PL128225B1 - Method of obtaining the aglycone derivative of plasma thromboplastin component of the a-35512 antibiotic mixture - Google Patents

Description

The present invention relates to a method for the preparation of an aglycon derivative of factor B of the A-35512 antibiotic mixture. A-35512 antibiotics are closely related glycopeptide antibiotics. Factor B of the antibiotics A-35512, the most thoroughly characterized component of the antibiotic complex A-35512, is a novel antibiotic of the group of peptide-containing antibiotics such as vancomycin (US Patent No. 3067099), antibiotics A-4696, H, B, and C (U.S. Patent No. 3,952,095), Avoparcin (U.S. Patent No. 3,855,410), Reistoimycin A / N, Lonnakina, 7th International Symposium of Chemistry of Natural Products, Riga, 1970, p. 625) and ristocetin A (US Pat. No. 2,990,329. Antibiotics A-35512 differ from the above known antibiotics, including motility in various chromatographic systems and amino acid and sugar compositions. antibiotics, there is still a need to search for new, better antibiotics. One of the problems of modern antibiotic therapy is the fact that different antibiotics have different efficacy against pathogenic microorganisms. On the other hand, microbial strains are resistant to the effects of currently used antibiotics. In addition, individual patients often experience severe side effects after administration of certain antibiotics, such as hypersensitivity and / or toxicity. Therefore, it is preferable to produce new antibiotics active in diseases caused by microorganisms. It is also preferable to obtain feed additives increasing the feeding efficiency of animals and poultry. Increasing the efficiency of feeding is becoming increasingly important. Diethyletylbestrol and other estrogens are agents which increase the feed efficiency of many animals and poultry, but there is a risk that the residues of these feed additives will end up in the meat for consumption. Therefore, there is a real economic need to search for new ways to increase the efficiency of the limited feed resources available for the production of meat from animals and poultry. The use of the antibiotic mixture A-35512 or the individual agents and their derivatives is advances in the art. * The method according to the invention produces an agli-3 <conium derivative of factor B of the antibiotic mixture A-35512, designated as the antibiotic factor A-35512-B. The feature of the method according to the invention is that the microorganism Streptomyces candidus NRRL 8156, are grown in a medium containing a source of digestible carbohydrate and nitrogen, and unlimited salts, in the direction of indirect aerobic fermentation to obtain significant antibiotic activity, and then the A-35512 antibiotic mixture is isolated from the culture Factor B of the antibiotic mixture A-35512 is separated from this mixture and this factor is hydrolyzed with a moderately strong acid. Factor A-35512-B algal and its fermentable salts are used as a feed additive. for poultry in an amount that enhances the efficiency of feeding. In addition, factor A-35512-B aglycone and its pharmaceutically acceptable salts by the oral route of feeding animals a developed function of the term, in a dose increasing the amount of propionate, increases the food absorption of these animals. Factor A-35512-B glyconate and its pharmaceutically acceptable salts can also be used to increase the absorption of food in poultry, providing For these animals, an orally effective dose of factor A-35512-B aglycone or a salt thereof. The infrared spectrum of factor A-35512-B aglycone suspended in paraffin oil is shown in Fig. 1. The individual factors of A-35512 are closely related. relationships. As an antibiotic mixture, A-35512 is isolated from the fermentation broth up to seven antibiotic agents. The individual factors are separated and the factors A, B, C, E and H are separated. A-35512 mixture is soluble in water, partially soluble in alcohols such as methanol and ethanol, but insoluble in other organic solvents. such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile and dioxane. Factor A-35512-B is a white, amorphous, basic substance with the empirical formula of Coy. ^ e ^ gCl and the following approximate elemental composition: C - 53.97%, H - 4.75%, N - 5.25%, O - 34.29%, Cl - 1.59%. In