described in Applied Microbiology 4: 243-250, 1956.

Spore-chain form

On various media, the chain of Pfizer F. D 23604 was 6-10 spirals or less and was similar to the chain of Streptomyces hygroscopic picus CBS mentioned above. Electron microscopy showed that the spores of Pfizer F. D 23604 matched the spores of the CBS strain of Streptomyces hygroscopicus, registered in Applied Microbiology 18: 695-696, 1969. It has a wrinkled surface with many holes in isotropes. Spores of both cultures appear almost identical.

[Color of Kyun colony]

Both cultures appear as gray aerobic mycelium on most media, but the exact shades appear slightly different. Pfizer F. D 23604 on Chapec-Sucrose agar grows thick, irregularly overlapping, off-white, but grows the CBS strain of Streptomyces hygroscophycus flat, thin and gray. Pfizer F. D 23604 was incubated on oat agar for 4 weeks to become yellow in the aerobic gray mycelium. Pfizer F. D 23604 has yellow growth spots in the gray mycelium.

[Melanin formation]

None of the cultures on peptone-iron agar forms melanin.

Carbon utilization (Freedam and Gotoliv method)

Pfizer F. D 23604 contains glucose, L-arabinose, dextrin, D-fructose, D (+) lactose, glycerol, inositol, inulin, lactose, maltose, D-mannitol, salixrapinose, rhamnose, sodium Acetate, D-sorbitol, sorbose, starch, saccharose, trehalose and D-xylose are used but do not use dulcitol. These results were reported in Streptomyces hygroscopicus strains and the Journal of Industrial Microbiology 10: 183-221, 1969, except that CBS strains do not use inositol. It has been demonstrated that the CBS strains of Streptomyces hygroscopicus are identical.

[Hydrogen sulfide generation]

Both cultures are positive on the peptone-iron agar slope using lead acetate.

[milk]

The two cultures showed similar results. It does not solidify and after 12 days it is fully or almost hydrolyzed. Both wore yellowish brown melt pigment.

[gelatin]

The growth rate, color and liquefaction rate of the two cultures are similar.

[characteristic]

CBS strains of Streptomyces hygroscopicus form black marshes on glucose-asparagine agar, oat agar and yeast extract-malt extracts after 3 weeks of culture, but Pfizer F. D 23604 is yeast extract-malt Black spots appear only on the extracted agar. After one more incubation period, the blackness of Pfizer F. D 23604 is increased when stored on a Petri dish at 4-10 ° C for 4 weeks. On oat agar, chapec-sucrose and yeast extraction-malt agar, Pfizer F 23 D forms onions or garlic with a slightly earthy odor, while Streptomyces hygroscopicus CBS produces only earthy odors There was no smell. Pfizer F.D 23604 strain of Streptomyces hygroscophytes produces compound 38295, a novel polycyclic ether antibiotic, but does not produce Streptomyces hygroscophycus and any other Streptomyces hygroscophytes Curse strain also does not produce compound 38295.

The culture (F. D 23604) was deposited in American type culture collection and named ATCC 31050.

Streptomyces Hygrococcus ATCC 31050 is readily cultured in aqueous nutrient media under agitation conditions under agitation with a temperature of 28-36 ° C. Nutritious media useful for this purpose include organic carbon sources such as sugar, starch and glycerol: casein, casein digestive enzymes, soy flour, cottonseed soy flour, peanut flour, bran, meat flour and fish flour. Growth materials such as grains and yeast extracts, as well as salts such as salt and calcium carbonate, and inorganic elements such as iron, manganese, zinc, cobalt and magnesium have good results. If bubbles are excessively produced during fermentation, vegetable oils or silicones may be added to the fermentation medium as foam inhibitors. The amount of aeration of the medium in the tank during the deposition growth is preferably maintained at a volume of 1 / 2-2 of the glass air with respect to 1 volume of the juice per minute. Agitation can be performed using the stirrer normally used in fermentation industry. Preservative conditions should, of course, be maintained during the movement of the organism and during its growth.

