SU576966A3 - Method of preparing antibiotic complex a-28086 - Google Patents

Description

d) specific rotation 54, (s - 0.2 methanol), determined at 25 ° С; e) IR absorption spectrum in chloroform by its following absorption maxima; 2.85; 3.34; 5.83; 6.84; 7.22; 7.53 (weak), 5.78 (weak), 8.76 (strong), 8.95 (strong), 9.15; 9.50 (strong), 9.55 (strong), 9.60; 9.85; 10.15; 10.45 and 10.70 (weak) µ; e) in its UV - spectrum in ethanol only the terminal absorption is below 200 MMK; g) NMR spectrum in chloroform with the following characteristics: 66.01; 4.21; 4.11; 3.99; 3.89; 3.80; 3.67; 3.65; 3.57; 3.55; 2.83; 2.66; 2.58; 2.56; 2.30; 2.22; 1.96; 1.90; 1.85; 1.70; 1.62; 1.31; 1.25; 1.18; 0.95; 0.93; 0.77; 0.75; 0.73; 0.68 and 0.66 ppm; h) a titratable group with a pKd of 7.9 in 80% aqueous dimetypformamide; i) X-ray diffraction pattern obtained by X-ray diffraction on a sample in a powder (Cu - radiation, 1.5405Х, nickel filter), having the following interplanar spacings j in angstroms (d); Rf value is 0.11 Water saturated with 0.41 Water saturated with acid and 1% pip Water-methanol-NH4OH to pH 10.5 1% MIBK, 0.5% S 17.4 g K2HP04, 30 Benzene, saturated Water Water-MIBK-ethyl m) acidic function capable of forming salts and ether; n) one at least hydroxyl group capable of esterification. The invention also provides for. Cj-Cs-acyl esters and physiologically acceptable salts of these factors. Ii. Factor B of the antibiotic A-28086 is a white crystalline compound, when crystallized from a mixture of acetone and water, soluble in lower alcohols, dimethylformamide, dimethyl sulfoxide, ethnyl acetate, chloroform, acetone and benzene; but little soluble in hexane and insoluble in water. The compound has the following characteristics: a) TP. 150-153C; b) molecular weight 762, determined by high resolution mass spectrometry; c) empirical formula С4зН-; оО11. determined by high resolution mass spectrometry; d) IR spectrum in chloroform with the following different absorption maxima:, 82; 3.30; Race system dRelative intensity 12.00100 10.1050 9.2590 8.0040 7.5015 6.9290 6.4040 5.9805 5.6815 5.2040 4.9840 4.6240 4.2120 3.4810 k) Rf 0.24 during chromatography in a thin layer on silica gel in the solvent system benzene-ethyl acetate (3: 2) using Bacillus subtilis ATCC 6633 as a detecting organism; l) RfjB value of the systems indicated below for chromatography on paper using Bacillus subtilis ATCC 6633 as a detecting organism: body or isobutyl ketone (MIBK) K, plus 2% paratoluene sulfoin (12: 3: 1) with added NzGO4 to pH 7.5 water ethanol per liter of water ode am (98: 1: 1), 77; 5.85; b, 80; 7.20; 7.50 (weak), 7.72 (weak, 57 (strong), 8.68; nd), 7.80 (weak), 9.50; 9.83 (strong), 9.90; , 90 (strong), 9,10; 10.43 (weak), 0.10; 10.17 (strong), 11.35 (weak). 0.80 (weak), 11.20 (weak), 11.73 (weak) and 12.03 (weak) µ; e) the observed maximum in ethanol in its UV spectrum at 220 μM (E 137.5; 477); e) NMR spectrum in deuterochloroform with the following characteristics: 5 7.20; 7.09; 6.26; 6.15; 4.19; 4.12; 4.05; 3.95; 3.89 3.52; 3.48; 2.81; 1.91; 1.84; 1.71; 1.18; 1.11; 0.96: 0.94; 0.90; 0.74 and 0.68 ppm; g) the value of fif 0,42 in chromatography in a thin layer on silica gel in a mixture of benzene - ethyl acetate (3.2) using Bacillus subtilis ATCC 6633 as a detecting organism; h) Rf values in the following systems with paper chromatography using Bacillus subtilis ATCC 6633 as a detecting organism: G

Rf Solvent System

Water saturated with MIBK, plus 2% para-toluenesulfonic acid

and 1% piperidine Water-methanol-acetone (12: 3: 1) with the addition of

NH40H to pH 10.5, then HzP04 to

рН7,5

1% MIBK, 0.5% NH40H in water

Benzene saturated with water

Water

Water-MIBK-ethyl acetate (98: 1: 1) and) acidic function, capable of forming salts and esters; k) two ketogroups; l) one at least gadroksilnug group. Physiologically acceptable salts of the factor III are envisaged. Factor D of the antibiotic A - 28086 is a white crystalline compound, when crystallized from a mixture of acetone and water, soluble in methanol, ethanol, dimethylformamide, dimethylsulfoxe, ethyl acetate, chloroform, acetone and benzene, but only slightly soluble in hexane and insoluble in water; m.p. 96-98 ° C. The compound has the following characteristics: a) molecular weight 778, determined by high resolution mass spectrometry; b) approximate elemental composition: 67.59% carbon, 9.38% hydrogen and 22.77% oxygen; c) empirical formula C4 H740i 1, defined by high-resolution spectrometry; d) specific rotation - 56 ° (c 0.1 methanol), determined at 25 ° C; e) IR spectrum in chloroform with the following absorption maxima: 2.89; 3.39; 3.43; 3.50; 5.88; 6.90; 7.25; 7.60; 7.84; 9.00; 9.26; 10.31; 10.58; 11.10 and 11.49 microns; e) UV-absorption in 95% aqueous ethanol is not observed; g) NMR spectrum in chloroform with the following characteristics: 5 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; 2.37; 2.29; 2.21; 2.15; 2.10; 2.04; 1, 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; Rf value Water, nat 0.10 Water saturated 0.26 and 10% Water-meth NH4OH pipa to pH 7.5 1% MIBK, 17.4 g K2H Benzene, per

576966 h) a titratable group with a pKc value of 8.67 in 80% total dimethylformamide; i) X-ray diffraction pattern obtained by X-ray diffraction on a sample in a powder (radiation, 1.5405Х, nickel filter), having the following interplanar spacing, in angses: femah (d): Relative intensity 100 k) Rf values in the following systems with thin layer chromatography on silica gel using Bacillus subtilis ATCC 6633 as a detecting organism: Rf value Solvent system 0,26Benzene-ethyl acetate (3: 2) 0.66 Ethyl acetate-diethlamine (95: 5) l) Rf values in the same systems specified during chromatography on paper with Vasya $ subt ilis ATCC 6633 as a detecting organism: solvent matschshobutyl ketone (MIBK) MIBK, plus 2% para-toluenesulfonic acid and cetone (12: 3: 1) with 5, then HzP04 H4OH in water, 0 ml of ethanol per liter of water, m-acid; a function capable of forming; salts and ethers; and at least one hydroxyl group capable of esterification. Cj-Cg are aniline esters and physiologically acceptable salts of this factor. Factors A, B and D of the antibiotic A - 28086 Lye members of the group of polyester antibiotics. The members of this group include monperzin, dianemicin (nigercin) and salinomycin. The term antibiotic complex means a mixture together; formed individual antibiotic factors. The ratio of indium-derived factors in the antibiotic complex may vary depending on the application of the fermentation conditions. The antibiotic complex A-28086 is obtained by growing a new strain of Streptomyces aureofaciuns-NRRL 5758 during fermentation under aerobic conditions in a thick layer until a significant level of antibiotic activity is reached. This antibiotic is also obtained by growing another new strain of Streptomyces aureofaciens NRRL 8092. The antibiotic obtained with S. aureofaciens NRRL 5758 or S. aureofaciens NRRL 8082 is extracted from the fermentation broth and the organic solvents are separated from the mycelium. The extracted mixture is separated by concentrating the solutions, adding petroleum ether to the concentrate to precipitate impurities, followed by filtration and evaporation of the filtrate to obtain an antibiotic A mixture - 28086. This mixture is further purified and separated into individual factors by column chromatography. Composites A-28086 inhibit the growth of organic gas pathogenic to animals and plants. Compound A-28086 are agents that inhibit the growth of coccidia and, in addition, they are antibacterial, antiviral, active against organisms that cause pneumonia, insecticidal acaricidal agents, as well as substances that increase the efficiency of feed utilization of ruminant animals. FIG. 1-8 depict IR absorption spectra in chloroform. FIG. 1 - factor A antibiotic A - 28086; in fig. 2 - factor B antibiotic A - 28086; in FIG. 3 — an acetyl ester of factor A of the antibiotic A — 28086; in fig. 4 - propionyl ether of factor A of antibiotic A - 28086; in fig. 5 - butyryl ester of factor A antibiotic A - 28086; in fig. 6 - antibiotic A factor valeral ether A - 28086; in fig. 7 - Caproil ether of factor A of antibiotic A - 28086 and in FIG. 8 - IR spectrum of absorption of factor D of antibiotic A - 28086. L v BC-ethyl acetate (98: 1: 1). Factors A - 28086 are structurally similar to each other. During fermentation, at least four factors are co-produced in the BH / te mixture. Factors are separated from one another and factors A, B, N O are isolated as individual compounds. The mixture of factors A - 28086 is soluble in the pain of a part of organic solvents, but is insoluble in water. Factor A is crystallized from a mixture of acetone and water. It has a melting point of V8100 ° C, solidifies and melts again at 195-200 ° C. Elemental analysis of factor A gives the following composition: carbon 66.69%, hydrogen 9.85; and oxygen 23.10%. The empirical formula for this factor is С4зН72011 Factor A has a molecular weight of approximately 764, as determined by mass spectrometry. FIG. 1 shows the IR spectrum of factor A. The following absorption maxima were observed: 2.85; 3.34; 5.83; 6.82; 7.22; 7.5z (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. . The UV spectrum of factor A in ethanol shows only the terminal absorption below 220 microns. The NMR spectrum of factor A in deuterochloroform showed the following characteristics: 66.01; 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 Factor A, crystallized from a mixture of acetone and water. Had the following X-ray diffraction pattern obtained by X-ray diffraction on a sample in the powder (radial, 1.5405Х, nickel filter, d - interplanar spacing in angstroms): dRelative intensity 12.00100 10.1050 Specific rotation of factor A - 54 ° (s 0.2 methanol), determined at 25 ° C.

