CS209848B2 - Method of preparation of polyetheric antibiotics a-2808 - Google Patents

Description

(54) A method for preparing A-28086 polyetberic antibiotics

The invention relates to a process for the preparation of A-28086 factors A, B and D polyetheric antibiotics and to a process for the preparation of the A-28086 antibiotic complex from which factors A, B and D are derived. The antibiotic compounds are useful as antibacterial, fungicidal, antiviral agents, agents against pleuropneumonia-like organisms, as well as anticoccidial, insecticidal and acaricidal agents, and for increasing the utility of food in ruminants.

The present invention relates to a process for the preparation of a complex of A-28086 antibiotics comprising Factor A, Factor B and Factor D, characterized by culturing Streptomyces aureofaciens NRRL 5758 or Streptomyces aureofaciens NRRL 8092 in a nutrient medium containing assimilable sources of carbohydrates, nitrogen and inorganic salts. submerged aerobic fermentation conditions until a significant amount of antibiotic activity is produced by the organism in said nutrient medium.

Antibiotic A-28086 Factor A is a white crystalline compound that is soluble in lower alcohols, dimethylformamide, dimethylaulinoxic, ethyl, acetone and benzene, but is only slightly soluble in hexane and is insoluble in water after crystallization from acetone-water.

The compound melts at about 98 to 100 ^° C, solidifies again, and then melts again at 195 to 200 ° C and shows

(a) molecular mass 764, as determined by mass spectrometry;

b) approximately elemental composition 66.69% C, 9.85% H and 23.10% O,

(c) empirical formula C13H72O11 as determined by mass spectrometry;

(d) specific rotation 5454 ° (c = 0.2, mestanol), determined at 25 ° C;

(e) infrared absorption spectrum in chloroform with the following distinct absorption maxima: 2.85, 3.34, 5.83, 6.82,

7.22, 7.53 (weak), 7.78 (weak), 8.75 (strong), 8.95 (strong), 9.15, 9.50 (strong), 9.55 (strong), 9, 60, 9.85, 10.15, 10.45 and 10.70 (weak) microns,

(f) the ultraviolet spectrum in ethanol only has a terminal absorption below 200 IU;

(g) The NMR spectrum in deutero-chloroform has the following characteristics: S S 6.01, 4.21, 4.11, 3.99, 3.89, 3.80, 3.67, 3.65, 3.57, 3, 55,

2.83, 2.76, 2.74, 2.68, 2.66, 2.58, 2.56, 2.30,

2.22, 2.17, 2.10, 2.05, 1.96, 1.90, 1.85, 1.70,

1.62, 1.60, 1.47, 1.39, 1.31, 1.25, 1.18, 0.95,

0.93, 0.90, 0.88, 0.85, 0.77, 0.75, 0.73, 0.68 and 0.66 ppm,

h) titratable group with pK ₃ = 7.9 in 80% aqueous dimethylformamide,

i) X-ray powder diffraction (Cu ^{+ +} radiation, 1.5405 λ, nickel filter) with the following interplanar distances in angstroms (d), d Relative intensity

12.00 100 ALIGN! 10.10 50 9.25 90 8.00 40 7.50 15 Dec 6.92 90 6.40 40 5.98 05 / 5.68 15 Dec 5.20 40 4.98 40 4.62 40 4.21 20 May 3.48 10

}) Rf value 0.24 when thin-layer chromatography on silica gel in benzene-ethyl acetate (3: 2) using Bacillus subtilis ATCC 6633 as the detection organism,

k) the following Rf values in paper chromatography on the systems below using Bacillus subtilis ATCC 6633 as the detection organism

Rf value Solvent system

0,11 water saturated with methylisobutyl ketone (MIBK)

0.41 MIBK saturated water and 2% p-toluenesulfonic acid and 1% piperidine

0.54 water: methanol: acetone (12: 3: 1) - adjusted to pH 10.5 NH 3 OH and then to pH 7.5 H5PO4

0.48 1% MIBK, 0.5% NH 4 OH in water

0.15 17.4 g of K2HPO4, 30 ml of ethanol per liter of water

0.24 water saturated with benzene

0.24 water

0.75 water: MIBK: ethyl acetate (98: 1: 1),

(l) an acid function capable of forming salts and esters; and

m) at least one esterified hydroxyl group and acyl esters of 2 to 6 carbon atoms in the acyl group and their physiological salts.

Antibiotic A-28086 factor B is a white crystalline compound after crystallization from acetone-water, which is soluble in lower alcohols, dimethylformamide, dimethylsulfoxide, ethyl acetate, chloroform, acetone and benzene, but is only slightly soluble in hexane and insoluble in water and shows :

(a) a melting point of about 150 to 153 ° C;

(b) molecular weight 762, as determined by high resolution mass spectrometry;

(c) the empirical formula C43H70O11 as determined by high resolution mass spectrometry;

(d) an infrared absorption spectrum in chloroform with the following distinguishable absorption maxima: 2.82, 3.30, 5.77, 5.85,

6.80, 7.20, 7.50 (weak), 7.72 (weak), 7.80 (weak), 8.57 (strong), 8.68, 8.90 (strong), 9.10 , 9.50, 9.83 (strong), 9.90, 10.10, 10.17 (strong), 10.43 (weak), 10.80 (weak), 11.2.0 (weak), 11.35 (weak), 11.73 (weak) and 12.03 (weak) microns,

(e) ultraviolet absorption maximum in ethanol at 220 mu ( ^E | = 137,5, ε = 10477),

f) NMR spectrum in deuterochloroform having the following characteristics: δ 7.20, 7.09, 6.26, 6.15, 4.19, 4.12, 4.05, 3.95, 3.89,

3.78, 3.62, 3.59, 3.52, .3.48, 2.81, 2.73, 2.63,

2.54, 2.52, 1.99, 1.91, 1.71, 1.67, 1.64, 1.55,

1.43, 1.33, 1.18, 1.11, 0.96, 0.94, 0.90, 0.87,

0.84, 0.77, 0.74 and 0.68 ppm,

(g) an Rf value of 0,42 by thin-layer chromatography on silica gel with benzene-ethyl acetate (3: 2) using Bacillus subtilis ATCC 6633 as the detection organism,

h) the following Rf values for paper chromatography in the systems below, using Bacillus subtilis ATCC 6633 as the detection organism

Rf value Solvent system

0.16 water saturated with MIBK and 2% p-toluenesulfonic acid and 1% piperidine

0.46 water: methanol: acetone (12: 3: 1) - adjusted to pH 10.5 with NH4OH and then to pH 7.5 with H3PO4

0.36 1% MIBK, 0.5% NH 4 OH in water

0.51 water saturated with benzene

0.11 water

0.61 water: MIBK: ethyl acetate (98: 1: 1),

(i) an acid function capable of forming salts and esters;

j) two ketone functional groups,

k) at least one hydroxyl functionality, and physiologically acceptable salts thereof. Antibiotic A 28086 Factor D is a white crystalline solid after crystallization from acetone-water, which is soluble in methanol, ethanol, dimethylformamide, dimethylsulfoxide, ethyl acetate, chloroform, acetone and benzene, but is only slightly soluble in hexane and insoluble in water, it has a melting point of about 96 to 98 ° C and shows:

A) molecular weight 778, as determined by high resolution mass spectrometry,

(b) an approximate elemental composition of 67.59% C, 9.38% H and 22.77% O,

(c) the empirical formula C44H74O.11, as determined by high resolution mass spectrometry;

d) specific rotation -56 ° (c = 0.1, methanol J, determined at 25 ° C),

e) infrared absorption spectrum in chloroform with the following absorption maxima: 2.89, 3.39, 3.43, 3.50, 5.88, 6.90, 7.27, 7.60, 7.84, 9, 00, 9.26, 9.62, 10.31, 10.58, 11.10 and 11.49 microns, fj not observed ultraviolet absorption in 95% aqueous ethanol, gj NMR spectrum in chloroform with the following characteristics: δ 6.00 , 4.20, 4.10, 4.00, 3.98, 3.92, 3.86, 3.83, 3.79, 3.67, 3.64,

3.57, 3.54, 2.88, 2.81, 2.71, 2.62, 2.58, 2.48,

2.43, 2.37, 2.29, 2.21, 2.15, 2.10, 2.04, 1.97,

1.89, 1.83, 1.76, 1.68, 1.61, 1.58, 1.55, 1.47,

1.39, 1.30, 1.25, 1.18, 0.95, 0.90, 0.88, 0.84,

0.74 and 0.68 ppm,

(h) a titratable group with a pKa value of 8,67 in 80% aqueous dimethylformamide;

(i) characteristic X-ray powder diffraction (Cu ^{+ +} radiation 1,545 λ, nickel filter) with the following interplanar distances in angstroms (d):

d Relative intensity

12.40 100 ALIGN! pathogenic organisms. Another problem 10.20 70 is to develop strains of organisms that are 8.85 90 resistant to standard antibiotics. 7.80 30 Yet another problem is the fact that 6.80 10 Individual patients often have severe reactions to certain 6.30 100 ALIGN! pure antibiotics, which is caused by 5.70 20 May knowledge and / or toxic effects. Considering 5.35 20 May to these problems are in current therapy 5.10 20 May new antibiotics still needed. 4.90 10 In addition to the demand for new antibiotics, 4.65 20 May which would be useful for treating diseases 4.45 40 In humans, antibiotics are also needed 4.20 30 in the veterinary field. One important 3.30 10 for which better antibio- 3.15 10 tics, antibiotics are needed to accelerate growth 2.99 05 / poultry and cattle. Acceleration of growth 2.77 05 / for example by reducing the disease and 2.28 05 / increasing the usability of food.

Well known economic disease

(j) the following Rf values for silica gel thin-layer chromatography in the systems listed below, using Bacillus subtilis ATCC 6633 as the detection organism:

Rf value

Solvent system

0.26 benzene-ethyl acetate (3: 2)

0.66 ethyl acetate-diethylamine (95: 5),

(k) the following Rf values for paper chromatography in the systems listed below, using Bacillus subtilis atcc 6633 as the detection organism:

Rf value Solvent system

0,10 water saturated with methylisobutyl ketone (MIBK)

0.26 water saturated with MIBK and 2% p-toluenesulfonic acid and 1% piperidine

0.36 water: methanol: acetone (12: 3: 1) - adjusted to pH 10.5 NH4OH and then to pH 7.5 H3PO4

0.29 1% MIBK, 0.5% NH 4 OH in water

0.25 17.4 g K 2 HPO 4, 30 mL ethanol per liter water

0.26 water saturated with benzene

0.09 water

0.64 water: MIBK: ethyl acetate (98: 1: 1),

(i) an acid function capable of forming salts and esters;

m) at least one hydroxyl group capable of esterification, and acyl esters of 2 to 6 carbon atoms in the acyl and physiologically acceptable salts.

Although a large number of antibacterial agents are currently known, there is a continuing need for new improved antibiotics. One problem with current antibiotic therapy is that antibiotics differ in efficacy against reach from the veterinary field, more specifically from the poultry industry, is protozoan coccidiosis. Coccidiosis is caused by infection with one or more species of Eimeria or Isospora (for review see Lund and Farr "Diseases of Poultry" 5th edition Biester and Schwarte, Eds. Iowa State University Press, Ames, Ia., 1965, pp. 1056-1096). In view of the large economic losses caused by coccidiosis and the disadvantages of some known coccidiosis agents, research into better coccidiosis agents is still ongoing.

Enteritis and other diseases can cause significant economic losses to animal producers. Enteritis occurs in chickens, pigs, cattle and sheep and is mainly caused by anaerobic bacteria, particularly

289648

Clostridium perfringens and viruses. Enterotoxemia in ruminants, exemplified by the “ruminant disease” in sheep, is a condition caused by C. perfringens infection.

Accelerating growth in ruminants, such as cattle, is another economically desirable goal of the veterinary sciences. The mechanism for utilizing the main nutrients (carbohydrates) of ruminant food is well known. Microorganisms in the stomach of ruminants degrade carbohydrates to monosaccharides and then convert these monosaccharides to pyruvate compounds. Pyruvates are metabolized by microbial processes to acetates, butyrates or propionates, commonly known as volatile fatty acids. For a detailed discussion, see Leng in "Physiology of Digestion and Metabolism in the Ruminant", Phillipson et al., Eds. Ctriel Press, Newcastle-upon-Tyne, England, 1970, pp. 408-410.