particular, they follow the preferred empirical formula of C9H10H104NftC47Cl (calculated: C - 53.60, H - 4.75, N - 5.47, O - 34.30, Cl - 1.61). Another preferred empirical formula is CgH10aNg-O47Cl (calculated: C - 54.00, H - 4.75, N - 5.15, O - 34.50, Cl - 1.60). In the ultraviolet absorption spectrum of the factor A-35512-B has a maximum in acid and neutral methanol at 282 nm (s = 15,000) and in basic methanol at 292 nm (* = * = 16,000), the molar exposure coefficient being calculated for a molecular weight of 2000. in the ultraviolet there is also a final absorption band 5 at 225 nm. The A-35512-B factor shows the following values of specific rotational behavior: [a] ^ * »—423 ° (c = 1, water) and [a] JJB = ^ 446 ° -fc = 1, water). Electrometric titration of A-35512-B in a 66% aqueous solution of dimethylformamide shows the presence of four titratable groups with pKa values of about 7.15, 8.81, 10, 20, 12.00 and the possible presence of a group with a pKa greater than 13.50. The probable molecular weight of factor A-355H2-B, according to a titration, is around 2143. After crystallization of factor A-35512-B from 50% aqueous methanol, it is a white crystalline substance. Although factor A-35512-B dihydrochloride is hygroscopic and does not have a pronounced melting point, the thermogram shows a weight loss starting at 25 ° C and a 7.4% loss at 121 ° C and decomposition. temperature 135 ° C. Factor A-35512-B dihydrochloride has the following elemental composition: C - 52.57%, H - 30 4.80%, N - 5.66%, O - 32.8%, Cl - 4.51%. The above results of elemental analysis are in particular consistent with another alternative empirical formula CgsH ^ NgC ^ TCl • 2HCl (calculated: C - 51.93, H - 4.65, N - 5.57 , O - 33.20, Cl - 4.65). In the ultraviolet absorption spectrum of factor A-35512-B dihydrochloride * there is a maximum in acid and neutral meltanol at 282 40 nm (e = 12,000) and in basic methanol at 292 nm (e = 14,000), the molar extinction coefficient was calculated for a molecular weight of 2,000. There is also a final absorption band at 225 nm in the ultraviolet spectrum. Factor A-35S12-B dihydrochloride exhibits the following specific curvature values: [] d ≤ -128 ° (g * 1, water) and WL- -475 ° (c = 1, water). 50 The electrometric titration of factor A-35512-B dihydrochloride in a 66% aqueous solution of dimethylformamide shows the presence of four titratable groups with pKa values of about 7.15, 8.87, 10.30 and 12.10 and Possible presence of a group with a pKa higher than 13.1. Probable molecular weight of factor A-35512-B dihydrochloride is titrated around 2027. 60 In the magnetic resonance spectrum of 18C carbon dihydrochloride factor A-35512-B made in D20, there are the following signals listed in Table 1.5 Table 1 No. 2 3 4 5 6 7 8 9 10 U 12 13 14 15 16 17 18. 19.. 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 152 63 ¦54- ¦ 55 56 57 58 69 [60 ppm 173.0 171 , 9 171.6 171.0 170.8 169.6 159.0 157.9 157.5 156.6 155.6 155.3 154.9 154.3 151.7 144.3 136.7 136.2 135.4 135.2 133.6 133.3 129.8 129.3 128.8 127.6 126.1 124.2 122.4 122.0 120.7 116.5 109.5 108.2 107, 7 104.5 101.8 100.9 98.2 76.9 76.1 74.1 73.5 72.7 72.3 71.0 70.3 69.7 67.4 64.6 62.0 58 , 0 56.8 55.4 \ 54.3 24.5 17.9 17.2 16.3 height (%) 4.1 3.7 3.3 5.8 5.0 3.6 ¦ 4.1 4.4 3.7 4.8 6.1 4.2 3.3 4.2 3.3 3.1 3.5 4.9 4.0 4.4 4.2 4.1 1.7 3, 0 2.6 1.5 3.9 5.6 1.4 4.4 3.3 2.7 0.8 1.1 2.7 1.7. 2.9 1.6 1.0 1.2 1.8 2.0 2.7 ¦ 2.4 4.0 7.1 2.5 '. 2.5 '"74.7 * 1.2 1.5 1.3 1.7 3.9 2.5 2.0. 3.0. 2.0 2.5 | * Signal used as standard of dioxane 6 Factor A-35512-B dihydrochloride, crystallized from a mixture of methanol-water, has the following X-ray spectrum made by the powder method (Cu ++ radiation, 1.5405 X, nickel filter, d denotes the interplanar distances in angisters) with the following characteristics: ¬ in Table 2 Table 2 d 17.15 12.90 10.85 9.25 8.87 8.22 7.86 6.93 6.20 5.62 5.04 4.02 3.54 Relative 100 80. 