GB1587089A - Poly-ether antibiotics - Google Patents

Description

(54) IMPROVEMENTS IN AND RELATING TO POLY-ETHER ANTIBIOTICS (71) We, TAISHO PHARMACEUTICAL CO. LTD., a Japanese Corporation organised and existing under the laws of Japan, 6f 24-1, Takata 3-chome, Toshimaku, Tokyo, Japan, do hereby declare the invention for which we pray that a patent may be granted to us and the method by which it is to be performed to be particularly described in and by the following statement:- The present invention relates to poly-ether anitbiotics and has particular reference to antibiotic TMB-481 which is a novel compound of a group of polyether antibiotics which have four methoxy groups. Examples of compounds of this group include A-28695A (U.S. Patent No. 3,839,558), septamycin [J. Antibiotics, 28 854 (1975)], TMH81 (lonomycin) (U.S. Patent No. 3,950,514), DE-3936 [J.

Antibiotics, 29, 91 (1976)], 31559RP (emericid) (U.K. Patent No. 1,495,858) and A-218 [J. Antibiotics, 29, 10 (1976)].

Among them, antibiotic TM-481 is identified with DE-3936 and 31559RP, different from A-218 in terms of its molecular formula, and also different from septamycin which is identified with A-28695A in terms of whole structures and physico-chemical properties.

Antibiotic TMB-481 of this invention has the same structural formula as that of antibiotic TM-481. Antibiotic TMB-481, however, is different from antibiotic TM-481 as compared with their physico-chemical properties such as melting point, specific rotation, Rf value on thin layer chromatogram and spectrum of 13C-NMR. Antibiotic TMB481, therefore, is elucidated to be a novel compound of a group of polyether antibiotics.

This invention relates to a novel antibiotic TMBW81 and its alkali metal salts, and to a method for the preparation thereof.

More particularly, this invention is concerned with a novel antibiotic TMB-481 represented by the formula

and its alkali metal salts, derived from a strain of microorganism Streptomyces ribosidificus TM-481, and concerned with a process for the preparation of said novel antibiotics.

The antibiotics of this invention exhibit anticoccidial activity and are highly effective in controlling coccidiosis in poultry.

The antibiotics of this invention also exhibit antibiotic activity against pathogenic microorganisms. Especially, these antibiotics are useful for inhibition against Treponema hyodysenteria which causes swine dysentery.

Furthermore, the' antibiotics of this invention show lower toxicity than antibiotic TM "81.

Following is a description with reference to the accompanying drawings of methods of carrying the invention into effect.

In the drawings: Figure 1 shows the ultra violet absorption spectrum of antibiotic TMB--481 sodium salt in methanol.

Figure 2 shows the infrared absorption spectrum of antibiotic TMB--481 sodium salt with tablet of KBr.

Figure 3 shows the nuclear magnetic resonance spectrum of antibiotic TMB "81 sodium salt in deuterochloroform.

The antibiotics of this invention can be prepared by the cultivation, under controlled conditions, of a strain of Streptomyces ribosidificus TM-481 (ATCC No.

31,051) mentioned in the specification of U.S. Patent No. 3,950,514 concerning antibiotic TM-481.

The microbiological properties of the strain, Streptomyces ribosidificus TM-481 are as follows: 1. General morphological findings A mycelium is formed with the display of slight curved hyphae on glucoseasparagine agar medium although deveiopment of aerial mycelium is poor. But aerial mycelia on oatmeal agar, yeast extract-malt extract agar or starch agar medium form abundant spores.

Microscopic examination of the cultures grown on yeast extract-malt extract agar reveals branched filaments and spore chains forming spiral. Mature spore chains generally contain about 10 spores per chain. Electron micrograph of the spore shows oval to spherical form (0.7-1.0 x 1.0-1.4 ) with spiny surface.