The relative utility of volatile fatty acids is discussed by McCullough in Feedstuffs, June 19, 1971, p 19; Eskeland et al., J. An. Sci. 33, 282 (1971); and Church et al. in "Digestive Physiology and Nutrition of Ruminants, Vol. 2, 1971, pp. 622 and 625. Although acetates and butyrates are used, propionates are used more. Furthermore, if too little propionate is available, ketosis develops in animals. Preferred compounds therefore stimulate the production of a higher propionate ratio from carbohydrates in animals, resulting in a higher utility of carbohydrates as well as a reduction in cases of ketosis.

Antibiotics A-28086 factors A, B and D are new members of polyetheric antibiotics. Examples of this group are: monensin (US patent 3,501,558); dianemycin [R. L. Handli, Μ. Hoehn M., Pittenger G. E., Chamberlin J., and Gorman M., J. Antibiotics 22, 161 (1969)]; nigericin [K. L. Steinrauf, Mary Pinkerton and J.W. Chamberlin, Biochem. Biophys. Res. Comm. 33 29 (1968)]; and salinomycin [Japanese Published Patent 47-25392, 20, X. 1972, Derwent No, 7-960Τ, U, S. Patent No. 3,857,948 and H, Kinashi, N. Otake, H. Yonehara, S. Sato and Y. Salto, Tatrahedron Lett. 49, 4955-4958 (1973)].

The term "antibiotic complex" as used in fermentation does not mean a chemical complex, but is a mixture of individual antihiotic factors produced simultaneously. As is known to those skilled in the art of fermentation, the ratio of the individual factors produced in the antibiotic complex varies depending on the fermentation conditions.

The A-28086 antibiotic complex is produced by culturing a new strain of Streptomyces aureofaciens NRRL 5758 under submerged aerobic taffeta fermentation conditions until a significant level of antibtotic activity is produced. The A-28086 antibiotic complex may also be produced by another strain of Streptomyces aureofaciens NRRL 8092.

When the A-28086 antibiotic complex is produced by either S. aureofaciens NRRL 5758 or S. aureofaciens NRRL

8092, then extracted from the fermentation solution and mycelium with polar organic solvents. The extracted antibiotic mixture is separated by evaporation of the solvents, the concentrate is added to an excess of petroleum ether to precipitate impurities, and after filtration and evaporation of the filtrate an antibiotic mixture is obtained.

A-28086. The antibiotic mixture is further purified and separated into individual factors by column chromatography.

Compounds A-28086 show growth inhibition of organisms that are pathogenic to animal and plant life. In one aspect of this inhibitory activity, Compound A-28086 acts as an anti-coccidiosis agent. In addition, compounds A-28086 are antibacterial agents, antiviral agents, anti-PPLO (pleuropneumonia-like organism) agents, insecticidal agents, and acaricidal agents, and increase food utilization in ruminants. *

The following infrared absorption spectra in chloroform are shown in the drawings, wherein Fig. 1 is A-28086 factor A, Fig. 2 is A-28086 factor B, Fig. 3 is A-28086 factor A, acetyl ester Fig. 4 Fig. 5 is A-28086 factor A propionyl ester, Fig. 5 is A-28086 factor A butyryl ester, Fig. 6 is A-28086 factor A valeryl ester, Fig. 7 is A-28086 factor A caproyl ester, giant. 8 shows the antibiotic A-28086 factor D.

A-28086 antibiotic factors are structurally related. At least four antibiotic factors are produced simultaneously during the fermentation and a mixture thereof is obtained.

The factors are separated from each other and factors A, B, and D are isolated as individual compounds as described below. The mixture of factors A-28086 is soluble in most organic solvents, but is insoluble in water. ψ

The following paragraphs describe the physical and spectral properties of A-28086 factors A, B, and D. *

Antibiotic A-28086 factor A crystallizes from acetone-water. A-28086 Factor A melts at about 98-100 ° C, solidifies again, and melts at 195-200 ° C again. Elemental analysis of factor A gave the following average percent composition: carbon 66.69 percent, hydrogen 9.85%, and oxygen .23.10%.

The empirical formula designed for factor A is C43H72O11.

Factor A has a molecular weight of 764 as determined by mass spectrometry.

The infrared spectrum of factor A in chloroform is shown in Figure 1 of the accompanying drawing. The following absorption maxima were observed: 2.85, 3.34, 5.83, 6.82, 7.22,

7.53 (weak), 7.78 (weak), 8.75 (strong), 8.95 (strong), 9.15, 9.50 (weak), 9.55 (strong), 9.60,

9.85, 10.15, 10.45 and 10.70 (weak) microns.

The ultraviolet spectrum of factor A in ethanol shows only a terminal absorption below 220 ιημ.

The NMR spectrum of A-28086 factor A in deuterochloroform has the following characteristics: δ 6.01, 4.21, 4.11, 3.99, 3.89, 3.80, 3.67, 3.65, 3.57, 3.55, 2.83, 2.76, 2.74, 2.68, 2.66,

2.58, 2.56, 2.30, 2.22, 2.17, 2.10, 2.05, 1.96,

1.90, 1.85, 1.70, 1.62, 1.60, 1.47, 1.39, 1.31,

1.25, 1.18, 0.95, 0.93, 0.90, 0.88, 0.85, 0.77,

0.75, 0.73, 0.68 and 0.66 ppm.

Antibiotic A-28086 Factor A crystallizes from an acetone-water mixture and exhibits the following characteristic X-ray powder diffraction (Cu ⁺⁺ radiation 1.5405 Å, nickel filter (d = interplanar distances in angstroms (10 ^{10 10} m)) d Relative intensity

12.00 100 ALIGN! 10.10 50 9.25 90 8.00 40 7.50 15 Dec 6.92 90 6.40 40 5.98 05 / 5.68 15 Dec 5.20 40 4.98 40 4.62 40 4.21 20 May 3.48 10

The specific rotation of the A-28086 factor A antibiotic is -54 ° (c = 2, methanol), determined at 25 ° C. This specific rotation is the average of several determinations.

Electrometric titration of factor A in 80% aqueous dimethylformamide indicates the presence of a titratable group pKa = 7.9.

Antibiotic A-28086 Factor A is soluble in various organic solvents such as methanol, ethanol, dimethylformamide, dimethylsulfoxide, ethyl acetate, chloroform, acetone and benzene, but is only slightly soluble in non-polar organic solvents such as hexane and is insoluble in water .

Antibiotic A-28086 factor A has an acidic functional group capable of forming salts and esters and at least one hydroxyl group capable of esterifying.

Based on the above physical properties, it is possible to design the structure of antibiotic A-28086 factor A. Since the structural determination is only designed, it is understood that the structure presented herein is only a working hypothesis. The structure design for A-28086 factor A is shown by Formula I.

Cf ~ f3 | OH

Antibiotic A-28086 factor B is a white crystalline compound (from acetone-water) having a melting point of 150-153 ° C.

As determined by high resolution mass spectrometry, factor B has a molecular weight of 762 and the suggested empirical formula is C43H70O11.

The infrared spectrum of factor B in chloroform is shown in Figure 2 of the attached drawing and has the following absorption maxima: 2.82, 3.30, 5.77, 5.85, 6.80, 7.20, 7.50 (weak) , 7.72 (weak), 7.80 (weak), 8.57 (strong), 8.68, 8.90 (strong), 9.10, 9.50, 9.83 (strong),

9.90, 10.10, 10.17 (strong), 10.43 (weak), 10.80 (weak), 11.20 (weak), 11.35 (weak), 11.73 (weak) , and 12.03 (weak) microns.

Formula I

The ultraviolet spectrum of factor B in ethanol shows an absorption maximum at 220 mju (E icm = 137.5, ε = 10.477).

The NMR spectrum of A-28086 factor B in deuterochloroform shows the following characteristics: δ 7.20, 7.09, 6.26, 6.15, 4.19, 4.12, 4.05, 3.95, 3.89, 3.78, 3.62, 3.59, 3.52, 3.48,

2.81, 2.73, 2.63, 2.54, 2.52, 1.99, 1.91, 1.84,

1.71, 1.67, 1.64, 1.55, 1.43, 1.33, 1.18, 1.11,

0.96, 0.94, 0.90, 0.87, 0.84, 0.77, 0.74 and 0.68 ppm.

Antibiotic A 28086 Factor B is soluble in various organic solvents such as methanol, ethanol, dimethylfluoride, and the like ^. ormamide, dimethylsulfoxide, ethyl acetate, chloroform, acetone and benzene, but is

209846 sparingly soluble in non-polar organic solvents such as hexane and insoluble in water.

Although the chemical structure of A-28086 factor B has not been established, available physicochemical data indicate that factor B has one carboxyl group and two keto groups and one or more hydroxyls.

Antibiotic A-28086 factor D in the production of S. aureofaciens NRRL 5758 is a minor factor. But if it is cultivated

S. aureofaciens NRRL 8092, A-28086 factor D is produced in an amount of up to 10% of isolated antibiotic activity.

Antibiotic A-28086 factor D is a white crystalline substance (water-acetone), the melting point of which is about 96-98 ° C. Antibiotic A-28086 factor D has a molecular potential of 778 as determined by high resolution mass spectrometry.

The elemental composition of the mass spectrum of the A-28086 Factor D sodium salt is 800.5050 (calculated for C 44 H 7 O 11 Na = 800.5050). A small band at 778 and a larger band at 760.5117 (calculated for C44H72O10 = 760.5125) are observed in the free acid mass spectrum of the A-28086 factor D m / e 760 in the free acid mass spectrum due to the loss of water from the molecular · ion. Thus, the composition of the molecular ion A-28086 Factor D of the free acid is C44H74O11.

The empirical formula designed for · A-28086 factor D is C44H74O11. Elemental analysis of Factor D gives the following composition: carbon 67.59%, hydrogen 9.38%, oxygen 22.77%.

The theoretical percentage for C44H74O11 is carbon 67.87%, hydrogen 9.51%, oxygen 22.77%,

The infrared absorption spectrum of A-28086 factor D (Figure 8) contains the following absorption maximum: 2.89, 3.39, 3.43, 3.50, 5.88,

6.90, 7.27, 7.60, 7.84, 9.00, 9.26, 9.62, 10.31,

10.58, 11.10 and 11.49 microns.

A-28086 factor D at 95o / _o aqueous ethanol shows no ultraviolet absorption.

The NMR spectrum of A-28086 factor D in deutero-chloroform shows the following characteristics: δ 6.00, 4.20, 4.10, 4.00, 3.98, 3.92, 3; 86, 3.83, 3.79, 3.67, 3.64, 3.57, 3.54, 2.88,

2.81, 2.71, 2.62, 2.58, 2.48, 2.43, 2.37, 2.29,

2.15, 2.10, 2.04, 1.97, 1.89, 1.83, 1.76,

1.68, 1.61, 1.58, 1.55, 1.47, 1.39, 1.30, 1.25,

1.18, 0.95, 0.90, 0.88, 0.84, 0.74 and 0.68 ppm.

Antibiotic A-28086 factor D crystallizes from an acetone-water mixture and has the following characteristic X-ray powder diffraction (Cu ⁺⁺ radiation, 1.5405 λ, nickel filter, d = interplanar distances in angstroms of 10 ^{10 10} m) d Relative intensity

12.40 100 ALIGN! 10.20 70 8.85 90 7.80 30 6.80 10 6.30 100 ALIGN! 5.70 20 May 5.35 20 May 5.10 20 May 4.90 10 4.65 20 May 4.45 40 4.20 30 3.30 10 3.15 10 2.99 Ό5 2.77 05 / 2.28 05 /

The specific rotation of the A-28086 factor D antibiotic is -50 ° (c = 0.1, methanol), determined at 25 ° O.

Electrometric titration of A-28086 Factor D in 80% aqueous dimethylformamide indicates the presence of a titratable pKa group of 8.67.

Antibiotic A-28086 factor D is soluble in many organic solvents such as methanol, ethanol, dimethylformamide, dimethylsulfoxide, ethyl acetate, chloroform, acetone, and benzene. A-28086 factor D is only slightly soluble in non-polar organic solvents such as hexane and is insoluble in water.

Antibiotic A-28086 factor D has an acidic functional group capable of forming salts and esters and at least one hydroxyl group capable of esterifying.