Inoculum used for the production of antibiotics can be obtained by growing using the slope of the medium. Such development is usually inoculated in a shake flask or an inoculation tank and cultured, and the inoculation tank can be seeded from the shake flask. Growth in shake flasks reaches their maximum in 3-5 days, but inoculations deposited in inoculation tanks typically reach their maximum in 2-3 days. Actual antibiotic activity is usually obtained in the final fermentation stage, usually within 3-5 days. The level of antibiotic is 50-500 mg per liter.

The preparation of antibiotics is preferably followed by fermentation by a biological assay of juice using susceptible strains of Staphylococcus aureus or Bacillus subtilis. Standard edition assays are used when the juice-saturated filtration species uses the prohibited area surrounding the disc as the potential for antibiotics.

Thin layer chromatography using silica gel is a useful tool for analyzing the composition of antibiotics and fermentation broth extracted from fermentation broth. After developing with ethyl acetate, thin layer chromatography was sprayed with 3% vanillin ethanol sulfate (98.5-1.5% V / V) and then heated at 60-80 ° C. for several minutes. The antibiotic then appears as reddish bright spots on a white background.

The antibiotic compound 38295 can be separated and recovered from the transparent fermented broth by extracting it with an organic solvent such as chloroform, ethyl acetate or methyl isobutyl ketone. Most of the antibiotics are contained in isolated mycelium, which can be easily extracted by slurrying the mycelium with a water-soluble solvent such as methanol.

Preferred separation and recovery methods for the antibiotic compound 38295 are as follows. Unfiltered whole growth juice is extracted twice with 1 / 5-1 / 2 volumes of methyl isobutyl ketone. The solvent extract is concentrated in vacuo to give an oily residue that is slurried with a chloroform: hexane (1: 1) solvent and then deposited on a silica gel column (silica gel 60 covered with silica gel PF 254: all of these silica gels The reagents were used). The silica gel column is continuously treated with hexane, chloroform: hexane (1: 1) mixed solution, and chloroform. The main antibiotic portion elutes with ethyl acetate. The eluate is concentrated in vacuo and diluted in acetone, then activated carbon (Darco G60) is added and stirred for 30-60 minutes. Activated charcoal is removed by filtration, and the remaining solution is concentrated in vacuo, and a small amount of water is added, followed by caustic soda to adjust the pH to 8.0-8.5. The resulting crystalline material is filtered off, washed with water and dried.

The antibiotic isolated in this step is a mixture of sodium or potassium salts of compound 38295, produced by purifying the fermented broth and then adding sodium hydroxide solution. When analyzed by thin layer chromatography with the above material, it is often contained at least two small amounts of antibiotics similar to compound 38295, which shows reddish-red spots when sprayed with vanillin solution of ethanolic sulfuric acid.

Compound 38295 can also be obtained as a single substance by column chromatography on silica gel PF 254 consisting of hexane. The mixture of sodium and potassium salts was mixed to add ethyl acetate or chloroform, and the column was developed at 80 psi with a mixture of hexane with an increased ethyl acetate component. Separation is performed by thin layer chromatography. Fractions are mixed and vacuum distilled and the antibiotic crystallized as acetone-water.

The free acid of compound 38295 can be obtained from the mixed sodium and potassium salts by adjusting the pH of ethyl acetate or chloroform to 4.5 with dilute phosphoric acid. The organic layer was dried over anhydrous forget-me-not, vacuum distilled, and the residue was crystallized with chloroform-hexane.

The sodium salt of compound 38295 can be obtained by adjusting the free acid of ethyl acetate or proroform solution with 1N caustic soda solution to pH 8.5. The product is crystallized from acetone-water. Crystalline potassium salt can be obtained by the above method using potassium hydroxide instead of sodium hydroxide.

Crystalline silver salts can be prepared by adding silver nitrate aqueous solution to an ethanol solution in which sodium and potassium salts of compound 38295 are mixed. In a similar manner, copper, zinc, ammonium, calcium, magnesium and lithium salts of compound 38295 can be obtained.

Since the final use of the compound is the treatment and prevention of poultry cosidium disease, fermented broth containing compound 38295 (preferably spray-dried) can be used by mixing an appropriate amount with poultry feed.