2. Cultural characteristics The cultural characteristics of Streptomyces ribosidificus TM--481 are listed in Table 1.

TABLE 1 Cultural characteristics of Streptomyces ribosidificus TM-481

Medium Growth Aerial Mycelium Soluble Pigment Sucrose-nitrate Poor, yellowish Poor, pale yellow None agar cream to yellowish green Glucose-asparagine Poor, colorless to Powdery, grayish None, pale yellow agar yellowish cream brown after 2 weeks Glycerol-asparagine Good, yellowish White to light gray None agar cream or yellowish green Starch agar Good, yellowish Abundant, pale None cream to light yellowish green to gray dark grey Tyrosine agar Good, yellowish Abundant, white to None cream light gray or pale yellowish green Nutrient agar. Poor, colony shape, None None yellowish cream Yeast extract-malt Good, grayish yellow Abundant, pale None extract agar to yellowish green yellowish green to dark gray Oatmeal agar Good, yellowish Abundant, yellowish None cream to light gray green to dark gray All cultures were incubated at 30 C.

3. Physiological properties The physiological properties of this strain are as follows: Growth temperature range: 15-450C on oatmeal agar, Optimal growth temperature: 30--35"C, Liquefaction of gelatin: slightly positive around the growth in 5-7 days at 20"C, Hydrolysis of starch: positive, Coagulation of skimmed milk: negative, Peptonization of skimmed milk: negative at 300C, positive at 370 C, Melanin production: negative, Liquefaction of Loeffler's coagulated serum: positive, and Reduction of nitrate: positive.

Carbon source utilization test by method of Pridham and Gottlieb shows that this strain is able to utilize moderately or well D-glucose, L-arabinose, D-fructose, sucrose, inositol, rhamnose, ratfinose, and D-mannitol, but not at all D-xylose.

From the above results, the microbiological characteristics of this strain may be summarized as follows: The strain TM--481 forms aerial mycelium with spiral and spore surface is spiny. Growth on synthetic media is poor and mycelium is yellowish cream to yellowish green. Soluble pigment is not generally observed but the culture on glucose-asparagine agar medium produces pale yellow soluble pigment. Cultures on organic media show good growth with yellowish cream to yellowish green color and abundant spore formation. But soluble pigment is not produced on the media.

This strain has been deposited to the Institute for Microbiological Industry and Technology, Japan, as FERM-P No. 2267, and at American Type Culture Collection, Rockville, Maryland as ATCC No. 31051.

Antibiotic TMB481 is usually obtainable by inoculating an aqueous nutrient medium with a culture of the strain of Streptomyces ribosidificus To481, culturing said strain by the shaking culture method or aerated submerged culture method and separating the thus produced antibiotic TMB481 from the culture medium.

As well as the media in which other microorganisms are grown for the production of antibiotics, the nutrient medium for culturing the strain TM--481 usually contains sources of assimilable carbon and nitrogen. As sources of assimilable carbon, various carbohydrates such as ordinary starch, glycerol and sugars, for example, glucose and sucrose are preferably used. Various lipids and vegetable or animal oil may be used for the same object. Suitable sources of assimilable nitrogen include a wide variety of substances such as peptone, amino acids, casein, fish meal, soya bean meal, meat extract, yeast extract and various other nitrogenous substances of vegetable or animal origin. Chemicals such as urea nitrates and ammonium compounds may also be added to the nutrient media as a nitrogen source. Corn steep liquor, because of the wide variety of both organic and inorganic substances contained therein, is a valuable additive to the fermentation media. In some cases, essential mineral salts such as sodium chloride or antifoam may be added also.

The pH value of the medium is brought substantially to neutrality before sterilization, preferably to about pH 7. Fermentation is preferably carried out at a temperature of 28--35"C. The development of the culture is comparatively rapid, and so it can be observed that the active substance was produced in the culture media after 30 hours under suitable aerated submerged culture condition. The maximum production of antibiotic TMB1181 is usually attained after 48-72 hours in jar fermentation.

The fermentation medium is basified with an alkali metal base such as sodium hydroxide and potassium hydroxide, and centrifuged to separate a broth and a mycelium. The broth is extracted with a water-immiscible organic solvent such as ethyl acetate, benzene and chloroform and the mvcelium is extracted with a watermiscible organic solvent such as acetone and methanol. These extracts are combined, and then concentrated to dryness to give a crude antibiotic mixture.

Antibiotic TMB481 mixed sodium and potassium salts is separated from the crude antibiotic mixture and further purified by the efficient combination of silica gel column chromatography, gel filtration on Sephadex (Registered Trade Mark).

LH--20, and crystallization from a n-hexane-benzene solution.