Electrometric titration of factor A in an 80% aqueous solution of dimethylformamide shows the presence of a titrated group with a pK of 7.9.

Factor A is soluble in organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone, and benzene, but slightly soluble in non-organic solvents such as hexane and does not dissolve in water.

Antibiotic factor A 28086 has an acid function capable of forming salts and esters, as well as at least one hydroxyl group capable of esterifying.

Factor B of the antibiotic A - 28086 is a white crystalline compound (during crystallization from a mixture of acetone and water), and so on. 150 × 53 ° C.

Using high resolution mass spectrometry, it has been established that factor B has a molecular weight of approximately 762 and an assumed empirical formula of C4 3 H 7 0 0 1.

FIG. 2 shows the IR spectrum of factor B in chloroform. The following absorption maxima were observed: 2.82; 3.30; 5.77; 5.85; 6.80; 7.20; 7.50 (weak), 7.72, (weak), 7.80 (weak), 8.67 (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), 10.20 (weak), 11.35 (weak), 11.73 (weak) and 12.03 (weak) micron

The UV spectrum of factor B in ethanol shows absorption maxima at 220 μM (E 137.5; 477).

The NMR spectrum of factor B in deuterochloroform showed the following characteristics; 6 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; 1.64; 1.55; 1.43; 1.33; 1.18; 1.11; 0.96; 0.94; 0.90; 0.87; 0.77; 0.74 and 0.68 ppm .

Factor B of antibiotic A - 28086 is soluble in organic solvents such as, for example, methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone, and benzene; slightly soluble in non-polar solvents, such as hexane, and insoluble in water.

Although the chemical structure of factor B is not clarified, however, the physical and chemical properties show that factor B has one carboxylic acid group, two ketone groups, and one or more hydroxyl groups.

When obtained with S, aureofaciens NRRL 8092, factor D comprises up to 10% of the total antibiotic activity; when obtained with S. aureofaciens NRRL 5758, it is the least of the factors.

Factor D of antibiotic A - 28086 is a white crystalline product (during crystallization from a mixture of water and acetone) with mp. 96-98 ° C. Kazuishis of molecules 1: 1n, 1st weight 778 (determined by high resolution mass spectrometry). The elemental composition of the peak in the mass spectrum of the sodium salt of factor D is 800.5050 (calculated for € 44473011 800.5050). In the mass spectrum of the factor O

in the form of the free acid, a small maximum was observed at 778 and a larger maximum at 760, 5117 (calculated for C44H720io); m / z 760 in the free acid mass spectrum is a result of the loss of water from the molecular

and she. Therefore, the composition of the molecular Io1I factor D in the form of the free acid C44H74Ol 1

The hypothetical empirical formula for factor D is C44H740i 1 Elemental analysis of the faktrr D gave the following composition: 67.59% carbon, 9% hydrogen and 22.77% oxygen.

Theoretically, for C44H74Oi, 67.87% carbon, 9.51% hydrogen and 22.77% oxygen are required.

Factor D at 95%

This aqueous ethanol does not absorb UV rays.

The NMR spectrum of factor D in deuterium chloroform has the following characteristics: 5 6.00; 4.20;

3.67; 4,10; 4.00; 3.98; 3.92; 3.86; 3.83; 3.79;

2.71; 3.64; 3.57; 3.54; 2.88; 2.91; 2.62; 2.58; 2.48; 2.43; 2.37; 2.29; 2.21; 2.10; 2.04; 2D5; 1.97; 1.89; 1.83; 1.76; 1.68; 1.58; 1.55; 1.61; 1.47; 1.39; 1.30; 1, 18; 0.90; 0.88; 0.95; 0.84; 0.74 and 0.68 ppm

Factor D, crystallized from a mixture of acetone and water, has the following X-ray diffraction pattern obtained by X-ray diffraction on a sample in the powder (Cu - radiation, 1.5405 X, nickel filter), d - interplanar spacing in angstroms:

dRelative

intensity

100 70 90 30 10

100 20 20 20 10 20 40 30 10 10 05 05 05

The specific rotation of the factor D - 56 (s - 0.1 methanol), determined at a temperature