Based on the physical characteristics listed above, the structure of antibiotic A-28086 Factor D was designed. The design of structure A-28086 factor D is shown in Formula II

wherein either R1 = CH3 and R2 = C2H5, or R1 = C2H5 and R2 = CH3.

Rf values of antibiotic A-28086 factor

A, B, and D in various paper chromatography systems using Bacillus subtilis ATCC 6633 as the detection organism are shown in Table I.

TABLE I

Factor A Rf values of factor B Factor D Solvent system 0.11 0.09 0.10 water saturated with methyl isobutyl ketone (MIBK) 0.41 0.16 0.26 water saturated with MIBK and 2% p-toluenesulfo- new acid and 1% piperidine 0.54 0.46 0.36 water: methanol: acetone (12: 3: 1) - pH adjusted to 10.5 with NH 4 OH and then to pH 7.5 by addition of H 3 PO 4 0.48 0.36 0.29 1% MIBK, 0.5% NH 4 OH in water 0.15 0.33 0.25 17.4 g of K 2 HPO 4, 30 ml of ethanol per liter of water 0.24 0.51 0.26 benzene saturated with water 0.24 0.11 0.09 water 0.75 0.61 0.64 water: MIBK: ethyl acetate (98: 1: 1) Table II shows the Rf values of an- boards E. Merck, Darmstadt F-254, layer thickness- tibiotics A-28086 factors A, B and D in two 0.25 mm thick) also using B. sufetilis chromatographic systems at chromá- ATCC 6633 as a detection organism. tografli to thin layer of silica gel {cash TABLE II R _f values Factor A Factor B · Factor D Solvent system 0.24 0.42 0.26 benzene-ethyl acetate (3: 2) 0.54 0.34 0.66 ethyl acetate-diethylamine (95: 5)

Along with the A-28086 antibiotic complex, another substance called A-28086-1 is produced. Although A-28086-I is not microbially active, it is structurally related to A-28086 antibiotic factors. A-28086-I is a white crystalline compound (from acetone-water) and has a melting point of about 160-162 ^° C. Comparative NMR spectral studies and other properties of A-28086-I and synthetically prepared A-28086 factor A methyl ester show A-28086-I is A-28086 factor A methyl ester or a closely related compound thereof such as a stereoisomer.

Although A-28086-I initially precipitates with active A-28086 factors, it is easily separated from them by silica gel chromatography. A-28086-1 has an approximate Rf value of 0.53 in silica gel thin layer chromatography using ethyl acetate as eluent and detection with vanillin detection reagent (3% vanillin in methanol + 0.5 mL concentrated H2SO4 per 100 mL solution). After spraying with vanillin and heating, the A-28086-I gives a blue spot, while the A-28086 factors provide bright pink spots that rapidly turn into dark brown-blue spots.

The antibiotic A-28086 factors A, B and D and said acyl esteryl factors A and D are capable of forming salts. "Physiologically acceptable salts" are pharmaceutically acceptable salts, ie, those in which the toxicity of the compound as a whole is not relatively higher in warm-blooded animals than in the free compound. Representative and suitable alkali metal and alkaline earth metal salts of A-28086 factors A and B are sodium salts, potassium salts, lithium salts, cesium salts, rubidium salts, barium salts, calcium salts and magnesium salts. Suitable ammonium salts of A-28086 factors A and B are ammonium salt, primary, secondary and tertiary C 1 -C 4 alkyl ammonium salts and C 2 -C 4 hydroxyalkylammonium salts. Suitable ammonium salts are those which form Factors A and B with A-28086 with ammonium hydroxide, methylamine, secbutylamine, isopropylamine, diethylamine, diisopropylamine, ethanolamine, triethylamine, 3-amino-1-propanol and the like.

The salts of A-28086 Factors A, B and D and the acyl esters of Factors A and D with alkali and alkaline earth metal cations are prepared by a process commonly used to prepare salts with cations. For example, the free acid of the antibiotic factor or the corresponding ester is dissolved in a suitable solvent such as hot methanol or ethanol, and to this solution is added a solution containing a stoichiometric amount of the desired inorganic base in aqueous methanol. The salt thus formed is isolated in a conventional manner by filtration or by evaporation of the solvent.

Salts formed with organic amines can be prepared in a similar manner. For example, gaseous or liquid amines can be added to a solution of the antibiotic in a suitable solvent such as acetone and solvent, and the excess amine is removed by evaporation.

It is well known in veterinary pharmaceutical practice that the form of the antibiotic is not critical in treating an animal with an antibiotic. In many cases, the animal changes the formulation to a form other than that in which the antibiotic is administered. The salt form in which the antibiotic is applied is not critical to the method of treatment. However, the salt form is often suitable for reasons of economic, applicability and toxicity.

A-28086 factor A forms acyl esters. Esterification takes place at one of the hydroxy groups of A-28086 Factor A by reaction with anhydrides or chlorides of acids of 2 to 6 carbon atoms. These esters are prepared by reacting A-28086 factor A with, for example, the corresponding acid anhydride at room temperature. These esters are also useful as antibiotics and as food enhancers.

The following paragraphs describe the characteristics of these A-28086 factor A acyl esters.

Factor A acetyl derivative A-28086 is a white crystalline compound (from acetone-water) melting at 100-103 ° C. Factor A acetyl ester A-28086 has an empirical formula of C45H74O12 and a molecular weight of about 807 based on the empirical formula designed for A-28086 factor A. Elemental analysis of factor A acetylester has the following percentages:

For C45H74O12:

Calculated:

66.97% C, H 9.24, 23.79 0/0 O; found: 67.67% C, , 8.71 O / o H, 23.13 _0/0 O. Infrared spectrum of acetylester A- -28086 factor And in chloroform is mentioned

3 of the accompanying drawing. The following absorption maxima are observed in the spectrum: 2.85, 3.36, 3.38 (strong), 5.80, 6.83, 7.25, 7.52 (strong), 7.60 (weak), 7. , 80 (strong), 8.45 (strong), 8.80 (strong), 8.95 (strong), 9.10 (strong), 9.20, 9.63, 9.80 (strong), 10 , 12 (weak), 10.25 (weak) and 10.50 microns.

The ultraviolet spectrum of factor A acetylester A-28086 in ethanol shows only terminal absorption.

The electrometric titration of A-28086 factor A acetylester in 80% aqueous dimethylformamide indicates the presence of a titratable group with a pKa of 8.5.

Factor A propionyl ester A-28086 is a white crystalline compound (from acetone-water) melting at 96-98 ° C. The A-28086 factor A propionyl ester has an empirical formula of C46H76O12 and a molecular weight of 821 based on the empirical formula designed for A-28086 factor A. The elemental analysis of the propionyl ester of factor A has the following percentage composition:

»

For C46H76O12:

Calculated:

67.29% C 9.33 _0/0 H, O 23.38%; found:

% C, 66.06;% H, 9.17;% O. 23.41.

The infrared spectrum of the A-28086 factor A propionate ester in chloroform is shown in Figure 4 of the accompanying drawing. The following absorption maxima are observed in the spectrum: 2.85, 3.33, 3.38 (strong), 3.45 (strong), 5.75 (strong), 5.82, 6.81, 7.22, 7, 30 (strong, 7.50 (weak), 7.60 (weak), 7.80, 8.43, 8.75 (strong), 8.90, 9.05, 9.15 (strong, 9.50 (strong), 9.63, 9.83 (weak), 10.05 (strong), 10.13, 10.20 (strong), 10.45 and 10.68 microns.

. The ultraviolet spectrum of the A-28086 factor A propionate ester in ethanol shows only terminal absorption.

A-28086 factor A butyl ester is a white crystalline compound (from acetone-water j, melting at about 96-98 ° C. A-28086 factor A butyl ester has an empirical formula of C47H78O12, a molecular weight of about 835 and an approximate composition of 67.60 / ₀ C, 9.41% H and 22.99% O, as derived from the empirical formula proposed for, a-28086 factor A.

The infrared spectrum of factor A butyryl ester A-28086 in chloroform is shown in Figure 5 of the accompanying drawing. The following absorption maxima were observed: 2.89, 3.40, 3.45, 3.51, 5.85, 5.92 (strong, 5.97 (strong), 6.90, 7.30, 7.84) (weak), 8.55, 8.85 (weak), 9.01 (strong), 9.26, 9.76, 9.95, 10.31 and 10.64 microns.

Factor A valery ester A-28086 is a white crystalline compound (from acetone-water), m.p. 173-175 ° C. Factor A Valerylester A-27086 has an empirical formula of C18H80O12, a molecular weight of about 849, and an approximate composition of 67.89% C, 9.50% H and 22.61% O, as derived from the empirical formula designed for A-28086 Factor A.

The infrared spectrum of factor A valerylester A-28086 in chloroform is shown in Figure 6 of the accompanying drawing. The following absorption maxima are observed in the spectrum: 2.90, 3.40, 3.45, 3.51, 5.87, 5.92 (strong), 5.99 (strong), 6.91, 7.30, 7.69 (weak), 7.87 (weak), 8.16, 8.58, 8.85 (weak), 9.26, 9.76, 10.00 (weak), 10.31 and 10, 64 microns.

Factor A-28086 Kaproylester is a white crystalline compound (from a mixture of acetone and water), m.p. 163-167 ° C. Kaproylester A-28086 factor A has an empirical formula C49H82O12, a molecular weight of about 863 and an approximate composition of 68.18 _0/0 C, 9.58% H and 22.24% O, as derived from the empirical formula proposed for A-28086, Factor A.

The infrared spectrum of Factor A caproyl ester A-28086 in chloroform is shown in FIG. 7 in the drawing. The following absorption maxima are observed in the spectrum: 2.90, 3.40, 3.45, 3.51, 5.87, 5.92 (strong), 5.97 (strong), 6.90, 7.30, 7 , 66 (weak), 7.84 (weak), 8.16, 8.58, 8.85 (weak), 9.05 (strong), 9.17, 9.72, 9.95, 10.29 , 10.62 microns.

Acylation of A-28086 Factor A leads to the following changes in the proton nuclear magnetic resonance spectrum: The carbonyl resonance occurring at 4 ppm shifts down to approximately 5.3 ppm (the exact position varies slightly according to various acyl derivatives and vinyl protonic signals also shift). The following characteristic changes are represented by the partial formula:

wherein R is alkyl of 1 to 5 carbon atoms.

This partial structure is fully in accordance with decoupling experiments in the PMR spectrum and with ¹³ C NMR data for acetyl and propionyl esters.

Acyl esters of A-28086 factor A of 2 to 6 carbon atoms in the acyl are soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethylsulfoxide, ethyl acetate, chloroform, acetone and benzene, but are only slightly soluble in non-polar organic solvents such as is hexane and are insoluble in water.

Each of the C 2 -C 6 -alkyl esters has a carboxyl group and is capable of forming salts and esters.

The novel antibiotics are produced by culturing the A-28086 producing strain of Streptomyces aureofaciens under submerged aerobic conditions in a suitable nutrient medium until significant antibiotic activity is produced. The antibiotic is isolated by conventional techniques used to isolate and purify antibiotics.

One of the new organisms useful for the preparation of A-28086 antibiotics was isolated from a sample of soil collected on Mount Ararat in Turkey. This organism was classified as a strain of Streptomyces aureofaciens Duggar as described by E. B. Shirling and Mr. Gottlieb in the Cooperatlve Description of Type Cultures of Streptomyces. III. Additional Species Descriptions frorn First and Second Studies', Intern. Bull. Systematic Bactetiol. 18, 279-392 (1968). This classification is based on the method recommended by the International Streptomyces Project [E. B. Shirling and D. Gottlieb, "Methods for Characterization of Streptomyces Species," Intern. Bull. Systematic Bacteriol. 16, 313-340 (1966)] along with some additional tests. Color names were assigned according to the ISCC-NBS method (K.L. Kelly and D. B. Judd, "The ISCC-NBS Method of Designing Color and

Medium Characteristics

Diciionary of Color Names', U. S. Dept. of Commerce, Circ. 553, 1955, Washington, D. C.). Values in brackets refer to the Tresner and Backus color series (Tresner, HD and SJ Backus, " System of Color Wheels for Streptomyces Taxonomy ", Appl. Microhiol. 11, 335-378 (1963)); the color control designation is underlined. Paul color values (A. Maerz and MR Paul, "Dictionary of Color", McGraw-Hill Book Co., Inc., New York, NY, 1950) are shown in parentheses and cultured for 14 days at 30 °. C, unless otherwise stated.