In order to obtain the free acid of antibiotic TMB--481, antibiotic TMB--481 mixed sodium and potassium salts is dissolved in an organic solvent such as acetone and methyl ethyl ketone, and then acidified with an acid such as hydrochloric acid.

After addition of water, the mixture is extracted with ethyl acetate, benzene or chloroform to give the desired objective compound.

In order to obtain an alkali metal salt of antibiotic TMB48l, the free acid of antibiotic TMB-481 is dessolved in an organic solvent such as acetone and methyl ethyl ketone. To the resulting solution, an alkali metal base such as sodium hydroxide and potassium hydroxide is added. After addition of water, the solution is extracted with ethyl acetate, benzene or chloroform to give an alkali metal salt of antibiotic TMB-481.

Antibiotic TMB481 sodium salt obtained as described above has the following physico-chemical properties: (1) Appearance Colorless needle (2) Elemental Analytical Values C: 62.16, H: 8.81, Na: 2.60% (3) Molecular Weight 850 (4) Molecular Formula C44H75O14Na (5) Melting Point 181-182 C (6) Specific Optical Rotation [] 2D5 = +48.20 (C = 0.5%, methanol) (7) Ultara Violet Absorption Spectrum Antibiotic TMB--481 sodium salt dissolved in methanol shows only end absorption as shown in Fig. 1.

(8) Infrared Absorption Spectrum The infrared absorption spectrum determined with tablet of KBr illustrated in Fig. 2 shows characteristic bands at 3380, 3180, 2960, 2920, 2865, 2810, 2580, 1585, 1450, 1400, 1385, 1375, 1355, 1330, 1305, 1290, 1260, 1240, 1200, 1165, 1105, 1090, 1075, 1055, 1040,1015, 1000, 970,965,940,915,890, 870,855,825,790,745, and 700 cm-'.

(9) Nuclear Magnetic Resonance Spectrum The nuclear magnetic resonance spectrum at 60 MHz of antibiotic TM B48 1 sodium salt in CDCI3 shows the characteristic four singlets at a 3.25-3.S2 which mean four methoxy groups (Fig. 3).

(10) Solubility Antibiotic TMB--481 sodium salt is soluble in methanol, ethanol, butanol, acetone, ethyl acetate, chloroform, ethyl ether, benzene, n-hexane and petroleum ether, but practically insoluble in water.

(11) Color reaction Antibiotic TMB=48l sodium salt gives positive reaction with iodine, potassium permanganate and vanillin-sulfuric acid, but negative reaction with ninhydrin, Molisch and ferric chloride.

The acute toxicity of antibiotic TMB-481 sodium salt, administrated orally to ddY mice(male, 5-week-old) and expressed as LD50 is 150 mg/kg. The toxicity of antibiotic TMB-481 is lower than that of antibiotic TM-481(LD50: 48 mg/kg) coproduced from said microorganism.

Antibiotic TMB=481 and the salts thereof can be used as the anticoccidial agent. For the control of coccidiosis in poultry, a nontoxic anticoccidial amount of antibiotic TMB-481 or a salt thereof is administrated to the birds, preferably orally on a daily basis. In case of the oral administration, the compounds of this invention may be supplied with a substance capable of being consumed by the birds, preferably the feed of the birds. The rate of administration, which is effective against infection of coccidiosis, is generally in the range of from about 50 ppm to 250 ppm by weight of unmedicated feed. The compound of this invention may be used optionally together with another anticoccidial agent. The anticoccidial activity of antibiotic TMB-481 is further illustrated by test involving Eimeria tenella in chickens.

For these studies, groups of five 8-day-old chickens were fed a diet containing antibiotic TMB=48l sodium salt at the concentrations of 75 ppm and 125 ppm uniformly dispersed therein. The diet used has the following composition.

47.0% corn 3.0% lucerne meal 10.0% barley four 3.0% sucrose 10.0% defatted rice bran 3.0% CaCO3 10.0% soy bean meal 0.5%- NaCI 7.0% fish meal 0.5% Vitamin premix 7.0% wheat bran After having been on this ration for 48 hours, each chicken was inoculated with sporulated oocysts of Eimeria tenella. Other groups of five 8-day-old chickens were fed the diet which did not contain antibiotic TMB481 sodium salt. One of these groups was also inolculated with E. tenella after 48 hours and served as infected control. Other of these groups was not inoculated with E. tenella and served as normal control. Medication was continued 7 days after infective oocysts were administrated and then the results of treatment were evaluated.