Electrometric titration of the factor in 80% aqueous dimethylformamide showed the presence of a group with a pKa of 8.67. Factor D is soluble in organics such as methanol, ethanol, dimetelfer: dimethyl sulfoxide, ethyl acetate, chloroform, ac, and benzene; slightly soluble in non-organic solvents such as, for example, san, and insoluble in water. Factor D has an acidic function, and T is a variable Rf factor A factor B factor B 0.11 0.09 0.410.160.26 0.540.460.36 0.480.360.39 0.150.33. 0.25 0.240.510.26 0.240.110.09 0.750.610.64 Rf values for factors A, B and D in two systems with chromatography on a thin layer on silica gel (pre-coated plates Rf value FakyurA factor Y 0.240.420.26 0.540.340.66 Simultaneously with the antibiotic complex A-28086 forms another substance, arbitrarily named A-28086-1. This substance, not microbiologically active, is structurally related to the factors of antibiotic A-28086, A -28086 - 1 - a white crystalline compound (during crystallization from a mixture of acetone and water) has mp 160-162 ° C. Comparative study of CU NMR and other properties of A -28086-1 and synthetically prepared factor A methyl ester prove that A-28086 is methyl factor A ester or a very close compound, i.e., a stereoisomer. Although A-28086-1 precipitates together with active factors of antibiotic A is 28086, however it is easily separated by blister chromatography. A - 28086 - 1 has an Rf value of about 0.53 in thin layer chromatography on silica gel with ethyl acetate as eluent and using vinyl reagent for spraying ( 3% vanillin in methano le plus 0.5 ml of concentrated sulfuric acid per br. of solution) for the purpose of detection. After spraying with Vinyl and heating A - 28086 - 1 gives a blue spot, while factors of the antibiotic A - 28086 give bright pink spots that quickly become dark, tan-blue. Factors A, B and D, as well as the indicated esters of carboxylic acid and factors A and D, are capable of forming salts and firs and at least one hydroxyl group capable of esterifini MS; stay with Eton Values The Rf factors A, B, and D in different synthetic paper chromatography using Bacillus subtilis ATCC 6633 as an organism found are shown below. Solvent System Methyl Shzobutyl Ketone (MIBK) Water Saturated with MIBK, plus 2% naratoluenesulfonic acid and}% piperidine Water-methanol-acetone (12: 3: 1) was added with the addition of NH40H and then reduced to pH 7.5 HzRO4 . 1% MIBK, 0.5% MH40H inlet 17.4 g K2HP04.30 ml of ethanol per liter of water Benzene j saturated with water Water Water-MIBK-ethyl acetate (98: 1: 1). Merck, Darmstadt G - 254, thickness of a layer of 0.25 cm) when B. subtilis ATCC 6633 is used as a detecting organism with eaters. The solvent tilatsetat (3: 2) at-diethylamine (95: 5) to form salts Physiologically acceptable salts, which are also acceptable as pharmaceuticals, are salts in which the toxicity to warm-blooded animals does not exceed the toxicity of the non-salt form of the compounds. Suitable salts of factors A and B are sodium, potassium, lithium, desi, rubidi, barium, calcium and magnesium salts. Suitable amine salts of factors A and B include ammonium salts, primary, secondary to tertiary Ci-C4 - alkylammonium and hydroxy - - alkylammonium. Co and alkali and alkaline earth metals of factors A, B and D: also the acyl esters of factors A and D are prepared by conventional salt preparation methods. For example, the factor in the form of the free acid or ester is dissolved in a suitable solvent, such as heat of methanol or ethanol, and a solution containing a stoichiometric amount of inorganic base in aqueous methanol is added to the solution. The resulting salt can be isolated by conventional means, for example, by filtering BaiffleM or by evaporation of the solvent. In the same way, salts with organic amines can be obtained. For example, a gaseous or liquid amine can be added to a solution of a factor in a suitable solvent, for example acetone, and then the solvent and the excess amine can be evaporated. The formula of salt can be chosen depending on the use of the product, toxicity and 3jscomnos. Factor A forms carboxylic esters. Esterification occurs in one of the hydroxyl groups of factor A when treated with anhydride or an acid chloride Cj-C acid. Such esters are usually obtained by the reaction of factor A with an appropriate acid anhydride at room temperature. Such esters are also suitable as antibiotics and agents that increase feed utilization efficiency. Factor A acetyl ether - a crystalline compound of white color (during crystallization of a mixture of acetone and water) has so pl. 100-103 C and empirical formula € 4 j N, g Its molecular weight is about 807 according to the empirical formula proposed for factor A. Elemental analysis of factor A acetyl ether gives the following percentage composition: Calculated,% C 66.97; H 9.24; About 23.79. C4sH74O | 1 Found,%: C 67.67; H 8.71; About 23.13. The IR spectrum of factor A acetyl ether in chloroform is shown in FIG. 3. The following absorption maxima were observed: 2.85; 3.36; 3.38 (strong), 5.80; 6.83; ./7.25 (strong), 7.60 (weak), 7.80 (strong), 8.45 (strong) 8.80 (strong), 8.95 (strong), 9.10 (strong), 8 20; 9, 9.80. (Strong), 10.12 (weak) and 10.50 microns. The UV spectrum (factor A of acetyl ether) in ethanol shows only terminal absorption. Electrometric titration of factor A acetyl ether in 80% aqueous dimethylformamide indicates the presence of a titrated group | PY with a pKa of 8.5. Factor A propionyl ether is a white crystalline substance (during crystallization from a mixture of acetone and water) with m.p. about 96-98 ° C. Propionyl ether of factor A has an empirical formula of C47H7sOij and a molecular weight of about 821, calculated from the empirical formula proposed for factor A. Elemental analysis of the propionyl ether of factor A gave the following percentage composition: Calculated,%: C67.29 ; H9.33; About 23.38 S4bN7bO ,, Found,%: C 66.06; H 9.17; About 23.41. The IR spectrum of factor A propionyl ether in chloroform is shown in FIG. 4. The following maxima were observed: 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 UV spectrum of factor A propyl ether in ethanol showed only terminal terminus. The butnril ester of factor A is a white crystalline compound (with crnstl, chi: icium from a mixture of acetone and water) with m.p. s.colo96-98S. This ether has an empirical formula of C47H-) gOij, a molecular weight of about 835 and an approximate composition: C67.60%, P 9.41%, 022.397, calculated using the empirical formula facto {1a A. IR spectrum of factor A butyryl ether in chloroform shown in FIG. 5. The absorption maxima are as follows: 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 10.64µ. Valeryl ether from gA factor and valeric acid is a white crystalline compound (during crystallization from a mixture of acetone and water) with T.PL. about 173-175 ° C has the empirical formula G48HgoOij, molecular weight about 849 and approximate composition: C 67.89%; H 9.50%; About 22.61%, as calculated by the proposed empirical formula of factor A of antibiotic A - 28086. The IR spectrum of factorale valeryl ether in chloroform is shown in FIG. 6. The absorption maxima are as follows: 2.90; 3.40; 3.45; 3.51; 5.87; 5.92 (strong), 5.99 (strong), 6.81; 7.30; 7.69 (weak), 7.87 (weak), 8.16; 8, 8.85 (weak), 9.26; 9.76; 10.00 (weak), 10.31 and 10.64 microns. The ester of caproic acid and factor A, capric ester is a white crystalline compound (when crystalline is from a mixture of acetone and voy) with m.p. about 163-167 ° C has an empirical formula of C49H82Oj2, a molecular weight of about 863, and an approximate composition: C 68.18%; H9.58%; About 22.24%, determined by the empirical formula of factor A. The infrared spectrum of caproyl factor A is shown in m. 7. Absorption maximums: 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; 9.96; 10.29 and 10.62 microns The IR spectrum of factor D (Fig. 8) has 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; 10.57; 11.10 and 11.49 microns As a result of the acylation of factor A, the following changes in the spectrum of nuclear magnetic resonance I occur: a carbon dioxide gap that occurs at about 4 ppm shifts to about 5.3 ppm (the exact position varies slightly in different acyl derivatives); the signals of the viniproton are also shifted. These characteristic changes are represented in a particular structure: and where R represents Ci-Cs-alknl. These partial artworks fully correspond to aKcnopiiNiemaM no1 roNioMo; ieK; iHpnoMv separation and NMR to the spectrum of 13 ufyryz uussus c; and propionic. Factor A ester is soluble in organic compounds such as methanol, ethanol, dimethyl formamide, dimethyl sulfoxide, ethyl acetate, | chloroform, atsepsh and beieop, but dissolve little in the first non-polar and organic solvents, for example in hexane, and are soluble in water. Each Cj-Sb — acetyl ester of antibiotic A -28086 has an acidic salt, which can form salts and ethers. New anti & yutics are obtained by growing a strain of Streptomycer aureofacJens, which forms A 28086 under aerobic conditions by a deep method, using a suitable nutrient medium prior to the formation of significant antibiotic activity. Antibiotics are isolated using different, usually applied, methods of peeing and purification. NRRL characteristic 5 in different environments. JSP | 3 2 (yeast extract and malt extract) iSP / 33 (oats for flour) ISPj34 (inorganic salt - starch agar) JSPp5 (glycerin-assraphovy agar) Tomato paste - agar with oatmeal Glycerin-glycine agar Glucose asparopar cook cookar cookar from organisms suitable for the preparation of antibiotics A-28086, was isolated from a sample of soil collected on Ararat, Typimn. This opraiffl3M is classified as Dutgar strain Streptomy5 ces aureofaciens. Cultures were grown at 30 ° C for 14 days, except for the special cases. The characteristic of the strain NRRL 5758, forming A - 28086. {The hyphae morphologists, which form the spores, consist of blocks, loops and open spirals. A morphological representative of the recis yew elastic type was also observed. The spores are short and narrow, 15 form I chains from 10-50 spores. The dispute size is 1.3 X 1.75 /; in the range from 1.3 to 1.96 x 1.3 d. The cnoip surface, observed with an electron microscope, is smooth. Characteristics Abundant growth; the reverse side is yellow; air mycelium and sporulation from satisfactory to good; white mycelium (w) 13 La and dark gray (Gy), 3ih; j insoluble pigment. Growth is good; the reverse side is greenish yellow (11B2); satisfactory airy mycelium (Gy) dark green, 3ih; soluble pigment light brown. Abundant growth: the reverse side is light yellowish brown (12E5); air mycelium and spores (w) purple and white 13 b a to (gv) light gray d insoluble pigment. Growth is good; the reverse side is light, yellowish - green; good spawning mycelium and spores (Gy) yellowish gray 2dc to light grayish reddish brown 5fe; insoluble pigment. Abundant growth; the reverse side is light, yellowish-brown (13), aerial mycelium and spores from satisfactory to good (w), from white A to (Gy) medium gray. The soluble pigment is light brown. Abundant growth; the reverse side is dark grayish yellow (1216); good aerial mycelium and spores (Y), light yellow 2dbj insoluble pigment. Abundant growth; reverse grayish greenish yellow (12E2) abundance of aerial mycelium and spore (Gy) yellowish gray 2dc; light brown soluble pigment. Growth is good; the reverse side is greyish yellow (12B2), the color of the aerial mycelium and the dispute is not determined due to poor development; insoluble pigment. APS Bennett Agar with a calcium salt of malic acid Agar with a Czapek Agars Emerson solution Throzine Agar Trypton-yeast The NRRL 5758 organism determines some of the standard method.

Properties Effect on milk

Nitrate recovery of food gelatin Formation of melanin pigment on oblique agar with tyrosine

peptone difco - yeast extract - iron - oblique agar

on broth with tryptone and extract. Required temperature ISP medium 02 - spit medium from Schyuzhev and malt extracts

The results of the experiments to determine carbon utshgazavii conducted with the organism NRRL 5758 are as follows. The following designations are used for growth characterization:

+ good growth positive use of carbon;

(+) growth from poor to satisfactory

(-) poor growth, probably without disposal

- no growth, no recycling.

Carbon source

Reactions

-I- + + D - glucose L - arabinose D- xylose D - fructose

+ Sucrose

Specifications

Peptonized milk, white ring; a clear reddish brown area, pH 5.7.

Positive Thinning by 30% after 14 days

Very slightly positive (pigment after 4 days)

Negative Negative

Good growth at 26-30 ° C; excellent growth and sporulation at 37 ° C; weak vegetative growth at 45 ° C; reddish soluble pigment.