Characterization of strain NRRL 5758, producing A-28086 Morphology

Sporulating air hyphae consisting of hooks, eyes and open spirals. Morphology corresponding to the rectus flexis type was also observed. The spores are short and cylindrical and have 10 to 50 spores in the chain. Spores measure 1.3 μ x 1.75 μ in the range of 1.3 μ to 1.95 x 1.3 μ. Electron microscope surface spores are smooth.

Culture characteristics of NRRL 5758 on different media

Medium Characteristics

Tomato paste - oatmeal agar

Glycerol-glycine agar

Glucose-asparagine agar

Nutrient agar

Bennet agar

ISP (32 (yeast extract and malt extract))

ISP (33 (oatmeal) growth abundant, reverse slightly yellow [11K3]; nice to good airy mycelium and sporulation; white (W) 13ba and dark gray (Gy) ih; no soluble pigment.

Calcium maleate agar

ISP (34 (inorganic salts - - starch agar)

ISP 65 (glycerolasparagine agar) abundant growth, gray-yellow back [11B2j; nice airy mycelium (Gy) dark gray, 3 ih, light brown soluble pigment.

abundant growth, pale yellow-brown [12 E5], aerial mycelium and spores (W) magenta-white 13ba to (Gy) light gray d, there is no soluble pigment.

abundant growth, reverse light yellow to green [10 Bl], good aerial mycelium and spores (Gyj yellow-gray 2dc to light gray-red 5fe; no soluble pigment).

Agar s

Through the solution solution grow abundant, reverse light yellow-brown [13 H7]; nice to good airy mycelium and spores (Wj white and up to (Gy) medium gray g; very slightly brown soluble pigment.

abundant growth, gray-yellow rab [12 16 j; good aerial mycelium and spores (Y) light yellow 2db; no soluble pigment.

growth abundant, gray-green-yellow reverse (12E2]; abundant aerial mycelium and spores (Gy) yellow-gray 2dc; very faintly brown soluble pigment.

good growth, gray-yellow back [12B2]; aerial mycelium and spores, no color assignment due to poor growth; no soluble pigment.

abundant growth; gray-yellow back [12K3]; sparse air mycelium and spores (Gy) yellow-gray 2dc; no soluble pigment.

good growth; gray-brown back [15C8]; no aerial mycelium or sporulation; brown soluble pigment;

growth poor, no color assignment due to poor growth.

Emerson's growth abundant; reverse gray-yellow agar IH15]; no aerial mycelium or spores; no soluble pigment.

Tyrosine growth abundant; reverse light olive agar brown [14C4]; abundant aerial mycelium and spores from (W) b (center) to (Gy) light gray-gray 3fe (rim; very light brown soluble pigment).

Trypton-grow sparse, no colored when-yeast sorting.

agar

NRRL 5758 organisms were studied to select physiological properties according to standard procedures. The following properties and characteristics were found:

Characteristics

Milk nitrate reduction nutrient gelatin melamine pigment production on: sloping agar styrosine Difco peptone yeast extract sloping agar medium with Trypton and yeast extract temperature requirements (ISP medium β2 yeast extract, malt extract sloping soil)

The results of the carbon utilization tests performed with NRRL 5758 are shown below. The symbols used to assess growth are as follows:

+ good growth, positive use (+) poor to nice growth (-) poor growth, probably no use - no growth, no use carbon source rating

D-glucose +

L-arabinose +

D-xylose +

D-fructose + sucrose -

D-mannitol - i-inositol + rhamnose + raffinosis -

-C control (no carbohydrate) -

The second new A-28086 producing organism was derived from S. aureofaciens NRRL 5758 by a series of natural selection and subsequent chemical mutation. This organism has been identified as NRRL 8092 and is also classified as a Streptomyces aureofaciens Duggar strain. This classification is based on studies of NRRL 8092 using the previously described Streptomyces classification methods under similar growth conditions. The characteristics of NRRL 8092 observed in these studies are summarized in the following paragraphs.

Characterization of A-28086 producing strain NRRL 8092

Morphology

On ISP β7 (tyrosine agar), the culture occasionally produces hooks, but mainly produces short, direct savers. Spore strings are less than 10 spores per string, typically 4 to 7 spores per string. Short straight spore chains were observed in the following media: ISP / 33, agap with Czapka's solution, and ISP (35. Abundant coremia was observed in Emerson agar. , white growth rings, clear spots tan-yellow, pH reaction 5.7 positive% liquefaction in 14 days very slightly positive (pigment after 4 days) negative negative up to 30 ^C C - good growth, 30 to 37 ° C excellent growth and sporulation; 45 ° C slight vegetative growth, red soluble pigment.

gin-agar. The spores are smooth and have a size of 1.2 to 2.0 μ in length and a diameter of about 1.0 μ. The average spore size is 1.6 μ x 1.0 μ.

Culture characteristics of NRRL 8092 on different media

Medium characteristics ISP β2 (yeast extract, molasses extract) nice growth, reverse light yellow-brown (12H8); nice airy mycelium; poor sporulation; airy part light gray [11A1]; no soluble pigment. ISP / 33 (oat flour) growth sparse; reverse crystal; no aerial mycelium; no soluble pigment. ISP, 34 (inorganic salts, starch agar) moderate growth; reverse gray-yellow [11B2J; sparse air mycelium and sporulation; airy part light yellow-gray [10A1]; no soluble pigment. ISP, 35 (glycerol- asparagine agar) moderate growth; reverse light yellow (10F2); nice airy mycelium; sporá sporulace; airy part white [10A1]; no soluble pigment. Tomato paste - oat flour - agar moderate growth; reverse gray-green-yellow; airy mycelium nice; slight sporulation, slightly light gray [53A2]; no soluble pigment. Glycerol- -glycin- -agar abundant growth; gray-yellow [11E4]; slightly airy mycelium ,, white [10A10; no sporulation; no soluble pigment. Glucose- -asparagine agar moderate growth; reverse light yellow [10F21; mild aerial mycelium and sporulation, white [10A1]; no soluble pigment. Nutrient agar growth sparse; reverse light yellow [10B2]; no airwashers-

lium; no soluble pigment.

Medium characteristics

Medium characteristics

Bonnett agar

Calcium maleate agar to grow nice; yellow-pink medium back [11A7]; very sparse aerial mycelium; no sporulations; no soluble pigment.

growth very sparse; reverse crystal, no aerial mycelium; no soluble pigment.

Agar with growth very sparse; reverse crystal; no aerial mycelium; solution no soluble pigment.

Emerson agar growth moderate; gray-yellow back [1115]; air mycelium spots; no sporulations; no soluble pigment.

Tyrosine growth moderate; reverse light yellow-brown [12H6]; mild aerial mycelium; light slightly gray edges [53A2], center almost white and slight sporulation; no soluble pigment.

Trypton growth very scant; reverse crystal-yeast; no aerial mycelium;

agar no soluble pigment.

NRRL 8092 has also been studied to select physiological properties according to standard procedures. The following properties and characteristics were found:

Milk nitrate reduction nutrient gelatin melamine pigment production on: sloping agar with tyrosine medium with tryptone and yeast wedge extract from carrot wedge of potatoes

Temperature requirements (ISP medium β2 yeast extract, malt extract, sloping soil)

Characteristics milk peptones (90%) pale yellow growth rings, clear spots, pale yellow, pH reaction 4.6 positive

50% hydrolysis in 14 days very weak positive negative abundant growth, pale yellow, no aerial mycelium abundant growth, off-white, no aerial mycelium; no changes on the wedge 25 ° C - abundant growth; nice airy mycelium; reverse light brown; no soluble pigment ° C - abundant growth; nice airy mycelium; reverse light brown; no soluble pigment ° C - abundant growth; nice airy mycelium; reverse brown; soluble brown pigment 40 ° C - abundant growth; sparse air mycelium; rub red-brown; soluble pigment dark red-brown ° C - nice growth; no aerial mycelium; rub red-brown; slight reddish-brown pigment.

The results of the carbon utilization tests performed by the carbon source evaluation with NRRL 8092 are shown below. The symbols used to assess the growth of D-glucose + are as follows: L-arabinose +

D-xylose + + good growth, positive use D-fructose + (+] poor to nice growth sucrose - [-) poor growth, probably no you- D-mannitol - use i-inositol + - no growth, no use rhamnose + raffinosis -

-C control (no carbohydrate)

Some characteristics of S. aureo- ling and Gottlieb strains. These differences are summaries of the A-28086 producing products differing from those required in Table III. characteristics of the organism described SchirTABLE III

Carbon utilization NRRL 5758 NRRL 8092 published description sucrose i-inositol rhamnzza liquefaction gelatin effect on milk +

-t30% within 14 days milk peptones, white growth rings +

+% within 14 days milk peptones pale yellow growth rings limited or no limited and variable peptonation (often? no), limited growth and coagulation

The characteristics of NRRL 5758 and NRRL 8092 are summarized in Table IV. which are different from the characteristics of orgaTABLE IV

Characteristics

NRRL 5758

NRRL 8092 vegetative color sporulation grow on:

calcium maleate inorganic salts with starch

Benett's agar yellow on some media some spiral sporophores on tomato paste with oatmeal and on inorganic salts with starch grow nice, brown with ripples slight sporulation; airy mauve to gray reverse light yellow creamy to light yellow on some media short direct sporophores with possible hooks grow sparse clear, no conjunction sparse sporulation, airy part light yellow-gray, reverse pink light yellow-gray reverse pink

Streptomyces aureofaciens cultures used to produce A-28086 antibiotics were deposited as part of the Northern Marketing ang Nutrition Research Division, U. S. Dept. of Agriculture, Agricultural Research Service, Peoria, Illinois, 61,604, where they are publicly available under NRRL 5758 and NRRL 8092.

SUMMARY OF THE INVENTION The present invention provides a process for the preparation of complex-forming polyetheric antibiotics A-28086 consisting of Factor A, Factor B and Factor D, acyl esters of Factors A and D containing 2 to 6 carbon atoms in acyl or physiologically acceptable salts thereof. , A-28086 Factor B, A-28086 Factor D, and A-28086 antibiotic complex, characterized by: • culturing Streptomyces aureofaciens NRRL 5758 or Streptomyces aureofaciens NRRL 8092 in a nutrient medium containing assimilable sources of carbohydrates, nitrogen, and inorganic salts; submerged aerobic fermentation conditions at a temperature of 20 to 40 ^° C and a pH of 6.0 to 8.0 until sufficient antibiotic activity is produced by the organism in the nutrient medium and optionally separates the A-28086 antibiotic complex from the nutrient medium, optionally, factors A, B, or? are separated from the A-28086 antibiotic complex? D and then optionally converting these factors A, B or D into acyl derivatives or pharmaceutically acceptable salts.

Nutrient medium used for? the growth of Streptomyces aureofaciens NRRL 5758 or Streptomyces aurefaciens NRRL 8092 can be any of a variety of media. However, certain nutrient media are preferred for economical production, optimum yield and easy product isolation. For example, tapioca dextrin and sucrose are preferred sources of hydrocarbons for large-scale fermentation, although glucose, corn starch, fructose, mannose, maltose, lactose and the like can also be used. Corn oil, peanut oil, soybean oil and fish oil are other suitable carbon sources. A preferred carbon source is casein hydrolyzed by the enzyme, although peptones, soy flour, cottonseed flour, amino acids such as gluitamic acid and the like can also be used as nutrient inorganic salts which may be added to the nutrient medium to include soluble salts providing sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate, nitrate, etc.

The major trace elements necessary for the growth and development of the organism must also be added to the nutrient medium. These trace elements commonly occur as impurities in other media components in an amount sufficient to meet the growth requirements of the body.

It may be necessary to add small amounts, i.e. 0.2 ml / liter, of an antifoam such as polypropylene glycol to large-scale fermentation media where foaming becomes a problem.

Although not critical, the production of antibiotic strains of Streptomyces aureofaciens producing A-28086 is increased by the addition of a small amount of oil, such as soybean oil.

Submerged aerobic fermentation in tanks is preferred for the production of sufficient A-28086 antibiotics. Small amounts of A-28086 antibiotics can be obtained from shake flask cultures. Due to the time delay common to antibiotic production by inoculating large tanks with spores, the use of vegetative stitches is preferred. Preferably, the vegetative seed is prepared by inoculating a small amount of nutrient medium with spores or mycelium of the organism and obtaining a fresh, actively growing culture of the organism. The vegetative loop is then transferred to a large tank. The medium used for vegetative seed growth may be the same as that used for large fermentations, but other media may also be used.