Compound 38295 shows growth inhibition of a number of microorganisms (see Table 1). The test organisms were inoculated into a series of test tubes containing nutrient media and the minimum concentration of antibiotics required to inhibit the growth of the organisms for 24 hours with varying concentrations of compound 38295 was measured in m.c.g / ml.

TABLE I

Compound 38295 and its salts have good activity against pocidium in poultry. If the compound is mixed with poultry feed 15-250ppm, it is possible to excellently prevent the mixed infection of Ameria tenella, E, Acetbli, E, Makshima and the like and these bacteria.

The effect of compound 38295 and its salts on coccidium in chicks was demonstrated in the following manner. Groups 3-5 of 10-day-old Legohorn white chicks were fed a mixture of compound 38295 or a salt thereof uniformly. After providing the necessary food for 24 hours, each chick was orally inoculated with Omerist of Omeria. The rest of the chicks were fed an antibiotic-free diet and inoculated with the same strains for infection control. Non-infected, untreated chicks were considered normal control. E, 5 days for Aserbulina and 6 days for other E. coli strains.

Table II shows the results obtained using mixed sodium and potassium of compound 38295.

TABLE II

Note ^* anti Cauchy consists of 0-4 spores lesions for the reference temperature Te E. Nella used to measure the month, and active for the other species consists of 0-3 spores lesions.

Water ratio was evaluated by dividing treated follicles into infected follicles.

Note ^** The first number belongs to the colon lesion and the second number belongs to the appendix lesion.

The same result is obtained by taking a dry fermentation broth containing free acid, sodium salt, potassium salt or compound 38295 in an amount that can produce the desired antimicrobial efficacy.

The growth promoter of compound 38295 is explained by the cows tested below.

The Angus hyal calf was grazed in cages, and 5 calf per cage were added and orally administered with compound 38295 in a beverage. However, depending on the activity of the compound, a relatively high amount of 11-33 ppm was quickly and beneficially mixed in the feed. Compound 38295 can be taken in various forms, such as free acid, sodium salts, potassium salts, mixed sodium and potassium salts, or dried fermented broth produced by Streptomyces hygros Scopicas ATCC 31050.

Feed compositions of compound 38295 and feed compositions without antibiotics were randomly administered to test animals.

The feed composition contains 12.5% of protein and consists of animal feed consisting of corn, soy flour, alfalfa powder, calcium phosphate, molasses, vitamins and small amounts of minerals 75: herb feed 25. Compound 38295 was previously added when grinding corn, inorganic wheat and vitamins.

Basic feed (animal feed containing 12.5% protein)

Note ^* Sugar cane molasses on the part of the sugar cane stem: Crude protein is at least 3%, fiber is less than 15%, inert sugar is at least 42% and moisture is up to 6%.

Note ^{** As a} mineral, it contains 10 ppm of manganese, 6.25 ppm of iron, 1.25 ppm of copper, 0.025 ppm of oxo, 0.86 ppm of cobalt, and 45 ppm of zinc.

Note ^*** Each pound of feed contains vitamin A1497 IU, vitamin D 200 I. U and vitamin E25 I. U.

[Preliminary Additives]

Mixing one pound of preadditive to one tonne of finished feed results in a final concentration of compound 38295 of about 11 ppm.

Table III shows the in vitro results for 28 days for the same 3 cages with 5 calves per cage.

The initial weight of the test animals was approximately 3400 pounds.

TABLE III

Note ^* Daily average intake week ^** Daily average supply week ^*** Feed for intake (ℓb)

The same result can be obtained by using free acid, sodium salt, potassium salt or dried fermented broth containing compound 38295.

Similar results can be obtained with ruminants such as sheep and monogastric animals such as horses, pigs, and rats as test animals.

The active compound 38295 may be mixed with an animal feed or a beverage or mixed with a pre-addition used as a complex of animal feed. In order to increase the feeding efficiency in the animal body, the compound 38295 may be dissolved in an appropriate concentration in a suitable solvent such as ethanol or propylene glycol to prepare an antibiotic preconcentration concentrate for beverage addition.

[Preliminary Concentrate]

Antibiotic preadded concentrates for the addition of animal beverages can be prepared at a suitable concentration by dissolving an antibiotic of compound 38295 in a suitable solvent such as ethanol or propylene glycol.