D - Manchit Izoinozitol

Ramnoza +

Raffinosa

Control, without carbohydrate -

The second new organism, forming A-28086, was obtained from S. aureofaciens NRRL 5758 by selection, followed by chemical distillation. This organism, identified as NRRL 8092, is classified as a type of Duggar Streptomyces aureofaciens. This classification was based on the results of the NRRL 8092 study of the body, previously described by the Streptomices Classification methods under the same conditions of the post. Abundant growth; the reverse side is grayish yellow (1ЖЗ); poor air pollution and spores (Gy) yellowish gray 2dc; insoluble pigment. Good height; the reverse side is grayish brown (15С8); no aerial mycelium and sporulation; brown soluble pigment. Growth is good, color is not determined due to poor development. Abundant growth; the reverse side is greyish-yellow (11.5), there is no aerial mycelium or spore; insoluble pigment. Abundant growth; the reverse side is light, with brown brown (14.5) abundant aerial mycelium and spores from (w) b (center) to (SU) light brownish-gray; 3fe (on edge); very light brown soluble pigment. Weak growth; color not defined. Characteristics of the A 28086 Morpho pusher In the 1S nutrient sapling, the culture forms short and the straight line consists of at least 10 Short straight chain chains. Characteristic of NRRL (yeast and malt extract (oats for flour) 15РД4 (agar with inorganic salts and iSP / 35 (agar with gshscheri and asparagine) Agar with tomato paste and oat flour Agar with glycerin n glycine Agar with glucose and asparagine Food agar Bennet Agar with calcium maleate soda Agar with Emerson Agar solution Tyrosine agar with media, ISP: ISR: ISP: ISP: ISP: ISP with agar with glucose and asparagine Capes agar and ISP solution (35. An abundant shell was observed on Emerson agar, and electron microscopic observations were made on tyrosine agar (18RD7) and on glucose agar and asparagine agar. The spores were smooth from 1.2 to 2.0 in length and about 1.0 ac in diameter. pore 1, 0 micron Characteristic Growth satisfactory; back side light, yellowish brown (12H8); satisfactory aerial mycelium; poor sporulation; mycelium light gray; insoluble pigment. Growth scattered; the reverse side is transparent; no aerial mycelium; insoluble pigment. Moderate growth; the reverse side is greyish yellow (11B2); poor aerial mycelium and poor spore formation; aerial mycelium is light yellowish gray; (10A1); insoluble pigment. Moderate growth; the reverse side is light yellow (10Р2); satisfactory aerial mycelium; sparse sporulation; white mycelium (10A1); insoluble pigment. Moderate growth; the reverse side is greyish greenish - yellow; aerial mycelium satisfactory; average degree of sporulation: pale sulfur (55A2); insoluble pigment. Abundant growth; the reverse side is greyish-yellow (11E4), dead air mycelium, white (10A1); sporulation does not occur; insoluble pigment. Moderate growth; the reverse side is light yellow (lOF): moderate aerial mycelium and the same sporulation, white (10AI) mycelium; insoluble pigment. Growth is poor, the reverse side is light yellow (10B2); no aerial mycelium; insoluble pigment. Growth is satisfactory; the reverse side of the medium is yellowish - crimson; very poor aerial mycelium; sporulation does not occur; insoluble pygmy) t. Growth is scanty, translucent; no aerial mycelium; insoluble pigment. Growth is very poor; the reverse side is transparent; no mycelium; insoluble pigment. Moderate growth; the reverse side is greyish yellow (1115); mottled aerial mycelium; sporulation does not occur; insoluble pigment. Moderate growth; the reverse side is light yellowish - kortchina (12N6); moderate area of mycelium, light pale gray edge (53A2): center almost white; sporulation 21 Agar with tr ptox and yeast Phycologic sdrccg Properties Action w milk Nitrate reconstitution Nutritional gelatin Formation of melanin pigment on oblique agar with tyrosine broth with trilton and yeast extract - morknoy cork –potto tube — Spit Medium- {Yeast Extract and Malt Extract The results of the experiment, by definition, carbon ut carbon are given below. For growth, the indications are used to denote% to good growth; Positive (u) growth from bad to satisfactory (-) weak growth, probably), without growth there is no growth, there is no increase in weight. Carbon source Rezu D - glucose + c. - arabinose-iNRRL 5758

Yellow in several environments

Strellyk sporophores on the medium from the paste of tomato and oat flour; and on the environment: inorganic salts starch

Growth satisfactory, brownish with enlightenment

Moderate sporulation | aerial mycelium

NRRL 8092

From cream to light yellow

on multiple mediums.

Short straight sporophores

and random hooks

Growth is poor, light, without enlightenment

Poor sporulation; mycelium is light, yellowish - earrings 22 moderate; insoluble pigment. Growth is very scant, translucent; no aerial mycelium; pigment pigment. NRRL 8092 Features Peptonized (90%); light: yellow ring; transparent area is light yellow; Positive Hydrolyzed within 14 days at 50% Very weakly positive and Negative Abundant growth; light yellow; without air mivdli Abundant growth; grayish white; without aerial mycelium, without change in traffic jam. At 25 ° C plentiful growth; satisfactory aerial mycelium; the reverse side is light brown insoluble pigment. At 30 ° C plentiful growth; satisfactory aerial mycelium; reverse sides are light brown; insoluble pigment. At 37 ° C plentiful growth; satisfactory aerial mycelium; the reverse is brown; brown soluble pigment. At 40 ° C, abundant growth; scanty ws; Tsshshy mycelium i reverse side is reddish-brown; reddish brown soluble pigment. At 45 ° C satisfactory growth; without aerial mycelium; the reverse side is reddish brown; moderate reddish brown pigment. 1U D - Xylose + D - Fructose + Sucrose - D - Mannitol - Isoinositol + Ramnoza + Rafinose + Control. without carbohydrate The differences in the properties of the organism NRRL 5758 and the body NRRL 8092 are shown below:

Aiap Bennett

KyjtbTypbi Streptomyces aureofaciens, which are used to form antibiotics A-28086, are stored in the collections of the Northern Trade and Nutrition Research Department of the United States Department of Agriculture, Peori, Illinois, 61604, NRRL 5758 and NRRL 80.

For growing Strepfomyces: aureofaciens NRRL 5758 or Strepfomyces aureofaciens NRRL 8092 you can use any of these media. However, certain optimal media are preferred for optimal yield and easy product release. For example, preferred carbohydrate sources for fermentation processes on an industrial scale are tapioca dextrin and sucrose, although glucose, maize starch, fructose, mannose, maltose, lactose, and the like can also be used. Corny sources of carbon are also corn oil, peanut oil, soybean oil, and fish oil. The preferred nitrogen source is elzimatic hydrolyzate, but peptones, soy flour, flour {COTTON seeds, amino acids such as Glu {Pminova, and the like are also suitable. Inorganic salts that can be added to the nutrient medium include the usual soluble salts capable of producing chloride ions, carbonate, sulfate, nitrate, sodium, magnesium, calcium, ammonium, and the like.

For the growth and development of organisms, the nutrient Yared should also include trace elements.

Fermentation takes place on an industrial scale, if foaming occurs, it is necessary to add small amounts (i.e., 0.2 ml / l) of antifoaming agent, for example, lipropllenglycol.

The amount of antibiotic A - 28086, formed by the strain of Streptomyces aureofaciens, increases when a small amount of soybean oil is added to the medium.

To obtain significant quantities of antibiotics A 28086 It is desirable to apply aeration of the nutrient medium in the fermentation tanks. Small amounts of antibiotics A-28086 can be obtained by growing a culture in a shaking flask. Since the formation of antibiotics slows down during inoculation in large vats with spores of organisms, it is advisable to use vegetative seed. Such material is prepared by inoculating a small volume of nutrient medium with spores or fragments of the body’s mycelium to obtain a fresh, actively growing culture. The vegetative inoculum is then transferred to a large vat. For vegetative growth of inoculum, the same medium can be used as used for fermentation on a large scale, but other media are also suitable.

or light purple

to gray

The reverse side is yellow j The reverse side of the rose

Organisms forming A - 28086 can be grown at a temperature in the range of from about 20 to 40 ° C. Optimal growth of A - 28086 occurs, apparently, at a temperature of from 27 to 30 ° C.

In aerobic conditions, nutrient medium is rinsed with sterile air. For efficient growth of the organism, the preferred volume of air used in large vats is more than one volume per volume of medium per minute. To effectively form antibiotics A-28086 in vats, it is desirable to use aeration of more than 0.25 volume of air per volume of nutrient medium per minute. Large amounts of dissolved oxygen do not; inhibit antibiotic formation.

The formation of antibiotics can be monitored during fermentation by sampling broth or extracts of mycelium solids. In these samples, activity is determined for antibiotic sensitive organisms. One of the organisms suitable for testing antibiotics of the present invention is Bacillus subtil is ATCC 6633. The test is usually carried out using a paper disk on agar plates.

The initial pH of the inoculated culture medium depends on the application medium. In general, the pH should be between 6.0 and 7.5. At the end of the fermentation, the pH is usually somewhat higher and pezhit ranging from 6.5 to 8.0.

Antibiotic activity is manifested on the second day of fermentation. Maximum antibiotic activity is usually achieved on the sixth to seventh day.

The antibiotics A-28086 formed as a result of fermentation can be isolated from the fermentation medium by conventional means. Both the micelle mass and the filtered broth possess antibiotic activity. Therefore, the maximum release of antibiotics A-28086 is achieved with a combination of filtration, extraction and adsorption chromatography. The preferred solvent for separating these antibiotics from the whole or filtered fermentation broth is ethyl acetate, although other solvents commonly used for this purpose can be used.