The chickens were weighed, sacrificed and examined for evidence of coccidial lessions. Coccidial imvolvment was expressed on an arbitrary scale, increasing from minus (no evidence of coccidiosis) to four plus (maximum involvement for E. tenella). The results of the test is shown in Table 2.

TABLE 2 Treatment Fecals Lesion Relative Mortality scores scores wt. gain ~ # ++ +++ ++++ Non-medicated group, 5 5 0 0 0 0 100 0/5 non infected Non-medicated group. ++ ^ .0 0 0 5 31.4 2/5 infected Medicated group 75 ppm + I i D 3 0 87.4 0/5 125 ppm - 4 @ 0 0 0 94.3 0/5 Fecal scores -: normal +: diarrhea ++: blood diarrhea Lesion scores -: normal +: insignificant ++: slight degree +++: middle degree ++++: heavy degree The antibiotics of this invention also exhibit inhibitory activity against the growth of pathogenic microorganisms, particularly Gram-positive bacteria. The levels at which antibiotic TMB481 sodium salt shows inhibition against the growth of illustrative organisms are set forth in Table 3. The inhibitory levels were determined by the agar dilution test. In the agar dilution test, the test organism was streaked on a series of heart infusion agar plates containing various concentration of antibiotic TMBH81 sodium salt to determine the minimum inhibitory concentration of antibiotic TMB481 sodium salt in mcg/ml(micrograms per milliliter) in the agar substarate which inhibited the growth of the organism over a period of 24 hours.

TABLE 3 Antimicrobial Spectrum of Antibiotic TMB-481 Sodium Salt Microorganism MIC (mcg/ml) Staphylococcus aureus 209P 6.25 Staphylococcus aureus Smith 50 Staphylococcus aureus TPR--18 25 Staphylococcus aureus TPR-23 25 Staphylococcus aureus TPR-26 25 Staphylococcus aureus TPR-27 25 Staphylococcus epidermidis TPR-l3 25 Staphylococcus epidermidis TPR- 14 12.5 Staphylococcus epidermidis TPR-16 12.5 Staphylococcus epidermidis TPR-25 12.5 Staphylococcus epidermidis TPR-28 12.5 Bacillus subtilis PCI 219 12.5 Micrococcus luteus NIHJ 12.5 Escherichia coli B > 50 Escherichia coli 055 > 50 Proteus vulgaris HX--19 > 50 Pseudomonas aeruginosa P-32 > 50 Medium: Heart infusion agar Following is a description by way of example only of methods of carrying the invention into effect.

Example 1.

Shake flask production of antibiotic TMB-481 The production of antibiotic TMB-481 in shake flask cultures is illustrated by the following example: Spores of Streptomyces ribosidificus TM-481 (ATCC No. 31051) were inoculated on a nutrient agar slant made up of 20 g of oat meal 20 g of agar and sufficient water to make a total volume of one liter. The slant were inoculated with spores of Streptomyces ribosidificus TM--481 and incubated for three to four days at 30"C. The slants were covered with sterile distilled water and gently scraped to remove the organisms and provide an aqueous suspension thereof. One milliliter of the resulting spore suspension was used to inoculate each 100 milliliter portion of the production medium prepared as follows: The production medium was prepared by combining 10 g of glucose, 20 g of oat meal, 3 g of meat extract, 3 g of sodium chloride, 2 g of calcium carbonate, 400 mg of ferric sulfate, 400 mg of manganase chloride and sufficient distilled water to make the total volume one liter and adjusting pH at 7.0 with sodium hydroxide.

One hundred milliliter portion of the production medium were placed in five -hundred milliliter Erlenmeyer flask, which were then sterillized at 1200C for 15 minutes.

The production flasks were shaken for 72 hours at 300 C, on a rotary shaker operating at 200 r.p.m..

The whole broth (5 liters) obtained by the above precedure, was basified with sodium hydroxide and was filtered in vacuo with the aid of diatomaceous earth.