The A-28086 producing organism can be grown at a temperature between about 20 and 40 ° C. Optimal A-28086 production takes place at about 27-30 ° C.

In aerobic submerged culture procedures, it is preferred to introduce sterile air into the nutrient medium. For efficient growth of the organism, the volume of air used in production in the tank is preferably greater than 0.1 volume of air per volume of nutrient medium per minute. For efficient production of A-28086 antibiotics, the volume of air produced in the tank is preferably greater than 0.25 volume of air per volume of nutrient medium per minute. High levels of dissolved oxygen do not reduce antibiotic production.

Antibiotic production can be monitored during fermentation by testing samples of nutrient medium or extracts of mycelium solids for antibiotic activity against known antibiotic-sensitive organisms. One of the test organisms used in the testing of the antibiotics of the invention is Bacillus butilis ATCC 6633. The biological tests are preferably performed using paper discs on agar plates.

The initial pH of the inoculated nutrient medium is within the range of the medium used. In general, the pH should be in the range of 6.0 to 7.5. The pH at the end of the fermentation is usually somewhat higher, in the range of 6.5 to 8.0.

Generally, antibiotic activity is detectable on the second day after fermentation. Maximum production of antibiotic activity usually occurs on days 6 to 10.

For production under submerged aerobic conditions, the A-28086 antibiotics described above can be isolated from the fermentation medium in a manner commonly used in fermentations. Antibiotic activity produced during fermentation of A-28086 producing organisms occurs both in the mycelium mass and in the filtered medium. Maximum isolation of A-28086 antibiotics can be achieved by combining the methods used including filtration, extraction and adsorption chromatography. A preferred solvent for isolating A-28086 antibiotics from both whole and filtered fermentation broth ^; and is ethyl acetate, although the other commonly used solvents are sufficient.

A particularly preferred method for isolating A-28086 factors A, B, and D is to lower the pH of the entire fermentation medium to about pH 3.0. At this pH of 3.0, A-28086 factors A, B, and D are well separated from the mycelium mass by filtration. Another preferred method of isolating A-28086 factors is to add bicarbonate per se, for example sodium bicarbonate, to the total nutrient medium in an amount of about 1 g per liter. The A-28086 factors are thereby preferably separated from the mycelium mass in the form of a salt. Methanol is preferably used to separate the antibiotics from the mycelium mass, but other lower alcohols and ketones are also suitable.

In the isolation of the A-28086 antibiotics, azeotropic distillation may also be advantageously used. In this method, an organic solvent is added to the aqueous fermentation medium which forms the appropriate azeotrope with water. This solvent-medium mixture is subjected to azeotropic distillation to remove at least half of the water from the medium leaving a water-solvent mixture in which the A-28086 antibiotics are in solution in an organic solvent. Insoluble byproducts can be removed by a suitable method, such as filtration or filtration. The A-28086 antibiotics can be isolated from the organic solution by a conventional method, such as evaporation of the solvent, precipitation by addition of a non-solvent, or extraction.

The organic solvents that form the appropriate azeotropic mixtures with water for carrying out the process are butyl alcohol, amyl alcohol, hexyl alcohol, benzyl alcohol, butyl acetate, amyl acetate, 1,2-dichloroethane, 3-pentanone, 2-hexanone, benzene, cyclohexanone, toluene, xylenes and the like.

Isolation by azeotropic distillation is particularly advantageous in the case of large fermentation processes. Both the water and the solvent collected during azeotropic distillation can be separated in a conventional manner and then recycled for further use. The water thus separated is not contaminated and does not require waste water treatment. The solvent thus separated can be recycled to the process.

Further purification of the A-28086 antibiotic involves additional extraction and adsorption processes. As adsorbent materials, silica gel, coal, magnesium silicate (Florisil), and the like can be used.

Alternatively, culture solids, including media and mycelium components, can be used as the source of the A-28086 antibiotics without extraction and separation, but preferably after separation of water. For example, after producing the antibiotic activity of A-28086, the nutrient medium can be freeze dried and mixed directly into the premix of food.

According to another feature, after production of A-28086 activity in the nutrient medium, the mycelium can be separated and dried to give a product which can be directly added to the premix. Once the mycelium has been distilled off for such use, a better dry product is obtained by adding calcium carbonate [about 10 g / l] as filter aid.

Under the conditions previously used, the previously described strains of Streptomyces aureofaclens and referred to as NRRL 5758 and NRRL 8092 produce the antibiotic A-28086 factor A as a predominant factor. Although the ratio of the factors varies depending on the fermentation conditions used, the amount of factor A is generally greater than 99% of the total isolated antibiotic activity in producing NRRL 5758 strain and more than 90% of the total antibiotic activity in producing NRRL 8092 strain. A-28086 factor B forms almost all of the remaining antibiotic activity in strain NRRL 5758 and factor D is a minor factor. In contrast, A-28086 factor D accounts for about 10 percent of total isolated antibiotic activity in strain NRRL 8092 and factor B is a minor factor.

Antibiotics A-28086 factors A, B, and D are separate compounds from each other and are isolated as individual compounds using commonly known methods such as column chromatography, thin layer chromatography, etc. For example, silica gel chromatography is used to separate factors A, B and D eluting the column with various mixtures of solvents, such as a mixture of benzene and ethyl acetate. Using a mixture of benzene and ethyl acetate, factor B is first eluted from the silica gel column and later factors A and D are eluted later. Thin layer chromatography as described above is a preferred method for monitoring the separation process.

The A-28086 compounds inhibit the growth of bacteria and fungi that are pathogenic in animals and plants. The relative microbial activity of A-28086 factors A and B is described below in Table V.

The disc diffusion method is preferably used as the test method (6 mm inserts are soaked in a solution containing 1 mg of compound / ml solution; the inserts are placed on agar plates inoculated with organism).

TABLE V Test organism

Staphylococcus aureus

Bacillus subtilis ATCC 6633

Sarcina lutea ATCC 9341

Mycobacterlum avium ATCC 7992 - Saccharomyces pastorianum ATCC 2360 - Candida tropicalis

Fusarium moniliforme zone of inhibition (mm)

A 28086 factor A A 28086 factor B

19 Dec 21 28 31 26 16 12 12 17 - 14 14 12 netestováno

-O

According to another important feature, compounds A-28086 inhibit the growth of auerobic bacteria. The minimum inhibitory concentrations (MIC) at which A-28086 factor A inhibits various anerobic bacteria, as determined by a standard agar diffusion assay, are summarized in Table VI. The inhibitory concentration is read after 24 hours of incubation.

Bacteroides fragilis ssp. Fragilis 8.0

Bacteroides fragilis ssp.

thetaiotacmicron 8.0

Bacteroides fragilis ssp. Vulkagis 1563 8.0 Bacteroides fragilis ssp. VUlgatis 11211 2.0 Fusobacterium symbiosum 0.5

Fusobacterium necrophorum 8.0

Veillonella alcalescens 16.0

TABLE VI Tested organism MIC (jug / ml) Actinomyces bovis <0.5 Clostridium inocuum <0.5 Clostridium perfringens <0.5 Clostridium ramosum 4.0 Clostridium septicum <0.5 Clostridium septicum bovine <0.5 Eubacterium aerofaclens 1.0 Peptococcus anaerobius <0.5 Peptostreptococcus intermedius 16.0 Propionibacterium acnes 44 16.0 Propionibacterium acnes 2,0

Acyl esters of A-28086 factor A having 2 to 6 carbon atoms in the acyl also have antibacterial and fungicidal activity. In one aspect, these compounds have increased activity per gram-positive bacteria. The relative grarn-positive activities of certain of these derivatives were compared to that of factor A. The compounds were tested by a turbidometric method on a semi-automatic system (Autoturt microbiological assay system,

Elancoj, described by N. R. Kužel and F. W. Kavanaugh in J. Pharmaceut. Sci. 60 (5), 764 and 767 (1971).

The following test parameters were used to test A-28086 antibiotics after 209848. Staphyloeoccus aureus (H-Heatley® NRRL B-314 in nutrient medium (pH 7), incubation for 4 hours at 37 ° C. Test samples and standard are dissolved in methanol: water (10:90). Standard A-28086 factor A was present in the Autoturb ® conveyor in concentration

2, 3, 4, 5 mcg / ml. The test compound was diluted to give an activity of 3 or 4 mcg / ml. The relative activities of the test compounds compared to the standard are as follows:

Relative G ⁺

Compound activity

A 28086 factor A (standard) 1

A 28086-A acetyl ester 2.5

A 28086-A propionyl ester 7

A 28086-A n-butyl ester 8

A 28086-A n-valeryl ester 14

A 28086-A n-Kaproylester 20

Another important feature of the antimicrobial activity of compounds A-28086 is activity against Mycoplasma species. Mycoplasma species, also known as pleuropneumonia-like organisms (PPLO), are pathogenic in humans and various animals. Agents active against PPLO organisms are particularly desirable in poultry farming. Minimum Inhibitory Concentration (MIC) of the A-28086 factor A antibiotic against various Mycoplasma species, determined by in vitro dilution assay. These are summarized in Table VII.

TABLE VII organism MIC (mcg / ml)

M. gallisepticum 12.5 M. hyorhinis 12.5 M. synoviae 6.25 M. hypopneumoniae 6.25 M. hyosynoviae 6.25

Solutions Containing about 10 ml programs of the antibiotic Α-2808Θ per ml of solution are useful for surface disinfection to protect the algae from various Mycoplasma species.

Compounds A-28086 are also antiviral agents. A-28086 factor A is active against type III pollavirus, vakeinia virus, herpes virus, and Semliki Forest virus, as shown by the in vitro assay described by Siminoff Applied Mierobiofog 9 (1J 66-72 (1961). A-28086 factor A is also active against Transmissible Gastro-enteritis virus, Newcastle Disease virus, and Infectious Bovine Rhinotracheitis virus as demonstrated by similar tissue culture assays.

Thus, in one aspect, the A-28086 compounds can be administered orally, topically, or parenterally to an animal to achieve virus control. A useful dosage level for the prevention or treatment of viral diseases ranges from about 1 to about 5 mg / kg animal body weight, depending on the virus and whether the drug has been administered by the prophylactics or therapeutically.

Further, solutions containing the A-28086 compound, preferably with a surfactant, may be used to decantaminate in vitro sites where viruses such as polio or herpes may occur. Solutions containing from about 1 to about 1500 mcg / ml of the A-28086 compound are effective in controlling viruses.

The acute toxicity of A-28086 factor A antibiotics administered intraperitoneally to mice, expressed as · LD 50, is 7.15 mg / kg.

Compounds A-28086 are also insecticides and acaricides. The A-28086 compounds are active against insects such as Epilachna varivestis, Oncopeltus fasciatus, housefly and mites such as the worm whale when applied at about 500 ppm. In addition, the A-28086 compounds are effective against mosquito larvae in an amount of 1 ppm.

Anticoccidial activity is an important property of A-28086 compounds. For example, feeding experiments show that A-28086 compounds present in the diet of young chickens in an amount of 0.003% improve weight gain, 0.005% preventively prevent death and reduce injuries in chickens that may be caused by coccidia. The results of factor A tests in chickens caused by different Eimeria species are summarized in Tables VIII to X.