70 70 60 50 50 40 40 40 05 02 a 02 The infrared spectrum of A-35512-B dihydrochloride made in a potassium bromide tablet is shown in Fig. 2. The most important absorption bands appear at the following frequencies ( cm-1): 3420 (strong), 3300 (bending), 2950 (weak), 1725 (weak), 1675 (strong), 1630 (bending), 1605 (strong), 1520 (strong), 1470 (weak) ), 1440 (weak), 1410 (weak), 1345 (bend), 1312 (average), 1225 (average), 1180 (weak), 1135 (weak), 1080 (strong) and 1020 (weak). Amino acid analysis of acid-hydrolyzed factor A-35512-B showed that the factor contains at least five amino acid residues, one of which is glycine. The four remaining amino acid residues for factor A-35512-B form a complex and appear to be identical to that found in factor A-35512-A. One of these amino acid residues is probably structured as shown in the figure. acid hydrolysis indicates that factor A-355L2-B dihydrochloride contains% of the following sugars: glucose, fucose, mannose, rhamnose, and 3Hamiin, o-2,3,6-ttrdisoxy-3 ^ C- {methyl-L-xy- lo-hexopyranose.7 188 22S * - As a result of mild acid hydrolysis, glucose, fufeesis, mannpza and rhamnose 4 are removed and the characteristic agglycan derivative is obtained. The dihydrochloride of factor A-35512-B has at least one esterifiable hydroxyl group. Factor A-35512-B dihydrochloride is soluble in water, partially soluble in alcohols such as methane and ethanol, and insoluble in other less polar organic solvents such as benzene, chlorine form, aeetone, diethyl ether, ethyl acetate, thorium, hexane, acetonitrile and dioxane. Factor A-35312-B dihydrochloride is stable up to 72 hours in normal solutions with a pH of about 3 to about 10. Factor A-35512-B glycone. Factor A-35512-B aglycone hydrochloride is a white, amorphous p substance with the following approximate elemental composition: C - 54.29%, H t ~ 4.34 %, N - 7.40%, Cl - t - 5.02%, O - ^ 28.95% (by difference). The infrared spectrum of the aglycone hydrochloride of factor Ar35512-B is shown in Fig. 1. The spectrum obtained from suspension in paraffin oil. The most important absorption bands are at the tapping frequencies (cm_l) and 3440 (bend), 3340 (bend), 3215 (strong), 2950 (bend), 2916 (strong), 284P (strong), 2640 (bend) , 1735 (weak), J655 (strong), 1590 (medium), 1500 (sdlme), 1460 (strong), 1378 (medium), 1365 (strain), 1298 (medium), 1115 (average), 1155 (average) , 1120 (weak), 1105 (weak), 1060 (weak), 1040 (weak), 1008 (medium), 925 (weak), 875 (weak), 765 (bend), and 718 (weak). Factor A-35512-B aglycone hydrochloride in a 68% aqueous solution of dimethylformanide indicates the presence of three titratable groups with pKa values of around 7.5, 9.25 and 11.0 and the possible presence of two further groups with pKa values above 11 , 6.Pfawdo | p4ot) ny cjejar (jgastecz ^^ wy phlorpwp- ^ Ojrku. Agli & pnu conilca A =? 5 ^ 1? TB, calculated on the basis of titration, take, and ejcofo 12 £ 2. Factor A aglycone hydrochloride = 35512- B will delete the following real value of fcrecalality, - civ: [«] g = -178« (e = 5, methanol) and [a]% = == ^ 716.8 ° (c = 5, methanol). (? l £ Ci £ chlpro-y / adnricu. factor A- aglionium, 35512-B occurs in ^ J ^ imuin in neutral and light methanol pr ^ m nm (ElVtcm = JQg, € 3) oi: aa m ^ imum in / basic methanol at 302 nrn (fifcm ^ 182.09 In the nuclear magnetic resonance spectrum of MC carbon of the aglycone of factor A-35512-B, made in dimethyl sulfate-de at the temperature of 90 ° C, the following signals appear, summarized in Table 3 Table 3 1 • No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 ppm 187.8 172.0 170, 7 170.1 169.6 168.4 166.7 157.4 156.6 • 155.7 155.6 155.4 154.3 149.3 138.8 136.9 136.3 135.2 134.7 133.8 128.2 126.2 123.0 121.3 117.7 117.0 108.6 106.7 105.7 103.2 93.6 74.9 71.8 68.9 63.2 60, 4 57.1 55.2 53.2 51.8 40.7 39.9 39.1 23.8 17.1 0.0 height (%} 37.2 40.9 45.2 46.9 47.7 57.6 52.5 49.9 45.5 55.8 71.5 56.7 50.5, 43.0 38.8 54.3 40 ,? 31.7 28.4 40.9 102.9 77 ,? 57.5 38.1 44.4 31.5 31.0 47.9 81.3 26.5 33.4 39, Q 33.9 40.3 43.1 33, § 57, a 33.1 30.1 36, Q 43.4 * 60.3 * 43.9 * 55.7 47.8 43.7 *) Diirnetyl sulfoxide-de signal 55 The 13C nuclear magnetic resonance spectrum shows that in the factor A-35512-B aglycone there is still 3ramino--2 , 3,6-trioxy-3-C-methyl-L-xylorhex (iranose, one of the sugars found in the molecule of factor A-35512rB. Amino acid analysis of the acidic hydrolysis of eayennic aglycone hydrochloride) A-35512-B shows that this aglycone contains glycine and has the least abrasive ressability. 