A particularly advantageous way to isolate factors A, B and D of antibiotic A - 28086 is to lower the pH of the whole fermentation broth to about pH 3.0. For this pH, factors A, B, and D are usually separated by filtration together with the micelle mass. Another way of isolating the A-28086 factors is to add to the whole bu; thion of bicarbonate, for example, sodium bicarbonate, in an amount of about 1 g / l. Usually, the factors L-2L0 are separated together with the micelle mass in the form of salts. The precursor solvent for separating antibiotics from the micelle mass is coytanol, but other lower alcohols are also suitable for ketones. Azeotropic distillation can be successfully used to isolate antibiotics A-28086. In this case, an organic solvent is added to the aqueous fermentation broth, forming the corresponding A.: etropes, and the resulting mixture is subjected to azeotropic distillation to remove at least half of the water from the broth. As a result, a mixture of water and a solvent remains, while A 28086 antibiotics are dissolved in an organic solvent. Insoluble by-products can be separated by a suitable method, HanpHMejii, by filtration or centrifugation. Antibiotics can then be isolated from an organic solution by well-known methods, for example, by evaporation of the solvent, by precipitation upon addition of a non-solvent, or by extraction. Organic solvents that form the required azeotropes with water during the extrusion procedure include, for example, butyl alcohol, amyl alcohol, hexyl alcohol, benzyl alcohol, butyl acetate, amyl acetate, 1,2 - dichloroethane, 3 -pentanone, 2 - hexanone, benzene , cyclohexanone, toluene, xylenes, etc. Azeotropic distillation is particularly advantageous in fermentation processes on a large scale. The water and solvent taken from the top of the column can be separated by known methods and returned for further use. The separated water does not contain contaminants. The selected solvent can be returned to the process. Further purification of antibiotics A - 28086 includes additional extraction and adsorption. Jishkagel, coal, Florisil (magnesium silicate from Floridin Comp.), Etc. can be used as adsorbents. On the other hand, culture solids, including medium components and mycelium, can be used without separation, but preferably after removal of water, as the source of antibiotic A 28086. For example, the nutrient medium can be dried in a mixed vein and mixed with feed. On the other hand, after the end of fermentation in a nutrient medium, mycelium can be separated and, after drying, it can be used to obtain the product used directly for preparing the pre-feed mixture. When separating the mycelium for this purpose, the addition of calcium carbonate (about 10 g / l) blends filtering and gives a better-quality dry product. Strains of Streptomyces aureofaciens, called NRRL 5758 and NRRL 8092, form predominantly factor A of antibiotic A - 28086. Although the ratio of factors varies depending on the fermentation conditions used, but in general factor A accounts for more than 99% of the total antibiotic activity isolated from NRRL 5758 and npiaiepHo 90% of the antibiotic activity isolated from NRRL 8092. Factor B accounts for most of the rest of the activity isolated from NRRL 5758, factor D is minor. On the other hand, factor D is about 8-10% of the antibiotic activity isolated from NRRL 8092, and factor B is minor. Factors A, B and D are separated and isolated as individual compounds by known methods, such as, for example, a chromatograph on a column, chromatography on a thin layer, and the like. For example, silica gel column chromatography is used to isolate factors A, B, and D, and the column is eluted with mixtures of various solvents, such as benzene and ethyl acetate. When using such a mixture in a silica gel column, factor B is first fused, and then factors A and D. Typically, thin layer chromatography is used to control the elution process. Compounds A-28086 inhibit the growth of bacteria and fungi pathogenic to animals and plants. Below are the activities of factor A and B of antibiotic A - 28086 in relation to a number of microorganisms. The test was carried out using a conventional disk-diffusion method (6-millimeter toltons were immersed in solutions containing 1 mg of compound in 1 ml; the tampons were placed on agar plates, filled with test organ):

3 994ATCC

Growth area, mm factor A factor B 1921

2831

2616

1212

2366 17

1414

12Not tested

27

Conjunct A-28086 | o1b 1show effect ao inhibit the growth of anaerobic bacteria. Yayainayu ko1sTsentrnn, 11ri kotssa | factor A detains rayonny anaerobic Gaxispwiit

Subjects oftnaHaiijai

AsItutpusev bovCs Cto & iridiiuin tnocuutn CCostridium perjrtng-ens Ctostridiutn ramofteum CCo & triciium septicum

CtoftiridLuTn septicum bovine Eubacterium aet4) acUns Peptoeoccus anacrobius Peptostreptococcus intermediut PropiotiCbacterCum aene "i

PropiOnibaclerCum acne & 79 BocteroCdes fisp. Bacteraide "/ raoriCis ssp. tKetaiotac bacteroldes frag-ceit “sp. Vvtyarit Bocteroidet ssp. vuCg-ari "Fusobacteriutn si mCiObum

Pu «0bacterium necropkorum VeilConctta aCeaCeseens Cj-Ce - acyl efnry of factor A also have antibacterial and anti-bacterial activity. These derivatives have an increased activity against Gra11 “positive microorganisms. The activity of some of these derivatives is comparable to that of factor A. The compounds were tested by the nephelometric method on a semi-automated system. When testing antibiotics A-28086, the following parameters were used: Sl phylococcus aureus (H-Hitley) NRRLB-314 in a nutrient medium (pH7 was incubated for 4 hours at 37 ° C. Samples and the standard was dissolved in methanol / water (10:90) The reference, factor A, was at a concentration of 2.3.4 and 5 µg / ml. The compounds were diluted to about 3 or 4 µg of activity in ml before being placed in the apparatus. The relative activities of the compounds are shown in comparison with the activity of the standard. activity of C 1 Factor A (standard) ester ether A - 28.086 - A Propionyl A polyglycol ethers - 28086 - A Butirilovy ester A - 28086 - A Valerilovgy ester A - 28086 - A Kaproilovy ester A - 28086 - A

28

Lena standard Zethodzzh agar - dilution, the following figures are obtained after 24 - hour .-.

Smash concentration, g / ml

0, S 0.5 0, S

4.0 0.5 0.5

1.0 0.5 16.0 16.0 2.0 8.0 8.0 8.0 2.0 0.5

4.0 16 Compounds A 28086 also have anti-microbial activity against mycoplasms. Agents active against PPLO organisms are especially needed when breeding poultry. Below are shown the minimum concentrations of factor A of antibiotic A - 28086, which retard the growth of mycoplasma varieties, which were determined in vitro in a broth dilution study. Organism Minimum concentration, µg / ml M. galiseptjcum 12,5 M. hyorhims M. synovicil M. hypopneumoniae M. hyosynoviae, Solutions containing only 10 µg of antibiotic A - 28086 in 1 ml were used to disinfect surfaces to protect animals. from different types of mycoplasmas. Compounds A-28086 also have the ability to neutralize the effects of viruses. Factor A is active against type III polyvirus, vaccine virus, lichen free virus and Semliki forest virus. Factor A is also active in relation to the transmitting virus, the most prevalent gastroenteritis, virus, Nyskkasl's disease and the virus causing bulls rhinotracheitis.

Therefore, the connection And 28086 can be given, places or minus the digestive tract.

Deza da prevention or lessena viral efbopevayn varies from about 1 to S mg / kg body weight of a mammal, depending on the virus

to the value of the drug, i.e. with prophylactic

nlv therapist purpose.

In addition, solutions containing compounds A-28086, preferably in conjunction with the surface, the active substance can be used to disinfect the areas affected by the {{dying} virus or your poliomyelitis. For the fight against the virus, solutions containing examples from 1 to 500 µg / l of compound A – 28086 are effective.

The toxicity of factor A of antibiotic A-28086, administered intraperitoneally to mice, is defined as LD5. About 7.15 mg / kg.

Compounds A 28086 are also insecticides and skarns. These compounds are active against insects such as Mexican bean beetles, milkweed beetles, and domestic flies, as well as against such mites as a two-tailed arachnid mite, when used in an amount of 500 ppm. In addition, compounds A-28086 are active against mosquito larvae when applied in an amount of 1 ppm.

The ability of compounds A – 28086 B71 to affect cocciindi is important. For example, experiments on its use in feeding show that with the content of compound A-28086 in the feed, even in such a small amount (0.003%) an increase in weight is observed, and with a content in the amount of 0.005%, the mortality of chickens decreases, is infected. coccidi.

In tab. Figures 1, 2 and 3 show the results of experiments on the use of factor A for the treatment of chickens infected with different adami E i meria.

Table 1

31

 Average four) exp: 6 young males in each.

) Four experiences in each case; 5 young males in every experience.

For preventing or treating a disease caused by coccidi, birds are given daily, preferably through a beak, a nontoxic amount of compound A = 28086. Such a compound can be used in different forms, but it is best to be given together with a physiologically acceptable carrier, preferably mixed with feed. D. determining the appropriate concentration of compound A-28086 should take into account a number of factors, but generally concentration will be from 0.003 to 0.4% or preferably from 0.005 to 0.02% by weight of the feed.