Example 1

A sterile aqueous medium having the following composition was prepared.

Cells obtained from the slope medium of Streptomyces hygroscopic picus ATCC 31050 were transplanted into a series of 300 ml flasks containing 50 ml of the sterile medium of the above composition, and then on a rotary shaker at a temperature of 28-30 ° C. for 3-4 days. Shaken. The mature culture inoculum was preserved, divided into 5 ml portions, and transplanted into a 300 ml flask containing 100 ml of sterile medium of the composition. After shaking for 3-4 days at 28-30 ° C., the mature inoculum is transplanted into a 4 L-fermenter containing 2 L of the following sterile medium.

It was fermented at 28-36 ° C. for 90-120 hours, with stirring speed maintained at 1700 r.P.m and venting one volume of air with respect to one volume of gravy per minute. Large fermentors containing 80-10,000 gallons of medium were inoculated with 2% of the culture. Ferment until the active ingredient of the antibiotic reaches 50 mg per liter (takes 90-120 hours).

About 100 liters of the whole unfiltered fermented broth are extracted twice with 1/5 volume of methyl isobutyl ketone. The separated solvent extract was concentrated in vacuo until it became an oily residue (769 g), and dispersed in 1 L of silica gel PF 254 in 2 L solution of chloroform, and then the solvent was removed under reduced pressure. The residue thus obtained was slurried with 2 liters of chloroform: hexane (1: 1) solution, and 300 g of silica gel PF 254 laminated with 60 g of 300 g of silica gel was added to the upper layer, and the silica gel was then mixed with 2 gallons of hexane and 2 gallons of chloroform. : Continuous washing with hexane (1: 1) and digalone chloroform. Antibiotics were eluted with 1 gallon of ethyl acetate.

Ethyl acetate is removed under vacuum and the residue (38 g) is dissolved in 400 ml of acetone. The acetone solution is stirred with 40 g of activated carbon (Darco G60) at room temperature for 45 minutes, the activated carbon is filtered off, the filtrate is concentrated to about 200 ml, and diluted with 100 ml of water. Adjusting the pH to 8.5 with 1.0 N caustic soda solution separates compound 38295 in the form of a mixed sodium / potassium salt. Crystalline material (13.8 g) is dissolved in ethyl acetate and chromatographed on a silica gel PF 254 column in hexane. The column is treated with hexaneethyl acetate mixed solution with increased amount of ethyl acetate at 80 Psi. Thin layer chromatography is used for separation. The appropriate fractions are collected and concentrated under reduced pressure to yield pure compounds from acetone-water.

=+36°이다.The melting point of the pure crystalline sodium / potassium heat of compound 38295 is 200 ° C. and the optical intensity is 1% of methanol.

= + 36 °.

After drying overnight with phosphorus pentoxide under vacuum at 70 ° C., carbon and hydrogen were analyzed to be 60.58% and hydrogen was 8.56%.

The compound is readily soluble in methanol, ethanol, acetone, chromoform, methylene chloride, diethyl ether, ethaneacetate and methyl isobutyl ketone, partially soluble in hexane but not in water.

The sodium / potassium mixed salt of compound 38295 does not exhibit ultraviolet absorbance properties.

The infrared spectrum of sodium / potassium salt is shown in FIG.

KBr pellets exhibit infrared absorption characteristics at the following wavelengths (μ). 2.92, 3.40, 6.27, 6.85, 7.22, 8.40, 9.00, 9.15, 9.25, 9.48, 9.98, 10.45, 10.68 and 11.45.

Example II

The process of Example I was repeated using a transparent fermented broth instead of the whole non-filtered fermented broth.

Example III

The mycelium isolated from the transparent fermented broth of Example II was slurried several times with methanol, the methanol extract was concentrated in vacuo and the residue was treated by the method of Example I.

Example IV

The fermentation process of Example I was repeated using a fermentation medium of the following composition.

After the fermentation process, the whole filtered fermented broth is dried by dispersion drying.