9 128 223 it. One of these amino acid residues is probably of the structure shown in the figure. Aglycone hydrochloride Factor A-35512-B has at least one esterifiable hydroxyl group. Aglycone hydrochloride of factor A-35512-B is soluble in water and methanol but insoluble in less polar organic solvents such as benzene, chloroform - acetone, ethyl ether, ethyl acetate, toluene, hexane, aceto nitrile and dioxane. The Rf value of factor A-35512-B aglycone hydrochloride is 0.80 * in thin layer chromatography on cellulose and developing a mixture of n-butanol * pyridine * acetic acid and water (15; 10: 3: 12). ) -. The preferred development method is by-autography using Sarcin Lutea. The Rf value of factor A-35518 * B aglycone hydrochloride during thin-layer chromatography on silica gel (development with a mixture of methanol, chloroform and concentrated NH 4 OH (3: 2: 1) is 0. 26. Aglifton of factor A * 35512-B (free base) jfesite white A, an amorphous substance with the following approximate elemental composition: C - 52.65% ^ H - * 457 * / l N - 6.9l «fo, Cl - 2, 94tyo, O - 27.04 ° / *, po0i & l * ^ 4.7 W infrared spectra of factor A-3S512-B aglycone (free base), in potash bromide tablets, there are more important absorption maxima at the following frequencies (cm-1): 3360 (strong), 3z6Ó (inflection 2940 (inflection), 1735 (inflection), ifafo (strong), 1598 (medium 1510 (strong), 1445 (medium), lz95 (weak), 1215 (medium ), 1169 (moderate), "lizz (weak), 1070 (weak), 1018 (strong), Ub (weak) and tófl (weak). Electrometric titration of factor A-35Mz * B aglycone (slow) j base) in a 66% aqueous solution of dimethylformamide indicates that the presence of five titratable groups with pga values of about 6.2, 8, z, 10.1, li, 4 and tears, 4 possible presence of further or three groups With values greater than 12.5. The factor A-35512-B glyconate (free base) has the correct [a] ^ = —64.5 ° (c = 3, dimethylsulfoxide). In the afoisor vacirri, peyjny in the ultraviolet aiglritoonu factor A »3'3512-B (free base), there is a maximum absorption in neutral and acid methanol at 232 nm (Ej ^ = 43.65) and maxi * ftttlm in basic methanol at 301 nni CBf ^ * mt.tf The approximate ftf values of the factor A- & 5512 * -B (free base) aglycone are the same as described previously for the hydrochloride salt. In addition to the free base and factor A-3S5lz-E aglycone hydrochloride, factor A-35512 aglycone can be obtained as acid addition salts acceptable in pharmaceuticals. The term "pharmaceutically acceptable" denotes salts whose toxicity in warm-blooded animals does not exceed that of the free factors A-35512. Suitable and representative salts of agl and tail of factor A-35S12-B are those obtained by conventional reaction with organic acids. and inorganic such as sulfuric, phosphoric, vinegar, amber, lemon, lactic, maleic, fumaric, palmitic, cholic, mucic, mucic, D-glutamic, d-camphoric, glutaric, glycolic, phthalic , tartaric, lauryl, stearic, salicylic, methanesulphonic, sulphonic, sorbic, picric, benzoic, cinnamon and the like. Antibiotics A-35512 are produced by growing, Producing A-3S512, SBceep Stefcfc - tomyces candidiis NRRL 8156, under the conditions of subsurface and aerated maturation, in an appropriate nutrient medium, led to an adequate amount of antibiotics until the otphysiman. Antibiotics The isolation and isolation techniques are distinguished. The new microorganism used in the production of A-35512 antibiotics was isolated from soil samples collected in the Eniwetok Atoll. The minor organism has been classified as a new strain of & Streptomyeed candidus (Krassilnikov) Waksman, according to the "description of EB Shirling and D. Gottlieb in, Coogierative Be" feription of Type Oultures of StreptomyceB; II. Speeies Bescriptioms from Second Studies " . Intern. J. Systemafóc BacterioL, 18 (4). 279-10 302 (1968) and S.A. Waksmana "The Actinomycetes.Vol 8 CJaatóflcaitóGB ^ Idenitiifieatiion and Deiseritotions of Genera anid Speeies", Williams and Wiilktm Co., Baltimore, 1961.It is based on the U methods recommended by Mtea-national Pro-Streptom. fi.ft. ShirUhi and D. Gottlieb, "Methods fot Characlerl & atlon of Streptomyces Speeies", Intern.Buli; gystematlc Bacteriol., 16, 313-340, 1966) and Other Supplementary Tests. Barwy * bpisyWalió ziodnte * ISCC-NBS method, according to K. L. Kelly, ego and D. B. Judde, "The ISCC-NB3 Method of Detefmming Colors and a Dieti ^ nary of Color Nam« etJ *,) \ J-BK Department of Gommerce CM1. S58, Washingtclh, D. C, 1955. The symbols in the hawissaeh ordinary refer to the series