The mycelium cake was extracted two time with acetone. The two mycelial extracts were combined and concentrated in vacuo to remove the acetone. The filtered broth and aqueous concentrate of the mycelial extract solutions were combined, and the solution was extracted two times with one-half volume of ethyl acetate. The ethyl acetate extracts were combined and concentrated to contain 1.5 g of the crude antibiotic mixture.

Example 2 Pilot plant production of antibiotic TMBH81 The production of antibiotic TMBW81 byisubmerged fermentation on a pilot scale is illustrated by the following procedure: Five liters of an aqueous culture medium containing 1% glucose, 2% oat meal, 0.3% meat extract, 0.3% sodium chloride and 0.2% calcium carbonate at pH 7.0 is divided into 10 parts and each 500 milliliter is poured into a 2 liter Sakaguchi flask.

The medium is sterilized at 1200C for 15 minutes.~ fter co̲ling,~~each medium is inoculated with 10 milliliters of culture of Streptomyces ribosidificus TM481 in a 500 milliliter Sakaguchi flask. The culture is shaken for 48 hours at 300 C.

Then, 5 liters of the obtained seed culture is transferred into a 250 liter fermentor containing 200 liters of the same aqueous medium. The culture in the fermentor is aerated and agitated for 60 hours at 300 C.

The crude antibiotic mixture was obtained as brown syrup by the same method as described ..in Example 1 except that centrifugation instead of filtration was carried out to separate the mycelium from the supernatant. Yield: 250 milliliters.

Example 3.

Isolation of antibiotic TOMB481 mixed sodium and potassium salts All of the crude antibiotic mixture obtained in Example 2 was dissolved in one liter of benzene, and the insoluble precipitate was removed by filtration with the aid of filter paper. After the benzene solution was passed through a column packed with 500 g of silica gel(Wakogel C-200) by the use of benzene, the column was eluted with one liter of benzene to remove impurities, and eluted with chloroform until all of antibiotic TM--481 had been collected in the eluate. And then, the column was eluted with chloroform-methanol (99:1) to obtain the objective compound in the elute fractions. These fractions were combined and evaporated in vacuo. Six hundred mg of the resulting solid was dissolved in methanol, and further purified by gel filtration on Sephadex LH-20 by the use of methanol as the eluant.

The fractions containing only the objective compound checked by TLC on silica gel (Kieselgel 60 F254) in benzene-acetone (2:1) and detected by vanillin-H2SO4, were pooled and evaporated to dryness. The resulting white powder was crystallized from n-hexane-benzene (4:1) to obtain 400 mg of the objective compound, antibiotic TMB--481 mixed sodium and potassium salts, as colorless needles.

Example 4, Preparation of antibiotic TMBC81 and its sodium salt To 100 mg of antibiotic TMB--481 mixed sodium and potassium salt obtained in Example 3, 10 milliliters of acetone was added. To the resulting solution, one drop of 1 N HCI and 5 milliliters of water were added. The solution was extracted three times with 5 milliliters of benzene. The extracts were combined, washed with water and evaporated in vacuo to give 85 mg of the free acid of antibiotic TMB "81 as powder. The powder thus obtained was dissolved in 5 milliliters of acetone. To the resulting solution, 7.5 milliliters of water and one drop of 5 N NaOH were added. The extracts were combined, evaporated and crystallized with the same method as described in Example 3 to give 75 mg of antibiotic TM B--48 I sodium salt.

Claims (4)

1. WHAT WE CLAIM IS: I. An antibiotic TMB--481 represented by the formula

   and its alkali metal salts.
2. 2. A method for the production of antibiotic TMB-481 and its alkali metal salts which comprises cultivating the microorganism strain Streptomyces ribosidificus TM481(ATCC No. 31051) in a culture medium containing assimilable carbon and nitrogen sources, extracting antibiotic TMBH81 thus obtained from the culture medium with an organic solvent and separating TMB-481 from the extract solution as such or as an alkali metal salt.
3. 3. An antibiotic as claimed in Claim 1 and substantially as described in any one of the specific examples hereinbefore set forth.
4. 4. A method of producing an antibiotic as claimed in Claim 1 substantially as herein described with reference to the accompanying drawings or in the specific examples hereinbefore set forth.