Example V

=+38°이다.A mixed salt of sodium / potassium of compound 38295 is dissolved in ethyl acetate and the pH is adjusted to 4.5 with dilute phosphoric acid. The solvent layer was dried over anhydrous forget-me-not in vacuo and the residue was crystallized with chloroform-hexane to form crystalline free acid. Its melting point was 135-138 ° C and its optical density was 1% of methanol.

= + 38 °.

After overnight drying with phosphorus pentoxide at 70 ℃, elemental analysis showed 61.65wt% of carbon, 8.94wt% of hydrogen, and 29.41wt% of oxygen. The free acid of compound 38295 does not exhibit ultraviolet absorbing properties.

The infrared spectrum of the free acid of compound 38295 is shown in FIG. KBr pellets exhibit infrared absorption characteristics at the following wavelengths.

2.92, 3.43, 5.74, 6.85, 7.25, 7.53, 8.28, 8.60, 8.80, 9.00, 9.15, 9.80, 10.10, 10.45, 10.68, 10.92 and 11.13. The negative salt of compound 38295 was analyzed by X-ray to obtain the following structural formula.

Example VI

=+38°이다. 이것을 오산화인으로 70℃에서 하룻밤 진공건조한 후에 나트륨염을 분석하여 보면 탄소가 60.28%이고 수소가 8.76%이다.When the pH was adjusted to 8.5 by adding 1.0 N caustic soda to the ethyl acetate solution of the free acid of Example V, the sodium salt of compound 38295 was obtained. The melting point of the material obtained by distillation of this as acetone-water is 197-198 ° C and the beneficiation is 1.03% of methanol

= + 38 °. This was vacuum dried overnight at 70 ° C. with phosphorus pentoxide, followed by analysis of sodium salts to give 60.28% carbon and 8.76% hydrogen.

The infrared spectrum of the sodium salt of compound 38295 is shown in FIG. KBr pellets exhibit infrared absorption characteristics at the following wavelengths (μ). 2.94, 3.40, 6.25, 6.85, 7.20, 7.45, 8.38, 9.00, 9.27, 9.48, 10.00, 10.45, 10.65, 10.90, 11.13 and 11.47.

EXAMPLE VII

=+38°이다. 이것을 오산화인으로 70℃에서 하룻밤 진공건조한 후에 분석하여보면 탄소가 60.58%이고 수소가 8.70%이다.Potassium of compound 38295 is prepared by the method of Example VI using potassium hydroxide instead of sodium hydroxide. It has a melting point of 196-198 ℃ and its beneficiation at 1.02% of methanol.

= + 38 °. After vacuum drying at 70 ° C. overnight with phosphorus pentoxide, the result was 60.58% carbon and 8.70% hydrogen.

The infrared spectrum of the potassium salt of compound 38295 is shown in FIG. KBr pellets exhibit infrared absorption characteristics at the following wavelengths (μ). 2.94, 3.42, 6.27, 7.20, 8.75, 9.00, 9.14, 9.30, 9.45, 10.45, 10.92, 11.15 and 11.47.

EXAMPLE VII

=+27°이다. 이것을 오산화인으로 70℃에서 하룻밤 건조 후 분석하면 탄소가 55.95%, 수소가 7.89%로 나타난다. 화합물 38295의 결정성 은염의 분자량은 X-선으로 측정하였을 때 원자단위당 1060±4이다.Silver salt of compound 38295 was prepared by adding silver nitrate aqueous solution to the methanol solution of the sodium / potassium mixed salt of Example I. The melting point of the material separated from solids and crystallized into methanol is 198-200 ℃, and the optical density is 0.982% methanol.

= + 27 °. After drying overnight at 70 ° C. with phosphorus pentoxide, the result was 55.95% carbon and 7.89% hydrogen. The molecular weight of crystalline silver salt of compound 38295 is 1060 ± 4 per atomic unit, as measured by X-ray.

Claims (1)

As described above in this article, at least 50 mg of antibiotics per liter of Streptomyces hygroscopic picus ATCC 31050 microorganism in aqueous medium under deep aerobic conditions containing anabolic carbon, nitrogen, and inorganic salts until 50 mg of antibiotics per liter A method for producing an antibiotic compound 38295 represented by the following general formula and a cationic salt thereof, characterized by growing at a temperature of ℃.

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