of colors according to Tresner and Backus (HD Tre- ^ sner 1 SJ feadtus, "System of Color Whecls for Streptomyces ltlait ^ tlbmy, ^ Appl. Microbiol., 11 ^ 335 — SS8, 1963), and the color codes from the tables are under * underlined. The square symbols are given the symbols of colors according to Maerz and Paul (A. Maerz 1 IM. R Paul, "Dictionary of Oalor" \ McGraw-Hill ^ New-Yorkj N. Y., 1950). Cultures were carried out at 30 ° C for 14 days, unless otherwise stated. Characterization of the antibiotic producing strain A ^ 35,512. Morphology. Long, wavy feporotors are brewed. Disputes * 6 fusing into chains containing 10 — S6 j ^ bottom, hiaya cylindrical in shape and dimensions 0.7 to 1.05 ^ 1.4 to 3.s microns. Spore-like structures are produced on various nutrients. In some cases, bifurcated or bunched hyphae are observed. The surface of the spores, as observed under the electron microscope, is smooth. The cultivation characteristics of the various media are shown in Table 4 Table 4 Bread 1 1 ISP No. 2 (yeast extract agar and malt extract) ISP No. 3 (agar from oatmeal ISP No. 4 (starch agar with inorganic salts) ISP No. 5 (aspartic agar with glycerol) Emerson Agar • Modified. Bennett agar 1 and Characteristics 2 Profuse growth, grayish yellow underside (11J5), abundant aerial mycelium and sporulation, white (w) a; no soluble pigments Moderate growth, pale yellow-green underside (10B1); nice aerial mycelium and sporulation, white (w) 13ba; no soluble pigments, spore-like bodies are observed. Growth good; underside. amber-white (10C1), good mycelium air and nucleation, yellowish gray (GY) 2dc; light brown soluble pigments; spore-like bodies are observed. Growth good; pale yellow green underside (10B2); good air and nucleation properties, white (w) a; no soluble pigments; the mycelium appears to be in bunches. Growth good; light olive bottom (14L6); no air mycelium and no sporulation; light brown soluble pigments. Profuse growth; moderately yellow underside (11J6); abundant aerial mycelium and sporulation, white (w) a; no soluble pigments. | 225 12 cont. Table 4 1 Czapek agar Tomato paste and oat flour agar Nutritional glucose aspartic agar. Tryptone agar and yeast extract Tyrosine agar Glycerine and glycine agar 1 2 Growth good; underside pale yellow green (10B1 "; good aerial mycelium and germination, white (w) b; no soluble pigments; mycelium appears to be bundled. Abundant growth; greyish yellow husk (1115); abundant aerial mycelium and sporulation, white (w) a; mycelium "rolls" off the surface of the agar; no soluble pigments. Moderate to good growth; pale yellow-green bottom (1 &D2); 1 good aerial mycelium and sporulation, white (w) a; light and brown soluble pigments. Good growth; pale yellow bottom (17E1); good airborne mycelium and germination, white and (w) 13ba; no soluble pigments. Clear growth; white bottom (10A1); distinct aerial mycelium and sporulation, white (w) b; no soluble pigments. Growth good, white underside (10B2); good aerial mycelium and sporulation, white (w) e; brown soluble pigments; spore-like and similar bodies are observed. 1 Growth good; white bottom (10B2); good aerial mycelium and sporulation, white (w) b; no soluble pigments. / 128 225 13 continued Table 4 1 Calcium Malate Agar 2 Growth Good; sp6d white (10B1); good aerial mycelium and sporulation, white (w) a; no soluble pigments; the substrate becomes transparent around the contaminated surface. The organism was tested for selected physiological properties using standard practice. The following properties were found as given in Table 5. Table 5 Property Observed Effects on Milk Nitrate Reduction Preparation of maleic pigment on: peptone iron agar skis; Tyrosine agar slants (medium containing tryptone and yeast extract Gelatine liquefaction Temperature requirements (ISP medium No. 2 - slants with yeast and malt extract) Characteristics Coagulation with some transitions within 14 days There is no weak pigment formation after 7 days; none; total within 14 days 26-30 ° C - good growth and sporulation 37 ° C - poor growth; no aerial mycelium and spores 40 ° C - poor vegetative growth 45 ° C - no growth. Table 6 shows the results of the tests for carbon absorption by microorganisms NRRL 8156. The meaning of the symbols used is as follows: + - good growth, absorption (+) - poor to clear growth (-) - poor growth, probably no absorption - - no growth, no assimilation 10 15 25 35 40 45 50 55 14 Table 6 Carbon source None (negative control) D-glucose (positive control) L-arabinose Sucrose i-Inosit 11-mannitol D-fructose Rhamnose Raffinose D-xylose Mark no (-) + (-) (-) + + + (-) (-) (+) • Some characteristics of S. can- didus strain NRRL 8156, producing antibiotics A- -35512, the characteristics of the microorganism described by Elwood B. Shirling and David Cottlieb in ,, Cooparaitive Deseription of Type Cultures of Stre, ptomyces III. Additional Spacies Descriptions from First and Second Studies ", Intern. J. Systematic Bacteriol., 18, (2), 69 ^ 189 (1968) and by Waksman SA in" The Actinomyce- tes. Classification, Identification and Descriptions of Genera and Species ", Vol. 2, The Williams and Wilkins Co., Baltimore, Ed., 1961. The differences are summarized in Table 7. Table 7 Carbon uptake L-arabinose Rhamnose and-Inosit Gelatin Liquefaction Milk Treatment NRRL 8156 - - + Total within 14 days Coagulation with some clarity within 14 days Published description, + and + - Slow No coagulation, good peptonization 65 Antibiotic producing Streptomyces candidus strain A-35512 has been deposited in the collection strains of the Northern Regional Research Center, USDepartment of Agriculture, Agricultural Research Service, Peoria, Illinois, 61604, the collection of which is publicly available under number NRRL 8156. As a growth medium for Streptomyces can¬ didus NRRL 8156, any of the from many possible. However, for reasons of economy and in view of optimal yield and ease of isolating the product, certain culture media are preferred. For example, sucrose is a preferred carbohydrate in industrial production, although felukbze may also be used, tapioca decafcrine, fructose, mannitol, maltose, lactose, and the like. A preferred source of nitrogen is also pungent, but soluble. use soybean flour, flour, amino acids such as glutamic acid and similar products »The nutritional inorganic salts included in the nutrients include commercial roapufaGal salts *, which are a source of zinc, sodium, magnesium * calcium, ammonium, chlorine, carbonates, sulphates Nitrates and similar ions. The nutrient solution may also contain the basic trace elements necessary for the growth and development of the microorganism. Such elements are usually found as impurities in other nutrient components in sufficient amounts to meet the growth needs of the microorganism. Razite fw ^ zefey (a small amount (0.2 ml / ntrj soya ^^^ such as glflcolcol ^^ r ^ yie ^ bwy, if during large-scale fermentation it flares up with sU * proolem. For the production of large amounts of A-SSbiZ antibiotics, aerated fernlenta and aerated fibrosis are preferred. Small amounts of A-SS5 antibiotics can be achieved. yellow ^ aC In kolbaen culture on troughs, and later in the production of antiquities1, there is a general connection with the production of large items by inoculating a small volume of the culture medium with spore-forming forms or fragments of the mycelium of the organism, in order to obtain a fresh, actively growing culture of the microorganism, a fertile inoculum for It then starts to dry fermenters. The microorganism that produces antibiotics A-3551Z can be cultivated in a temperature range of about & 0-40 ° C. Optimal production efficiency of A * dd612 antibiotics is achieved at a temperature of & 0 ^ 34 ° C. This SW ^ glue has sweetness. In semi-turtle cultivation, with aeration, the sterile coating is passed through the phloid in order to achieve the efficiency of fine organism growth in industrial fermenters, preferably above 0.1 air volume per minute is passed through the medium per volume of nutrient For efficient production of A-35512 antibiotics, it is advisable to pass around 0.25 volumes of air per minute per volume of nutrient solution. The production process of A-35512 | fermentation stages can be controlled by analysis of samples of the wort or graying effect on the content of antipyretifrium. A fine drink is used which is known to be harmful to the preparation of antibiotics. Because such dróbnóustrojllW belongfcy & e cUIub subtilfc ATCC SB33. The biological assay was successfully performed using paper discs on low-nutrient agar agar plates. After fermentation, the A-35512 antibiotics can be isolated