TABLE VIII

Activity of antibiotic A-28086 factor A against Eimeria Tenella.

amount in diet weight percent number chickens mortality weight percent increment average percentage rate injury reduction in injury rate 0.04 10 0 50 0 100 ALIGN! 0.02 10 0 82 0.2 95 0.01 10 0 94 0.2 95 infected control 20 May 35 68 3.65 - normal control 20 May 0 100 ALIGN! - - 0.2 20 May 0 84 0 100 ALIGN! 0.01 20 May 0 96 0 100 ALIGN! 0.005 20 May 0 91 2.8 27 Mar: 0,0025 20 May 10 87 3.85 0 infected control 20 May 40 74 3.85 - normal control 20 May 0 100 ALIGN! - - TABLE IX Activity of antibiotic A-28086 factor A against various Eimeria species ¹ infecting amount percent percent average percent Oocysts Expired Organism antibioti- morta- hm- rate in reduction total / percent ka ferocious nitnífoo injury injury /bird reduction in the diet increment (i x ιοη % by weight- security Eimeria acervulina 0.005 0 87 0.75 53 6.15 83 0,0025 0 82 1,25 - 4.91 86 infected controls - 0 63 1.60 - 36.32 - Eimeria maxima 0.005 0 85 0.2 85 1.95 40 infected controls -, 0 65 1.3 - 3.24 - Eimeria brunstti 0.005 0 88 1,2 - 2.31 58 infected controls - 0 62 1.7 - 5.55 -

¹ four times, 5 taps each

TABLE X

Activity of antibiotic A-28086 factor A against Eimeria species ¹

Infectious organism Antibiotic mass percentages by weight

Percent Percent Intestimortality Weight Gain Growth Injury Average Percentage Average Cecal Reduction Injury Percentage Reduction

E. necatrix a E. tenella 0.005 0 94 0.7 59 1.75 50 infected controls E. tenella, E. necatrix, E. maxima, 20 May 52 1.7 3.5 E. brunetti, E. acervulina a E. mivati 0.01 0 91 0.4 79 ' 0.9 73 infected controls - 50 12 1.9 - 3.3 - ¹ four times, 5 taps in every attempt.

For prevention or treatment of coccidiosis in poultry, a non-toxic anti-coccidial amount of Compound A-28086 is administered to the birds, preferably orally on a daily basis. Compound A-28086 can be administered in a variety of ways, preferably in a physiologically acceptable composition, preferably with food. Although a number of factors have to be taken into account in determining the appropriate amount, the applied amount of Compound A-28086 generally ranges from 0.003 to 0.04 percent by weight of the diet, preferably from 0.005 to 0.02 percent.

The ability to improve food utility in animals is another important feature of compounds A-28086. For example, compounds A-28086 improve food utility in ruminants that have developed a ruminant stomach system.

as mentioned above, the utility of carbohydrates in ruminants increases when the gastric flora of ruminants produces propionates rather than acetates or butyrates. Food utility can be monitored by determining the production and concentration of propionates in the stomach of ruminants as follows:

The gastric fluid is recovered from the ox by a surgically inserted tube into the stomach. The ox is kept well fed. The gastric fluid sample is passed through four layers of cheese pressing material and the filtrate is collected. The particles remaining on the substance are resuspended in a sufficient amount of physiological buffer corresponding to the volume of the original liquid from the stomach, and this suspension is resuspended. The buffer used is as follows:

Additives g / liter

NaaHPCU 0.316 KH2PO4 0.152 NaHCCb 2,260 KC1 0.375 NaCl 0.375 MgSCU 0,112 CaCl2.2 H2O 0.050 FeSCU.7 H2O 0.008 MnSCU. H2O 0.004 ZnSCU.7 H2O 0.004 CuSCU · 5 H2O 0,002 OoCl2.6 H2O 0.001

as described by Cheng et al. in J. Dairy Sci. 38, 1225-1230 (1955).

The two filtrates were combined and allowed to stand until the particulate matter was collected on top. The clear phase was separated, diluted with the same buffer (1: 1) and then adjusted to pH 6.8 to 7.0.

Dilute gastric fluid (10 ml) is placed in a 25 ml flask containing 40 mg of the above food and 1 mg of soy protein is added and the compounds tested. Four flasks are used for each experiment. In addition, two groups of four control flasks were also used. Control is used at 0 time and control after incubation for 16 hours. All test flasks were incubated at 38 ° C for 16 hours. After incubation, the pH is measured and 2 ml of 25% metaphosphoric acid is added to each flask. The samples were allowed to settle and the supernatant was analyzed by gas chromatography for propionate, acetate and butyrate. The active compounds significantly increase propionate production over controls.

*

The test results are statistically compared with the results of the controls. Table XI shows the ratio of volatile fatty acid concentration in test flasks to that in control flasks.

TABLE XI

The ratio of treated to control mole% mole% total VFA propionate butyrate mM / L

Compound ppm molar% in gastric acetate liquid ruminant

A-28086 factor A 1 0.94 0.87 1.34 1.26 A-28086 factor B 1 0.97 0.96 1.15 1.29 A-28086 factor A 1 0.79 0.76 2.04 1.04 acetylester A-28086 factor A 1 0.90 0.68 1.73 1,25 propionylester A-28086 factor A 1 0.91 0.81 1.56 1.19 butyryl ester A-28086 factor A 1 0.92 0.79 1.55 1.18 valerylester A-28086 factor A 1 0.86 0.97 1.57 0.99

kaproylester

The efficacy of carbohydrate utilization is further demonstrated by in vivo tests carried out in animals having a tube inserted into the stomach such that a sample of the ruminant stomach content can be removed.

The tests shown in Table XII were performed with adult oxen weighing about 45b kg. Two steers were fed normal food and four steers were fed the same food to which A-28086 Factor A was added. The samples were allowed to settle and the supernatant was analyzed by gas chromatography to determine the propionic acid concentration.

The results in Table XII show the average percentage increase in the concentration of propionic acid in the gastric fluid from five analyzes over a 14-day test period. The controls had approximately 20 mole percent propionic acid.

TABLE XII

A-28086 factor A Percentage increase (mg / dj vs control)

17 _r 4

100 54.0

In addition, in vivo tests have shown that A-28086 factor A increases the utility of food in sheep. The results of these tests performed over 56 days are summarized in Table XIII.

TABLE XIII

A-28086 factor A (g) per ton of food Number of animals Average daily increment (0.45 kg / day) Food intake (0.45 kg / day) Food / Feed growth Percent· improvement 9 13 0.44 3.29 7.40 12 18 13 0.38 3.15 8.31 1 control 17 0.42 3.56 8.38 -

Compound A-28086 factor D are antiviral agents. A-28086 factor D is active against Maryland B virus, type III poliovirus, COE virus, herpes virus, and Semliki Forest virus, as demonstrated by in vitro assays described by Siminoff Applied Microbiology 9 (1), 66-72, (1961). A-28086 factor D is also active against Transmissible Gastroenteritis virus Newcastle Disease virus and Infection Bovine Rhinotracheitis virus, as demonstrated by tissue culture assays.

The fact that the A-28086 Factor D compounds are active against both viruses and anerobic bacteria makes these compounds particularly useful for treating or preventing enteritis in chickens, pigs and cattle and sheep. The A-28086 factor D compounds are also useful for treating enterotoxemia in ruminants.

Anticoccidial activity is an important property of A-28086 Factor D of the compounds of the invention. In vitro experiments showed the anti-coccidial activity of A-28086 factor D against Eimeria tenella. The following method was used (for more details see L. R. McDougald and R. B. Galloway in Exper. Parasitology 34, 189-196 [1973]).

Host cell cultures were prepared in conventional manner using Leighton tubes, plastic dishes or plates or other suitable culture containers. After two to three days growth in a suitable nutrient medium (Eagle's minimal base medium, lactalbumin hydrolyzate in Earle's or Hank's balanced salt solution, medium 199, etc.), a suitable monolayer is usually formed which is suitable for infection and drug testing. Chicken kidney cells are used as primary culture for these studies.

Viable Eimerie tenella oocytes are obtained from faeces of infected chickens and cleaned and sterilized before use. The oocytes are sporulated by incubation at room temperature and bubbling air through suspension or gentle shaking in a shaker or other manner. Potassium dichromate or sulfuric acid (or other chemical) is used to prevent bacterial or fungal growth during sporulaoe.

Excystation of infected sporozoites from oocytes is accomplished by a combination of mechanical breakdown of the oocyte wall and then by the action of trypsin and bile acid salts, induction of sporozites from the spores is induced.

Cell cultures are infected by introducing viable sporozoites into a culture vessel. Test chemical compounds are introduced simultaneously or in succession at the desired concentration at 10 o / _o of dimethylformamide in physiological saline. The end of the assay is the inhibition of asexual developmental stages and is determined by microscopic examination of fixed cultures and color 95 hours post infection. Results are reported as active (A), inactive (N) or cytotoxic (C) according to the presence or absence of second-generation schizons 38 and possible toxicity to the cell monolayer.

The anti-coccidial activity of A-28086 factor D demonstrated by this assay is summarized in Table VII.

TABLE VII

Anticoccidial activity of antibiotic A-28086 factor D

Concentration of A-28086 factor D (mcg / ml) Evaluation 5 C 1 C 0.2 C 0.4 A, C ’ 0.03 AND 0.02 AND 0.01 ± A 0.008 N * results of two tests. Another useful feature of A-28086 Factor D compounds is their ability to improve food utility in ruminants with developed ruminant stomach. Mechanism for use of main nutrient carbohydrate parts ruminant grass is well known. Micro- organisms in the stomach of ruminants degrade

saccharides to monosaccharides and then convert these monosaccharides to pyruvate compounds. Pyruvates are metabolized by microbial processes to acetates, butyrates or propionates, collectively known as volatile fatty acids (VFAs). For further discussion, see Leng, "Physiology of Digestion and Metabolism in the Ruminant," Phillipson et al., Eds., Oriel Press, Newcastle upon Tyne, England, 1970, pp. 408-410.

The relative effectiveness of the use of volatile fatty acids is discussed in McCullough in Feedstuffs, 19th VI. 1971, p 19; Eskeland et al., J. An. Sci. 33, 282 (1971); Ghurch et al., "Digestive Physiology and Nutrition of Ruminants", Vol. 2, 1971, pp. 622 and 625. Although acetates and butyrates are also used, propionates are used with greater effect. Furthermore, when only a small amount of propionate is available, ketosis develops in animals. Thus, the advantage of the compounds of the invention is to stimulate the production of a higher proportion of propionates from carbohydrates, thereby improving carbohydrate utilization and also reducing ketosis cases.

The activity of these suitable compounds can be determined by monitoring the effect on production and concentration of propionate compounds in ruminants using the same methods as for factor A above.

The test results are statistically comparable to those of the controls. Table VIII shows the ratio of volatile fatty acid concentration in flasks treated with A-28086 factor D to that of control flasks.

TABULKAVIII

A-28086 D mcg / ml Molar% 'Molar% Molar% Total VFA propionate butyrate acetate mM / l 7θ 0.8715 0.7973 1.7981 1.1979

0.3 0.8976 0.8726 1.5871 1.0630

The compounds are typically effective in increasing propionate and thus the utility is higher at ruminant doses ranging from 0.05 mg / kg / day to about 5.0 mg / kg / day. Most preferred results are obtained when an amount of from 0.1 mg / kg / day to about 2.5 mg / kg / day is applied. A preferred method of administering the compounds is admixture with the animal's diet, but may be administered by other means, for example in the form of tablets, drinks, pills or capsules. The formulation of these different dosage forms is carried out by conventional methods used in veterinary practice. Each individual dosage unit form may contain a quantity of compounds directly related to the respective daily dose of the animal.

Compounds A-28086 can be used in feed mixtures adapted for cattle fattening, which include feed and from 1 to 30 grams of compound per tonne.

As is known, cattle feed is different from other feeds, such as poultry feed. Cattle feed contains coarse forage, such as cotton seed husks, and corn silage. In addition, cattle feed contains a large percentage of a portion of from 15 to 20 percent of non-caloric nitrogen sources. A common non-protein source is urea.

As described above, the compounds are antiviral agents and are also effective against aerobic bacteria, particularly Clostridlum perfringens. Compounds A-28086 are therefore useful for the treatment and prevention of enteritis in chickens, pigs and cattle and sheep. Compounds A-28086 are also useful for the treatment of enterotoxemia in ruminants.

The invention is illustrated by the following examples.

Example 1

A. Fermentation of A-28086 in shake flasks using S. Aureofaciens NRRL 5758

A culture of Streptomyces aureofaciens NRRL 5758 is prepared and maintained on sloping agar media of the following composition:

Ingredient Quantity agar 20 g dextrin 10 g enzyme hydrolyzed casein 2 g beef extract 2 g yeast extract 2 g distilled science to 1 liter

The slope was inoculated with Streptomyces aureofociens NRRL 5758 and the inoculated slope was incubated at 30 ° C for six to ten days. The mature slant culture is overlaid with bovine serum and scraped with a sterile loop to release spores. The resulting suspension of spores and mycelial fragments in bovine serum is lyophilized into six pills.