576966

32 Table 2

Table 3

50

1.75

3.5

73 0.9

3.3

An important property of compounds A 28086 is also their ability to improve feed efficiency. For example, compounds A-28086 improve feed utilization efficiency. For example, compounds A 28086 improve

the effectiveness of the use of feed of ruminants, with a developed function of the rumen.

The effect of the use of carbohydrates in ruminants is increased by stimulating the flora in the animal rumen to form

derivatives of propionic acid, not in combination. The efficiency of utilization of carbohydrates was shown in experiments with animals with fistulas in scars.

Chyty was performed on animals weighing about 454 kg each. Dvhotkhotnykh received normal feed and four animals in each sub-crypt group received the same feed containing factor A. Otiralized aliquot amounts (100 ml) of cicatricial fluid and added to it 1

10% metaphosphoric acid. Samples were allowed to settle and the supernatant layer was analyzed by gas chromatography to determine 55, the concentration of propionic acid.

The results of the experiments will show an increase in the concentration of propionic acid in the cicatricial acid. fluids determined by five analyzes over a period of 14 days. In addition, SI vLs in vivo showed that factor A increases Tx215-1a1sbrma ovtsmi. Produce Factor O1B11Biotssa A - 2SQ86 are anti-viral anti-virus Factor D is active in the Maryland B virus, a type III polyvirus, a SCHA virus, a hepatic virus, and Semini forest virus. Factor D is also active against the virus that causes gastroenteritis, the virus of Newcastle disease and the cattle rhinotracheitis virus. The fact that factor O derivatives are active against viruses and anaerobic bacteria makes these compounds particularly beneficial for preventing enteritis in chickens, pigs, cattle, and sheep. Factor O compounds are also useful for the treatment of ectotoxemia in ruminants. The important property of factor D compounds is their activity against coccidia. Experiments in yitro show that factor D is active against Eiineria tenella. When cell cultures were infected with viable sporozoites followed by the addition of factor D, the following results were obtained. Activity against coccidia factor D of antibiotic A-28086; Concentration factor. Characteristic 1 μg / ml Results of two tests. Another useful feature of factor O derivatives is their ability to improve the efficiency of feed utilization by ruminants. These compounds effectively increase the amount of propionates and thus contribute to the effective utilization of feed when administered orally with ruminant choleshats in quantities ranging from 0.05 to 5.0 mg / kg. day. The most favorable results are achieved with doses of about 0.1 to 2.5 mg / kg per day. The compound is pre-mixed with food, but it can also be used in the form of tablets, pellets, and capsules. Such preparations are prepared by methods well known in veterinary pharmacy. Each individual dose should contain an amount of the compound corresponding to the daily dose for the animal. Antibiotic A - 28086 can be used in the composition of the feed intended for fattening5 JQ 15 20 30 30 35 40 45 50 55 60 or cattle in an amount of from 1 to 30 g of compound n ton of feed As stated above, the compounds A - 28086 represent anti-viral agents are also active against anaerobic bacteria, in particular against Clostridium perfringeusiltome; such compounds are suitable for the treatment and prevention of enteritis, pigs, cattle and sheep. They are also suitable for the treatment of entero-toxemia in ruminants. Example. A. Fermentations A - 28086 with stirring in a flask using s. aureofaciens NRRL 5758 Spores were loosened with bovine short and sterile loops. The resulting suspension of blinders and scorpions of mycelium was lyophilized in bovine short. One of the pieces of lyophilized balls was used for inoculation 50 ml of vegetative medium having the following composition: Ingredients Amount, g Glucose 20 Soya groats15 Water infusion of maize 10 Calcium carbonate 2 Water flow 1 liter. in an arc with a diameter of 50.8 mm with a spindle of 250 rpm; B. A-28086 fermentation in a vat using S. aureofaciens NRRL 5758. To increase the volume of seed, 10 ml of the obtained incubated vegetative medium was used for inoculation and 400 ml of vegetative medium of the second stage of the same composition. This medium was incubated in a 2-liter flask at 30 ° C for 24 hours in an apparatus rotating in an arc with a diameter of 50.8 mm with a speed of 250 rpm. The vegetative medium of the second stage (1l) was used to inoculate 100 liters of sterile medium of the following composition: Ingredients Amount, g / l Dextrin tapioca60.0 Casein hydrolyzed with enzyme8.0 Molasses15.0, O 0.5 CiaCSO, 2.0 5.0 1% 1firmedirovany Kassh Deionized water ml liter After sterilization in an autoclave at 120 ° C for 30 minutes under a pressure of 1.05-1.4 kg1, the pH value was 6.7. In a 165-liter fermentation tank, the inoculated medium was fermented for 10 days at 29 ° C. The fermentation medium was aerated with sterile air supplied at a rate of 0.4 volume of air per volume of medium per minute. The medium was stirred with conventional agitators at a speed of 250 rpm. PRI mme R 2. Isolation of antibiotic complex A-28086, formed by S. aureofacie NRRL 5758. Fermented broth (132 l), prepared as described in Example 1, was filtered using a filter (Hyflo Supertz, infusorna earth, Jones McVille Products Corp.) and received 97 liters of filtrate, which was extracted with an approximately equal volume of ethyl acetate. The extract was separated from the aqueous phase and concentrated to a volume of about 500 ml. This concentrated ethyl acetate extract was added: a large excess of petroleum ether (Skelly Zolve; about 10 liters) to precipitate, and that samien to separate the undesirable product. The separated filtrate was evaporated in vacuo and the A-28086 antibiotic complex (6.9 g) was obtained. The mycelial part of this complex was obtained by double extraction of the filtered mycelium with approximately half the volume of methanol (62 and 59 liters). The extracts were combined and concentrated in vacuo to remove methanol. After concentration, approximately 10 L of the aqueous phase remained, to which a dilute solution of sodium hydroxide was added to a pH of 7.5. The resulting solution was extracted twice with approximately equal volumes of ethyl acetate (9 and Ell). The extracts were combined and concentrated to a volume of approximately 400 ml. This extract was added to a large excess of petroleum ether in order to remove unwanted products in the above-described procedure for concentrating the filtered bulashuy extract. The mycelium portion of the complex obtained from the filtrate weighed 20.6 g. EXAMPLE 3. Isolation of individual factors A and B. The myceleral portion of the antibiotic complex A; 280B6 (235 g, as described in Example 3) was dissolved in about 80 ml of benzene. This solution was injected into a Si jacugel column (9x130 cm, 8l liter, Mate zone grade 62). The column was eluted with various mixtures of benzene and ethyl acetate. Elution was followed by thin layer chromatography. When using the solvent system benzene-ethyl acetate (90:10), factor B was first eluted, which was isolated as an individual factor. After crystallization from acetone, this factor was 43 mg and had tll. 150-153 ° L. Continuing to mix the mixture of benzene and ethyl acetate, but gradually increase the amount of ethyl acetate, extract factor A. The fractions containing factor A were combined and concentrated in vacuo. The residue was dissolved in acetone (about 150 ml) and about 160 ml of water was added to the solution. By adding 1 n-hydrochloric acid, the pH of the solution was adjusted to 3. The acidified mixture was stirred for 1 hour, resulting in precipitation. This precipitate was filtered and recrystallized (ca. 150 ml) after adding water (ca. 60 ml). The product was dried overnight in vacuo and factor A was dried (approximately 6.6 g). After partial isparein acetone from the filtrate, an additional 1.2 g of factor A was obtained. Example 4. Factor A acetyl ether. Factor A of the antibiotic A-28086 (7.41 g) was dissolved in pyridine (150 ml) and to the solution add 50 ml of acetic anhydride. The resulting solution was thoroughly mixed and allowed to stand overnight at room temperature. Then 200 ml of water was added, mixed thoroughly, and the mixture was kept for 4 hours at room temperature. The resulting white solid was filtered, washed with water and dried in air. The resulting solid was dissolved in acetone (100 ml) and the solution was dried to dryness in vacuo (this procedure was repeated 3 times). The residue was crystallized from a mixture of acetone (100 ml) and water (50 ml) to obtain factor A acetyl ether (614 g) with mp. 100-103 ° C. Examples are 5-9. The propionyl ester of the announcer A, obtained by the reaction of factor A with propionic anhydride in the presence of pyridine by the method of Example 5, had a mp. 96-98 ° C. Factor A butyryl ester was obtained by the reaction of factor A with butyric anhydride in the presence of pyridine by the method described in Example 5; tsh1 79-81 ° C. Caproyl ester of factor A was prepared by the reaction of factor A with caproic anhydride in the presence of pyridane by the method described in Example 5; m.p. 16-167 ° C. Valerdlovy ether of factor A was obtained by the reaction of factor A with varianic anhydride in the presence of pyridine by the method described in Example 5; t.Sh1.173-175 ° C. PRI me R 10. Preparation of sodium salt of factor A. Factor A (500g) was dissolved in acetone (50 ml). Water (50 ml) and 5N were added to this solution. sodium hydroxide solution to pH 10.5-11. The semi-gene solution was stirred for 1 hour and then extracted with ethyl acetate. The extract was scrubbed to dryness in vacuo and the residue was re-precipitated from the solution in the mixture

39

acetone and yoai, received 378 mg of sodium salt of factor A with T.Sh1. 120-123 ° C.