from the fermentation boundary using methods known in the art, Antibiotic production. fermentation of the fine * organism producing A-35512 is almost exerted in the wort flow. Therefore, the best isolation efficiency of A-3 & 513 antibiotics is obtained by first draining the mycelium mass. Prsesaezona wort can be eyes * szezac to obtain the A-35512 mixture, using a variety of methods. A preferred method consists in adsorption on a polyamide column followed by elution with water and aqueous mercury alcohol. The fractions of the eluate of sawteraja, antibiotics, are equal to% and are obtained with a mixture of A-8061 & MoMa funtea &lt; RTI ID = 0.0 &gt; using the same technique &lt; / RTI &gt; All factors A- & 5512. Further purification of the individual factors A-35512 consists in additional a-dissipation, elution. As adsorbent materials it is possible to use alumina, silica gel, sweep and gfcdeibne. At & Slft factors. they occur in the fermentation broth "# in the form of hydrochlorides. In a preferred method of separating the polyamides into the material, the factor A-35912 and a mixture are obtained. other factors in the form of cfaOlOWwater6lko% and Each of the individual factors tfioznaffife. Use nfroin as a source of A-i5512 antibiotics, omitting the extraction or separation operations, but preferably to remove the water. For example, after the end of fer- * mentaejl brown, containing antibiotics A-S86I2, can be lyophilized and used directly as an additive to the Aglitia of factor A-i5i512-B, it is produced by a libdf * acidic hydrolley c * yn -3SSlz * B, which is the easiest factor: J «si Olfiy ^ washed in a two-chambered half-chine *. ^ StA & also duodenum Factor A-35512-B hydride is a preferred starting material for the preparation of A-39512- aglycone B. Free factor A-35512-B or another acid addition salt thereof can be used for this purpose. Acid hydrolysis is carried out using known methods, also for the purpose of Stottiwac wtele r * fc riyeh ltWasow, it is preferable to salt if hydrochloric acid is used, then aglycone of factor A-35512-B replaced by slfc in the thick of chlorine. The hydrolysis is preferably carried out in water at the boiling point - for a time from about 1 to about 2 hours. The slower reaction time leads to the degradation of the aglycone, resulting in less active and finally inactive products. The optimal time for specific reaction conditions can be determined by analyzing the reaction mixture for biological activity. Table 8 shows the activity of the agent A-35512-B aglycone as determined by the agar diffusion method. The test was performed by plating on infected agar plates. With the tested microorganism, paper discs 6 mm in diameter were previously immersed in a solution containing the test compound at a concentration of 1 mg / ml. 1S Table 10 Table 8 Test organism Staphylococcus aureus Bacillus subtilis Saroin lutea Bacillus subtilis * Size of the inhibition zone (in mm) of the A-35512-B factor aglycone free base 19 and 13 14 22 *) On low-nutrient agar. 9 presents the lowest inhibitory concentrations (MIC) showing the activity of A-35512-C aglycone hydrochloride against selected Staphylococcus aureus strains in the standard agar dilution test. Table 9 Test organism Staphylocopcus aureus 3055 aureus H290 * Staphylococcus aureus V92 * Staip ^ ylocciccus aureus VI04 * Staphylococcus aureus 3Q74 ** Staphylococcus aureus H43 ** Sitophylpcoccus aureus V57 ** S ^ aphylpooccus aureus £ i3SJ6 ** Staphylocopcus aureus 373ocQ *** Staphylocopcus aureus 373ocQ *** µg / ml) 0.5 0.5 0.5 0.25 0.25 0.5 0.5 1.0 0.5 0.5 *) Penicillin G sensitive **) Penicillin G resistant *** ) resistant to penicillin G and methicillin ****) resistant to penicyline G, methicyl ine and clinicamycine Table 10 lists the results of an additional agar dilution test showing the activity of factor A-35512-B aglycone hydrochloride against different strains of Group D Streptocopia. 