One pill thus prepared is used to inoculate 50 ml of vegetative medium of the following composition:

Ingredient Quantity of soya bran 15 g corn steep liquor 10 g

CaCCte 2 g water to 1 liter

The inoculated vegetative medium in a 250 ml Erlenbeyer flask was incubated for 72 hours at 30 ° C on a shaker with a 5 cm shift at 250 rpm.

B. Fermentation of A-28086 in a tank using S. aureofaciens NRRL 5758

To obtain a larger seed volume, 10 ml of the incubated vegetation medium described above is used to inoculate 400 ml of the second stage vegetative growth medium having the same composition as the vegetative medium. This second stage medium in a 21 liter flask is incubated for 24 hours at 30 ° C on a 5 cm shaker at 250 rpm.

The second stage vegetative medium (1 liter) is used to inoculate 100 liters of sterile production medium and the following composition:

Component Quantity

Tapioca dextrin 60.0 g / 1 enzyme hydrolyzed casein 8.0 g / 1 molasses 15.0 g / 1

MgSO 3. 7 H2O 0.5 g / l

CaCO3 2.0 g / l purified soybean oil 5.0 g / l deionized water up to · 1 liter

After sterilization, the medium was autoclaved for 30 minutes at 120 ° C and a pressure of 0.103 MPa to 1.373 MPa, pH 6.7. In a 1651 liter fermentation tank, the incubated production medium is allowed to ferment at 29 ° C for 10 days. The fermentation medium is aerated with sterile air at a rate of 0.4 volumes of air per volume of nutrient medium per minute. The medium is agitated with a conventional stirrer at 250 rpm.

Example 1 a d 2

A-28086 antibiotics are produced by the procedure of Example 1, but the production medium for flasks is as follows:

Component Quantity

glucose 10 g / 1 molasses 20 May g / 1 peptone 5 g / 1 CaCOs 2 g / 1 water to 1 liter

Example 3

Isolation of A-28086 complex of antibiotics produced by S. aureofaciens NRRL 5758

The entire fermentation medium (132 L) obtained by the method described in Example 1 is filtered through a filter aid (Hyflo Super-cel, diatomaceous earth), yielding 97 liters of filtered medium. The filtered medium is extracted with about an equal volume of ethyl acetate. The ethyl acetate extract is separated from the aqueous phase and then concentrated to a volume of about 500 ml.` This concentrated thickened ethyl acetate extract is added to a large excess of petroleum ether (Skelly Solve F, about 10 L) to precipitate the undesirable material. The separated filtrate was evaporated in vacuo to give a portion of A-28086 antibiotic complex (6.9 g) present in the medium.

Part of the A-28086 antibiotic complex contained in the mycelium is obtained by extracting twice the filtered, mycelium by about half the volume of methanol (62 L and 59 L). The two methanolic extracts were combined and concentrated in vacuo to remove methanol. After this concentration, about 10 liters of aqueous phase remains. This aqueous phase is adjusted to pH 7.5 with dilute aqueous sodium hydroxide solution. The resulting solution was extracted twice with an equal volume of ethyl acetate (9 L and 10 L).

The ethyl acetate extracts were combined and then concentrated to a volume of about 400 ml. The concentrated ethyl acetate extract is added to a large excess of petroleum ether and the undesirable materials are removed as described above for the concentrated extract from the medium. A portion of the A-28086 antibiotic complex present in the mycelium weighing 20.0 g was recovered from the filtrate.

Example 1 a d 4

Isolation of A-28086 individual factors A and B

A portion of the A-28086 antibiotic complex present in the mycelium (235 g, prepared as described in Example 3) was dissolved in 80 mL of benzene. This benzene solution was applied to a silica gel column (9 x 130 cm, 8 L, Matheson Grade 62 silica gel). The column is eluted with various ratios of benzene-ethyl acetate mixtures. Elution was monitored by thin layer chromatography. The benzene-ethyl acetate (90: 10) mixture was first eluted with factor B and isolated as an individual factor. Factor B (43 mg) was crystallized from acetone-water and had a melting point of 150-153 ^° C.

Further elution of the benzene-ethyl acetate mixture, with an increasing ratio of ethyl acetate present in the mixture, elutes factor A; the Factor A containing fractions were combined and concentrated in vacuo. The residue was dissolved in acetone (about 150 mL) and water (about 150 mL) was added.

The resulting mixture was adjusted to pH about 3 by addition of 1 N hydrochloric acid. The acidified mixture was stirred for about one hour, during which time a precipitate formed. This precipitate was filtered off and recrystallized from acetone (about 150 ml) after addition of water (about 60 ml). The product was dried under vacuum overnight to give Factor A (about 8.8 g). Partial evaporation of the acetone from the filtrate yielded a second crop of Factor A (about 1.2 g).

Example 1 a d 5

A-28086 - factor A acetylester

The antibiotic A-28086 factor A (7.4 g) was dissolved in pyridine (150 mL). To this solution was added acetic anhydride (50 mL). The resulting solution was thoroughly mixed and then allowed to stand overnight at room temperature.

Water (200 ml) was then added with stirring and the mixture was allowed to stand at room temperature for 4 hours. A white solid precipitates, which is filtered off, washed with water and air-dried. The resulting solid was dissolved in acetone (100 mL) and the acetone solution was evaporated to dryness in vacuo (this operation was repeated three times). The obtained residue was crystallized from a mixture of acetone (100 ml) and water (50 ml) to give A-28086 factor A acetylester (6.14 g), m.p. 100-103 ° C.

269848

Examples 6 to 9

The antibiotic A-28086 factor A propionyl ester is prepared by reacting factor A with propionic anhydride in the presence of pyridine, following the procedure of Example 5. The product has a melting point of 96-98 ° C.

The antibiotic A-28086 factor A n-butyl ester is prepared by reacting factor A with n-butyric anhydride in the presence of pyridine, according to the method of Example 5. The product has a melting point of 79-81 ° C.

The antibiotic A-28086 factor A n-caproyl ester was prepared by reacting factor A with n-caproic anhydride according to the procedure of Example 5. The product had a melting point of 163-167 ° C.

The antibiotic A-28086 factor A n-valeryl ester was prepared by reacting factor A with valeric anhydride in the presence of pyridine according to the procedure of Example 5. The product had a melting point of 173-175 ° C.

Example 10

Preparation of A-28086 Factor A Sodium

The antibiotic A-28086 factor A (500 mg) was dissolved in acetone (50 mL). To this solution was added water (50 mL) and the pH of the solution was adjusted to 10.5-11 with 5N sodium hydroxide solution. The resulting solution was stirred for one hour and then extracted with ethyl acetate. The ethyl acetate extract was evaporated to dryness in vacuo. The residue was precipitated from acetone-water solution to give 378 mg of A-28086 Factor A sodium salt, mp 120-123 ° C.

Examples 11 to 15

A-28086 factor A barium salt was prepared from A-28086 factor A antibiotic (500 mg) and saturated with barium hydroxide using the method of Example 10. 369 mg of A-28086 factor A barium salt was obtained, m.p. 188-190 °. C.

Potassium salt of A-28086 factor A was prepared from A-28086 factor A (500 mg) and 5 N potassium hydroxide using the method of Example 10. 333 µg of potassium A-28086 factor A was obtained, m.p. C.

A-28086 factor A cesium salt was prepared from A-28086 factor A (500 mg) and 1N cesium hydroxide using the procedure of Example 10 to give 540 mg of A-28086 factor A cesium salt, m.p. 190-210 ° C. .

Sodium salt of antibiotic A-28086 factor B and 5 N sodium hydroxide as described in Example 10.

Example 16

Fermentation of A-28086 in shake flasks using S. aureofaciens NRRL 8092

A culture of Streptomyces aureofaciens NRRL 8092 is prepared and maintained on sloping agar of the following composition:

Component Quantity

K2HPO4 2 G MgSO 3. 7 H2O 0.25 G NH4NO3 2 G CaCO3 2.5 G FeSCU.7 H2O 0.001 G MnCl2. 7 H2O 0.001 G ZnSOá. 7 HzO 0.001 G glucose 10 G agar 20 May G deionized water up to 1 liter pH (untreated) 7.7

The slope was inoculated with Streptomyces aureofaciens NRRL 8092 and the inoculated slope was incubated for seven days at 30 ° C. The mature slant culture is overlaid with sterile bovine serum and scraped with a sterile loop to prepare a spore and mycelium suspension from the slant culture. The resulting suspension is lyophilized to a maximum of six tablets.

One lyophilized tablet prepared in this way is used to inoculate 50 ml of the vegetative medium of the following composition:

Ingredient 'Amount of glycose 20 g soya flour 15 g corn steep liquor 10 g

CaCO3 2 g water to 1 liter pH adjusted to pH 6.5 with dilute NaOH

The inoculated vegetative medium in a 250 ml Erlenmeyer flask was incubated at 30 ° C for 48 hours on a rotary shaker at 250 rpm at a 5 cm shift.

The incubated vegetative medium described above (0.5 ml, 1%) is used to inoculate 50 ml of a fermentative medium of the following composition:

Component Quantity

Tapioca dextrin 60.0 g enzyme hydrolyzed casein 6.0 g casein enzymatic hydrolyzate 2.0 g

CaCO3 2.0 g

MgSCU. 7 H2O 0.5 g

Blackstrap molasses 15.0 g purified soybean oil 5.0 ml water to 1 liter pH (untreated 6.6

Example 17

Fermentation of A-28086 in tank · using

S. aureofaciens NRRL 8092

The initial procedure described in Example 16 for A-28086 shake flask fermentation was also used for tank fermentation. To prepare a large seed volume, 10 ml of inoculated vegetative medium is used to inoculate 400 ml of the second stage vegetative medium of the same composition as the first vegetative medium. This second stage medium in a 21 liter Erlenmeyer flask was incubated at 30 ° C for 24 hours on a rotary shaker at 250 rpm with a 5 cm shift.

This second stage vegetative medium (800 ml) was seeded with 100 liters of sterile fermentation medium of the following composition:

Component Quantity

Tapioca dextrin 60.0 g / l enzyme hydrolyzed casein 6.0 g / l enzymatic casein hydrolyzate 2.0 g / l

CaCO3 2.0 g / l

MgSOd. 7H2O 0.5 g / l

Blackstrap molasses 15.0 g / 1 purified soybean oil 5.0 mg / 1 water to 1 liter

The medium, after sterilization in an autoclave at 121 ° C for 30 minutes and at a pressure of 0.103 to 0.1373 MPa, has a pH of 6.8 ± 0.1. In a 16 L fermenter tank, the production medium is seeded and fermented at 28 + 1 ° C for 10-12 days. The fermentation medium is aerated with sterile air at a rate of 0.4 volumes per volume of nutrient medium per minute. The medium is mixed with a conventional 300 rpm stirrer.

Example 18

The A-28086 antibiotics were produced as described in Example 17, but a production medium of the following composition was used in the shaker and tank:

Component Amount Tapioca dextrin 30.0 g / l glucose 15.0 g / l enzyme hydrolyzed casein 3.0 g / l casein enzymatic hydrolyzate 1.0 g / l yeast extract 2.5 g / l CaCO3 2.0 g / l MgSO 3. 7HzO 1.0 g / l Blackstrap molasses 15.0 g / l purified soybean oil 5.0 mg / l water up to 1 liter

The medium after autoclaving as described in Example 17 has a pH of 6.4.

Example 19

Isolation of the A-28086 antibiotic complex produced by S. aureofaciens NRRL 8092

The fermentation medium (60 µl obtained by the method described in Example 17) was adjusted to pH 3 by addition of dilute HCl.

4δ is filtered using a filter aid (Hyflo Super cell, diatomaceous earth). The separated mycelium filter cake was extracted with 30 L of methanol and 1.56 kg of NaHCO3 was added with stirring. After separation of the extract, the mycelium cake is again extracted with an additional 30 liters of methanol. The two methanolic extracts were combined and concentrated in vacuo to remove methanol. The remaining aqueous solution (about 7 liters) was adjusted to pH 7.5 with dilute hydrochloric acid, extracted twice with ethyl acetate (71 L portions), and the combined ethyl acetate extracts were concentrated in vacuo to give an oily residue. To this acetone solution was added water (1500 mL), the solution was adjusted to pH 3 with dilute hydrochloric acid and stirred for one hour, and the resulting precipitate was collected by filtration and then dissolved in acetone (1500 mL). 400 ml of water, the solution was allowed to stand for 16 hours, the product crystallized, the crystals formed were filtered off and dried in vacuo to give 74 g of a crude crystalline product containing A-28086 factors A and D and other crystalline impurities.