Examples 11-15. The barium salt of factor A was prepared from factor A (500 mg) and a saturated solution of barium hydroxide using the method of Example 10. We obtained 369 mg of the barium salt of factor A with T.PL. 199-190 ° C.

Potassium salt of factor A was obtained from factor A (500 mg) and 5N. solution of potassium hydroxide by the method of example 10: received 363 mg of the potassium salt of factor A; m.p. 165-167 ° C.

Cesium salt of factor A was prepared from factor A (500 mg) and 1i. cesium hydroxide solution by the method of example 10. 540 mg of factor A cesium salt; m.p. 190-210 ° C.

Sodium salt of factor B was prepared from factor B and 5N. sodium hydroxide solution using the method of Example 10.

Example 16 Fermentation A-28086 in a shake flask using S. anreofaciens NRRL 8092.

Culture Streptomyces aureofaciens NRRL 8092 prepared and weathered on oblique agar, which had the following composition:

IngredientsNumber, g

Kg NRO42.0

MgSO4 7H2O0.25

NH4 MO2.0

СОСОз2,5

FeSO4-7H2O0.001

MnS - 7H2O0.001

ZnS047H2O0.001

Glucose10.0

Agar20,0

Deionized water 1 liter pH (spontaneous)

7.7.

Oblique agar was inoculated with Streptomyces aureofaciens NRRL 8092 and incubated at 30 ° C for approximately 7 days. Agar-grown culture was coated with sterile & 1x serum and sterile loop culture for preparing spore suspension and mycelium. The resulting suspension was lyophilized.

One of the resulting lyophilic spheres was used to inoculate 50 ml of vegetative medium with the following composition:

IngredientsNumber, g

Glucose20

Soya flour15

Aqueous corn infusion 10 CaCO 2,0

Tap water up to 1 liter, pH after

adding MAON

6.5.

The inoculated vegetative medium in a 250 milliliter Erlenmeyer flask was incubated at 30 ° C for 48 hours on a shaker with a diameter of 50.8 mm rotating at 250 rpm.

40

The described incubated vegetative medium was used (0.5 ml of 1%) to inoculate 50 ml of the fermentation medium of the following composition: Ingredients Amount, g

Dextin Topioca 60,0

Casein Hydrolyzed with Enzyme 6.0

Enzymatic Casein Hydrolyzate 2.0

СОСОз2,0

MgS047H2015

Cherna reed - sugar molasses15,0

Refined Soybean Oil5.0

Water supply water 1 l, rZhsam. arbitrary) 6, (

1) Dextrin steels N 11 of the company A. E. Steel, Decatur, pcs. Illinois

2) Amber ShS,. Amber Lab, Giuno, - scht. Wisconsin

3) NZ Amin A, Sheffield Chemical, Norwich, Schm. New York

Example 17. Fermentation in vat A - 28086 using S.ureofaciens NRRL 8092.

In this case, the A-28086 fermentation procedure originally described in Example 16 was used. To obtain a larger volume of seed, Schl of incubated vegetative medium was used to inoculate a second-stage vegetative medium (400 ml) having the same composition. The medium of the second stage was incubated in a 2-liter Erlenmeyer flask at 30 ° C for 24 hours in a rotating shaker at a speed of 250 rpm.

The incubated vegetative medium of the second stage (800 ml) was used to inoculate 100 liters of sterile fermentation medium, which had the following composition:

Ingredients Colic. g / l

Q Dextrin tapioca 60,0

Hydrolyzed zyzimy

6.0

casein

Enzymatic hydrolyzate

casein

2.0 2.0

СОСОЗ

0.5 g / l

MgSO4i-7H2O

Cherna reed - sugar

molasses 15.0

Refined Soybean Oil5.0

Plumbing water 1 l.

1) Dextrin steels number 11 of the company A. E. Steel, Decatur, scht. Illinois.

2) Amber NS, Amber Laboratory, Juno, pc. Wisconsin.

3) NZ Amin A, Sheffield Chemical, Norwich, pc. New York.

After sterilization in an autoclave (for 30 minutes at a temperature of 121 ° C and a pressure of 1.05-1.4 kg / cm), the medium had a pH of 6.8 + 0.1. Fermentation proceeded for 10-12 days at a temperature of 28 ±

4J

± 1 ° С in a 165-liter vat when aerating the medium with sterular air supplied in an amount of 0.4 volume of air per 1 volume of the cooking medium per minute. The medium was stirred with agitators rotating at 300 rpm.

EXAMPLE 18. AHTHtffloTHKM A - 28086 was obtained as shisano in Example 17, but using a fermentation medium in a flask and a vat that had the following composition: Ingredients Amount, g / l

Dextrin tapioca30,0

Glucose15,0 Casein, hydrolyzed

enzyme 3,0 Enzymatic hydrolyzate

casein1,0

Yeast extract 2 "5i

CaCOj2,0

MgSO47H2O1,0 Black reed sugar

molasses15,0

Rafi jervil soybean oil5.0

Well conductive water 1 l.

1) Dextrin Steel, AE Steel, Decatur,

PC. Illinois.

2) AmberN Laboratory Amber, Juneau, pc. Wisconsin.

3) NZ Amin A, Sheffield Chemical, Norwich, pcs. New York.

After sterilization in an autoclave as described in Example 17, q, the medium had a pH of 6.4.

Example 19. The selection of the complex antenna & otika A - 28086, obtained using S, aureofaciens NRRL 8092.

Whole fermentation broth (60 l), obtained in the manner described in example 17, had pH 3 after the addition of diluted hydrochloric acid. This solution was filtered through a filter (P1FLO Super - whole, infusible earth, John-Manville Products Corp.) . The mycelial residue on the filter was extracted with 30 liters of methanol and 1.56-1 sodium bicarbonate was added to the extract with stirring. After separation of this extract, the mycelial residue was extracted again in vacuo to remove methanol. The remaining aqueous solution (7 L) was acidified with dilute hydrochloric acid to pH 7.5 and the resulting solution was extracted twice with ethyl acetate (twice with 7 L). The extracts were combined and concentrated in vacuo to give an oily residue, which was dissolved in 1500 ml of acetone. Diluted hydrochloric acid was added to this solution to pH 3 and stirred for 1 hour. The precipitate was filtered off, dissolved in acetone (1500 ml) and 400 ml of water was added to the solution. The resulting solution was held for 16 hours for crystallization. The crystals were filtered, dried in vacuo, and 74 g of crystalline product was obtained containing factors A and D and crystalline impurities.

42

Crude crystalline p;) oduc1 (40 i) pacino or approximately 250 ml of benzene and pactBfjp proj cnuiH column with silica gel (9x120 cm; ilkkagel grade 62 Grice-Davidson). The column was luled in succession with 40 liters of each of its not very thinning solvents:

1) Benzene

2) Benzene-ztirate 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.

Each fraction was tested, determined activity against Bacillus subtilis, and chromatographed in a tog bed to identify zljonated compounds A-28086, eluted with a mixture of benzene and ethyl acetate (4: 1). Factor B was zoned with a mixture of benzene and ethyl acetate (7: 3). Factors A and D were zoned with a mixture of benzene and acetyl acetate (7: 3 and 1: 1), fractions 119-156. These fractions were combined, dried to dryness and the residue was dissolved in 500 ml of acetone. To the resulting solution was added 500 ml of water, acidified with dilute hydrochloric acid to pH 3 and stirred for 1 hour. The precipitate formed was filtered and crystallized from a mixture of acetone (500 ml) and water (180 ml). The crystals were filtered, dried in vacuo, and 20.1 g of a mixture of reactors A and D were obtained.

Example 20. Separation and purification by INDIA of dual factors A and D.

f A crystalline mixture of factors A and D, prepared as described in Example 18 (18.8 g), was dissolved in benzene (50 ml). The solution was placed in a silica gel column (7 × 100 cm) (silica gel, Grade 60, Merck, thinner than 230 mesh ASTM). The column was vented at a flow rate of 90 ml / hour successively with the following solvents:

1) 12 liters of benzene;

2) 12 l of a mixture of benzene and ethyl acetate (9: 1); 3) 12 l of a mixture of benzene and ethyl acetate (4: 1);

4) 32l of a mixture of benzene and ethyl acetate (7: 3);

5) Yul methanol.

After chromatography in a thin layer (Merck Cellulose, Darmstadt on an aluminum substrate), bioautography was performed using B. subtilis to regulate the elution process. The following solvent system was applied: water – methanol – cetone (12: 3: 1), first adding ammonium hydroxide to pH 10.5, and then hydrochloric acid to pH 7.6.