This crude crystalline product (40 g) was dissolved in about 250 mL of benzene. The benzene solution is then loaded onto a silica gel column [9 x 120 cm Grace-Davldson grade 62 silica gel. The column is eluted sequentially with 40 liters of the following solvents:

1. Benzene

2. Benzene: ethyl acetate (9: 1)

3. Benzene: ethyl acetate (4: 1)

4. Benzene: ethyl acetate (7: 3)

5. Benzene: ethyl acetate (1: 1)

6. Ethyl acetate

7. methanol

Fractions of 1 liter were collected. Each fraction was assayed for activity against Bacillus subtilis and thin layer chromatography. A-28086 was eluted with benzene: ethyl acetate (4: 1), A-28086 factor B was eluted with benzene: ethyl acetate (7: 3). A-28086 factors A and D were eluted in fractions of benzene: ethyl acetate (7: 3 and 1: 1), fractions 119-156. These fractions were combined and evaporated to dryness in vacuo. The residue thus obtained was dissolved in acetone (500 ml). Water (500 ml) was added to the acetone solution and the resulting solution was adjusted to pH 3 with dilute hydrochloric acid and stirred for one hour. The resulting precipitate was filtered off and crystallized from acetone (500 mL) - water (180 mL). The crystals thus formed are filtered off and dried in vacuo to give 20.1 g of a mixture of A-28086 factors A and D.

Example 20

Separation and purification of factors A and D

The crystalline mixture of A-28086 factors A and D obtained in Example 18 (18.8 g) was dissolved in benzene (50 ml). Load the benzene solution onto a silica gel column (7 x 100 cm, E-Merck grade 60 silica gel finer than 230 mesh ASTM). The column is eluted at a rate of 90 ml per hour with the following solvents in turn:

1. 12 liters of benzene

2. 12 liters of benzene / ethyl acetate (9: 1)

3. 12 liters of benzene: ethyl acetate (4: 1)

4. 32 liters of benzene: ethyl acetate (7: 3)

5. 10 liters of methanol

The elution procedure is followed by thin-layer cellulose chromatography (cellulose, on an aluminum support) and bioautography using B. subtilis. The following solvent systems are used for chromatography: water: methanol: acetone (12: 3: 1), the pH of the solution being first adjusted to 10.5 with NH 4 OH solution and then to pH 7.5 with hydrochloric acid.

One to two liter fractions are collected until detection of biological activity and then 200 ml fractions are collected. Fractions containing only A-28086 factor D were combined and evaporated in vacuo to a residue. This residue was crystallized from acetone-water (1: 1). The crystals were collected and dried in vacuo. 140 mg of crystalline A-28086 factor D are obtained.

Fractions containing A-28086 Factor D with a trace of A-28086 Factor A were treated in the same manner to give an additional 150 mg of crystalline A-28086 Factor D containing a small amount of A-28086 Factor A.

Fractions containing only A-28086 factor A were also treated in the same manner to give 4.7 g of crystalline A-28086 factor A.

Example 21

A-28086 antibiotics were produced by the procedure of Example 16, but the sloping soil was used as follows:

and incubating the inoculated sloped soil at 28 ° C for seven days.

Example 22

The A-28086 antibiotics were produced as in Example 17, but the following composition was used as the medium for flask production:

Component Quantity

Tapioca dextrin 80 g / l enzyme hydrolyzed casein 15.0 g / l Blackstrap molasses 15.0 g / l calcium carbonate 2,0 g / l ammonium sulfate 1.0 g / i magnesium sulfate 0.5 g / l purified soybean oil 4.6 g / l

Example 23

The A-28086 antibiotics were produced as in Example 21, but the following composition was used as the third stage vegetative medium:

Component Quantity

Cerellose 20.0 g / l corn steep liquor (wet weight) 10.0 g / l soya bran 15.0 g / l

CaCO3 2.0 g / l yeast 2.0 g / l sugar molasses 5.0 g / l

Example 1 a d 2 4

Chicken feed modified A-28086 to control coccidiosis

A balanced, high-yielding fast-food diet for chickens shall be prepared according to the following formula:

Component Quantity

beef extract 2.00 G dextrin 20,00 G yeast extract 2.00 G casein enzymatic hydrolyzate 4.00 G C0Cl2. 6H2O 0.02 G agar 20,00 G deionized water up to 1 liter pH adjusted to 7.0 KO Ή

folders % kg ground yellow corn 50 453.6 Unpeeled soya flour extracted with solvent and finely ground, 50:% protein 31.09 272 animal fat (beef tallow) dried fish meal with soluble proportions 6.5 57.9 (60 o / o protein) 5.0 45.3 maize distillery extract 4.0 36.3 calcium phosphate 1,8 16.3 calcium carbonate vitamin premix (containing vitamins A, D, E, K and Biz, choline, niacin, panthothenic acid, riboflavin, biotin with 0.8 7.2 glucose as carrier) premix of trace elements (MnSO 3, ZnO, 0.5 4,5 KJ, FeSO, CaCO3) 2-amino-4-hydroxybutyric acid (hyd- 0.2 1.6 methionine roxy analogs) 0.1 0.9 A-28086 factor A 0.01 0.09 These substances are mixed by a method commonly used for mixing feed. Chickens are fed Example 25 this feed and water ad libitum and are Cattle feed, improved protected against outbreaks of coccidiosis; weight gain is comparable to chickens without A-28086 coccidiosis that was fed similarly Balanced, high valuable feed for diet without the compounds of the invention. bovine animals are prepared as follows: folders % kg finely ground corn 67.8 615.0 ground corn cobs dehydrated lucerne flour, 10 90.7 17% protein without soya flour 5 45.3 solvent extracted, 50% protein 9,9956 90.68 cane molasses 5 45.3 urea 0.6 5.4 A-28086 factor A 0.0044 0,0397 calcium phosphate 0.5 4,5 calcium carbonate 0.5 4,5 sodium chloride 0.3 2.7 premix of trace elements 0.03 0.27 premix of vitamins A and D2 * 0.07 0.63 premix of vitamin E ” 0.05 0.45 calcium propionate 0.15 1.3

* containing, per 0,45 kg, 2 000 000 IU vitamin A, 227 200 IU vitamin E and 385,7 g soya feed with 1% oil;

** Maize distilled corn grains with extracts containing 20 000 IU / d-α-tocopheryl acetate per 0,45 kg.

210 84 8

The mixed feed is compressed into pellets. An average daily feed rate of 6.7 kilograms per animal contains about 300 mg A-28086 factor A per animal per day.

Example 26

Acetylderivatives of A-28086 factor D

Antibiotic A-28086 factor D is dissolved in pyridine. A stoichiometric amount of acetic anhydride was added and the resulting solution was stirred and then allowed to stand at room temperature overnight.

Excess water is then added, the mixture is stirred and allowed to stand at room temperature for several hours. The resulting precipitate was filtered off, washed with water and dried. The resulting solid was washed with water and dried. The solid was dissolved in acetone and evaporated to dryness in vacuo. Acetylderivative A-28086 Factor D is obtained.

Examples 27 to 30

The propionyl ester of antibiotic A-28086 factor D is prepared by reacting A-28086 factor D, with propionic anhydride in the presence of pyridine according to the procedure of Example 26.

The n-butyryl ester of antibiotic A-28086 factor D is prepared by reacting A-28086 factor D with n-butyric anhydride in the presence of pyridine, following the procedure of Example 26.

A-28086 factor D n-caproyl ester was prepared by reacting A-28086 factor D with caproic anhydride in the presence of pyridine, following the procedure of Example 26.

n-Valerylester of A-28086 factor D is prepared by reacting A-28086 factor D

Ingredients ground yellow corn Unpeeled soya flour, solvent extracted, finely ground, 50% protein animal fat (beef tallow) dried fish meal with soluble proportions (60% protein) maize distillery calcium phosphate calcium carbonate vitamin premix (containing vitamins A, D, E, K and Biz, choline, niacin, panthothenic acid, ribofavavine, biotin, with glucose as carrier) premix of trace elements MnSCh, ZnO, KJ, FeSCD, CaCO3

2-Amino-4-hydroxybutyric acid (hydroxy analogs of methionine)

A-28086 factor D with valeric anhydride in the presence of pyridine, following the procedure of Example 26.

Example 31

Preparation of A-28086 Factor D Sodium Salt

Antibiotic A-28086 factor D is dissolved in acetone. An equivalent amount of water and sufficient 5N sodium hydroxide are added to bring the pH of the solution to about 11. The resulting solution is stirred for about one hour and then extracted with ethyl acetate. The ethyl acetate extract was evaporated in vacuo to give Factor D sodium salt A-28026.

Examples 32 to 34

The potassium salt of antibiotic A-28086 factor D was prepared from A-28086 factor D and 5N potassium hydroxide according to the procedure of Example 31.

A-28086 factor D antibiotic barium salt was prepared from A-28086 factor D and saturated barium hydroxide solution using the method of Example 31.

A-28086 factor D antibiotic cesium salt was prepared from A-28086 factor D and 1N cesium hydroxide solution using the method of Example 31.

Example 35

Chicken food containing A-28086 factor D to control coccidiosis

A balanced, high-yielding fast-food diet for chickens shall be prepared according to the following formula:

% kg

50 453.6 31.09 272.0 6.5 57.9 5.0 45.3 4.0 36.3 1,8 16.3 0.8 7.2

0.5 4,5 0.2 1,8 0.1 0.9 0.01 0,0!

0.9 8 4 &

of proteins

These substances are mixed by a method commonly used for mixing feed. The chickens are fed this feed and water ad libitum and are protected against the outbreak of coccidiosis; the weight gain is comparable to chickens without coccidiosis fed a similar diet without the compounds of the invention.

Folders:

finely ground maize ground corn cobs dehydrated lucerne flour,% protein unpeeled soy flour extracted with solvent, 50% molasses from cane urea

A-28086 factor D calcium phosphate calcium carbonate sodium chloride trace element premix vitamin A and D2 premix * vitamin E premix "calcium propionate * containing 2,000,000 soy feeds with 1% oil per 0.45 kg;

** corn distillery dried rylacetate per 0.45 kg.

The mixed feed is compressed into pellets. The average daily dose is 6.7 kg per

Example 36

Cattle feed containing A-28086 factor D

A balanced, high-value cattle feed is prepared as follows:

% kg

67,8 615,0 '

90.7

45.3

9,9956 90.68 5 45.3 0.6 5.4 0.0044 0,0397 0.5 4,5 0.5 4,5 0.3 2.7 0.03 0.27 0.07 0.63 0.05 0.45 0.15 1.3

Vitamin A, 227,200 IU of vitamin D2 and 385.7 g of leached grains containing 20,000 IU of d-α-tocopheran contains about 300 mg of A-28086 factor D per animal per day.

Claims (3)

Subject A process for the preparation of complex-forming polyetheric antibiotics A-28086 consisting of Factor A, Factor B and Factor D, acyl esters of Factors A and D having 2 to 6 carbon atoms in acyl, or physiologically acceptable salts thereof, and a process for isolating Factor A-28086, A-28086 factor B, A-28086 factor D from the A-28086 antibiotic complex, characterized by culturing Streptomyces aureofaciens NRRL 5758 or Streptomyces aureofaciens NRRL 8092 in a nutrient medium containing assimilable sources of carbohydrates, nitrogen, and inorganic salts under submerged aerobic fermentation conditions at a temperature of from 20 to 40 ° C and at a pH of from 6.0 to 8.0 until the organism is in this? A sufficient amount of antibiotic activity is produced in the nutrient medium and eventually the A-28086 antibiotic complex is separated from the nutrient medium, or factors A, B, or D are separated from the A-28086 antibiotic complex, and where appropriate are these? factors A, B or? D in acyl derivatives or pharmaceutically acceptable salts. 2. The process of claim 1, wherein the complex is extracted from the nutrient medium with a polar organic solvent, the solution is concentrated, and the concentrate is added to excess petroleum ether to precipitate impurities, then the A-28086 antibiotic complex is filtered and evaporated. . 3. A method according to claim 1, wherein the factors A, B or D are separated from the nutrient medium by lowering the pH of the nutrient medium to a value of 3 and then filtering off the respective factors.