Fractions with volumes from one to two liters were collected while they were found to be active: then they collected 200 ml fractions containing only factor D, which were combined and evaporated in a vacuum to dryness. The residue K1) was galvanized from a mixture of acetone and water. The crystals are peeled off and blown out in a vacuum; 140 g of crystalline factor D were obtained. The fractions containing factor D and traces of factor A were also treated, and additional ISO mg of crystalline factor D was obtained, containing a small amount of factor A. Fractions containing only factor A, 5 were also obtained, 4 , 7 g crystalline; factor A. Example21. Antibiotics A - 28086 were obtained in the process of Example 16, but using the slurry medium of the following composition: i o Ingredients 1 Solo, dairy extract 2.00 Dextrin 20.00 Yeast extract 2.00 Enzymatic casein hydrolyzate, 4.00 CoClj, 02 Agar 20.00 Deionized water 1 lC using KOH, the pH was adjusted to 7.0 and the inoculated media was incubated at 28 ° C for approximately 7 days. Example 22. Antibiotics A - 28086 were prepared in the process of Example 17, but using a medium of the following composition: Quantity, g / l Ingredients Dextin tapioca Ingredae Chopped yellow maize Soyeva flour, extracted with a solvent, crushed, finely ground, 50% protein% 1w shsh fat (dry beef toasted fish meal with soluble substances (60% protein) P & soluble substances of maize of wine and vodka distillery / food grade calcium phosphate food grade Calcium carbonate Vitamin CMeci (vitamins A, D, E, K, and B | 2, cholium, niashshsh, pentothenic acid, {zhboflavin, biot with a glucose binding agent A mixture of trace minerals (representing magnesium sulphate, zinc oxide, potassium iodide, ferrous sulphate and calcium carbonate) 2 - Amine - 4 - hydroxybutyric acid (methianine analog) Factor A of antibiotic A - 28086 These composites were mixed by the method used for mixing feeds. Those who were fed this feed, resisted the effects of coccidia and put on weight as fast as the healthy ones that received the same feed, but without an antibiotic. Enzi an {ch gdrolizovanny casein 15,0 Reed - saharf molasses 15,0 Calcium carbonate 2,0 Ammonium sulfate, o Magnesium sulfate 0.5 Refined soybean oil4,6 1) Dextrin Steels N11, AE Steel, Decatur, IgG. Illinois . 2) Amber ENS lab Amber, Juneau, Wisconsin. PRI me R 23. Antibiotics A - 28086 was prepared in the process of Example 21, but using an intermediate vegetative medium of the second stage, which had the following composition: Ingredients Amount, r / f Tsereloea 20.0 Aqueous maize infusion (wet weight) Soyeva croup Calcium Carbonate Yeast Beet Molasses PRI me R 24. Modified A - 28086 food for treating chicken disease caused by cocci. A balanced, high-quality chicken feed, which provides rapid weight gain, was prepared according to the following recipe: Number of th) and Ex. Meat for livestock with the addition of A-28086. Balanced with a high content of grain, feeds for fed cattle were prepared according to the following recipe:

Issues Ji ° kg

Shredded Corn6,78617

Shredded core of pochapse

corn1090.7

Dehydrated flour from alfalfa, 17%

squirrel S45,3

Flour from a collapsed dream,

solvent extraction9,995690.0

Reed molasses545

Urea 65.4

Factor A - 280866,00440.04

Dicalcium Phosphate food0,54,5

Calcium carbonate 0.54.5

Sodium Chloride 0,32,7

A mixture of minerals in the form of traces0,030,27

A mixture of vitamins A and D20,070,63

Pre Blend

, vitamin A ,

Calcium propionate OD 51.36

1) Contains in 1 pound (0.453 kg) 2000000 units. vitamin A; 227200 units of vitamin Oz and 385.7 g of soy feed with the addition of 1% ma

2) Dry corn kernels with a solvent containing

about; a-tocopheryl ester of acetic acid in the amount of 20,000 Mixed kbpM were pressed into tablets. The average daily ration is 6.8 kg for one animal. This feed contains approximately 300 mg of factor A per animal. PRI me R 26. Atsilovo ester of factor D of antibiotic A - 28086. Factor O was dissolved in pyridane and a stoichnometric caustic soda was added to the solution; acetic acid ash / y. The resulting solution was thoroughly stirred and allowed to stand overnight at room temperature. Zater was added an excess of water, carefully shifted kept for several hours at room temperature. The precipitate formed was filtered, washed and dried. The resulting solid {sifted in acetone and evaporated to dryness in vacuo; received a131tetilovsh zfir factor O. Examples 27-30. Propionic factor O ether was obtained by the reaction of factor D with an anhydride by the presence of gaonic acid in the presence of a pyrene dactus using the procedure described in Example 26. Butridate factor O ether was prepared by reacting Factor D with butyric anhydride in the presence of pyridite using the procedure described in Example 26 .. J Caprylic zyphir of factor O is prepared by the reaction of factor D with kapron anhydride. - Ingredients

Yellow Corn Hammer Flour salt splinter, solvent extracted and finely ground; 50% protein

amount

453.5

50

282

31.09 acids in the presence of pyridine using the method described in Example 26. Valery ester of factor D was prepared by reacting factor D with valeric anhydride in the presence of pyridine using the method described in Example 26. Example 31. Preparation of the sodium salt of factor D Factor D was dissolved in acetone and an equivalent amount of water and 5N were added to the solution. a solution of sodium hydroxide in an amount sufficient to adjust the pH to 11. The resulting solution was stirred for about 1 hour and then extracted with ethyl acetate. The extract was evaporated in vacuo and the sodium salt of factor O was obtained. Examples 32-34. Potassium salt of factor D polush | and from factor D and 5n. solution of potassium hydroxide by the method of example 31. The barium salt of factor D was obtained from factor D and a saturated solution of barium hydroxide by the method of example 31. The cesium salt of factor D was obtained from factor O and} n. cesium hydroxide solution by the method of Example 31. Example 35. Food for animals containing factor O and suitable for the treatment of coccidiosis. A balanced, high-quality feed, suitable for quick weight gain, prepared according to the following recipe: Quantity% kg

47

, UKaBonojA fat (baked beef) Dry ry & w with paciBopijMbiMH; 60% protein

Residues of maize, waste of ogarto, vodka production

Dicalcium phosphate pshtsevaya Calcium carbonate Mix of vitamins (vitamins A, O, E, KiB22, choline, shshtsin, nantothenic acid, riboflavin, biotin with an agent that binds glucose) Mixture of minerals contained in the form of traces, 2 amine-4 — oxyhydric acid ( oxy-anion methion Factor D These components are mixed in the usual way. The ration to the laKOM diet is resisted by coccidia, their weight increases in the same way as the weight of chickens not infected with coccidia and voluchashchik the same food, but without an antibiotic. Ingredients Fine-grained corn Shredded rod corn cob Dehydrated flour from alfalfa; 17% protein Flour from collapsed soybered, extracted with solvent; 50% protein Reed molasses Urea Factor Dicalcium phosphate food Calcium carbonate Sodium blend Mixed minerals in the form of traces Smell of vitamins A and D Contains in 1 pound (0.45 227200 units of vitamin Dj and 385.7 2) Dry grains with soluble alcohol and vodka distillery containing 1 pound.

The mixed feed was compressed into tablets. With an average amount of feed equal to 6.8 kg per day, each animal received approximately 300 mg of factor D daily Formula of the invention

Claims (2)

1. A method for producing an antibiotic complex A-28086, characterized in that 48 58.9 6.5 5.0 45D 36.3 16.3 7.27 4.53 0.9 h0.09 The culture of the microorganism Streptomycesaureofaciens NRRL 5758 / or Streptomycesaijreofaciens NRRL 8092 is grown under aerobic conditions on a nutrient medium containing digestible sources of carbon, nitrogen and inorganic salts, followed by separation from the culture fluid of the target product. 2. The method according to claim 1. that is, that from, the separated complex of the antibiotic is isolated Example 36. Meat feed containing factor D. Balanced high-grade cat food going to m with, prepared according to the following recipe:. The number of 000000 units. vitamin A by adding 1% oil. 20,000 food-, a-tocopheryl acetate 49 fractions containing A, B and O factors of this antibiotic; Priority according to the, featured: . 10.06.74 (for the application N 477954) on the basis of p. I Streptomyces aureotaciens NRRL 5758 - and on the basis of ariana-. Kup. 2-factor A; 04/21/75 (for application N 569740) on the basis of item 1 of Streptomyces aurefaciens NRRL 5758 - n for a prize, according to clause 2 - facto B; 50 04/21/75 (for application N 569719) on the basis of p. 1 of Streptomyces aurefaciens NRRL 8092 - and on the basis of p. 2 - factor D Sources of information taken into account during examination; 1. Author's certificate N 121542. cl. C12D9 / 18. 1959. 2. US Patent M 3155578, cl. 424-115,1964. / 9nf 9fnOfi 2Olf Csi §R Th  fe 3t § § P l§ 5 5tJ I "M j -j I h evj : 6 "i YU 8 | § %  fif f 3 /} iJOCfi / CM § JX / 7f ffAfJ trcflf it I I "" N (5) elO Csi  / Vi ffn oj sAuocfi / m c: a "5" O O if Csl -l8 Cs Sa nj S 4i CM ““ K "5 NC " ts. 5s “R I 5- c.  irT X § I  "I "I "five fn "N / f- / oj / o / Ji / oef / s "five § 2 ig "N uh about "five ъ se oi "and hell "five TO ABOUT cha S g iv I I ъ I I l sa vrj csi % s / ivгfno (