10 15 40 45 50 55 60 65 Test organism Streptococcus sp. Shrigley Streptococcus sp. Mitis Streptococcus sp. 12253F Streiptoooccus sp. SS992 Streptococcus sp. 9933 Streptococcus sp. 9913 Streptococcus sp. 292 Streptococcus sp. 238 MIC (^ g / ml) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 20 25 30 35 Factor A-35512-B aglycone exhibits in vivo antibacterial activity against experimentally induced bacterial infections. Infected mice were administered two intradermal doses of factor A-35512-B aglycone hydrochloride and the observed activity was determined as the ED50. The ED50 values obtained for factor A-35512-B hydrochloride are given in Table 11. Table 11 Test organism Streptococcus pyogenes C203 Streptococcus pneumoniae Staphylococcus aureus 3055 ED50 5.8 7.0 1.04 Infection rate {intraperitoneal) 1570XLDSO 374X U) 5D 370XLD5Q Antibiotics A-35512 are relatively nontoxic, for example the LD50 for factor A-35512-B dihydrochloride when administered intraperitoneally to mice is about 1356 mg / kg and when administered intravenously 1000-1250 mg / kg Another valuable property of the mixture of A-35512 and its components is the ability to increase the feeding efficiency of animals. This has been found in the case of animals with a high velocity function of the lather. It is known that the efficiency of carbohydrate absorption in rewormers is increased by an action which stimulates the production of the bacterial phloem with propionate instead of acetates or butter. This is described in detail by Church and colleagues in Digestive Physiology and Nutrition of Ruminants, Vol. 2, 1971, pp. 622 and 625. Feed efficiency can be determined in vivo in cattle with an erectile fistula, using Using the method described by Arthur P. Raun in US Patent No. 3,794,732, especially in Example VIII. Table 12 shows the results of this test c) for factor A-35512-B dihydrochloride. The propionic acid concentration is the average of the five analyzes carried out over a 21 day period. Table 12 Control test Test with little A-35512-B (50 g / ton) Number of animals 5 5 Average mole percent of propionic acid. 19.3 26.0 Molar percentage of increase in relation to the control sample - 6.7 A-35512 mixture and its components increase the concentration of propionates and thus increase the feed efficiency when fed in daily doses of 0.15-10.0 mg / kg Better results are achieved with dosing 1.0-2.0 mg / kg / day. A preferred method of administering the A-35512 mixture or a particular compound is by mixing them with the animal feed. These antibiotics can also be administered by other means, for example in the form of tablets, throat enemas or capsules. The foregoing pharmaceutical forms are prepared by methods well known in the art of veterinary drugs. Each single dose of the drug should contain a mixture of A-35512 or a component thereof in an amount corresponding to the daily dose administered to the animal. Mixture A-35512 and its components are also useful as growth factors for animals. The following examples illustrate more precisely the method of the invention. Of these examples, Examples 1-6 relate to the preparation of starting materials. EXAMPLE IA Fermentation of A-35512 in swing flasks. The lyophilized pellets of Straptomyces candidus NRRL 8156 are dissolved in 1-2 ml of sterile water and blocked with a solution. Agar slants containing yeast and ice Bacto extract (ISP No. 2, manufactured by Difeo Laboratories, Detroit, Michigan). The contaminated stacks are incubated at 30 ° C for approximately 7 days. The mature culture on the slants is covered with 2 ml of water and a sterile spore pipette is scraped off. 0.1 ml aliquots of aqueous spore suspension are used to infect the next ISP # 2 agar slant. The infected slant is incubated at 30 ° C for about 7 days. The mature culture on the slants is covered with 5 ml of water and the contents are scraped off with a sterile pipette. 2.5 ml portions of the obtained spore suspension are used to infect 30 ml of the vegetative nutrient solution with the following composition: 225 20 Ingredient Triptych-soybean (Baltimore Biological Laboratory, Cockeysville, Md). Demineralized water Quantity 30 g to 1 1 liter............................ Erlenmayer flasks are incubated in 250 ml Erlenmayer flasks at 30 ° C for 48 hours on a rotating jitter of 250 revolutions per minute with a swing of 50 mm. 15 0.5 ml of incubated vegetative medium is used to contaminate 50 ml of production medium with the following composition: Ingredient Tapioca Dextrin Glucose NH4NO3 KCl MgSO4 FeCl2-4H20 ZnCl2 KH2PO4 L-glutamic acid DL-citrulline / Caulalized water liter 25.0 10.0 2.5 1.5 1.1 0.03 0.03 0.1 1.0 0.1 5.0 to 1 liter The banned production medium is incubated in 250 ml Erlenmayer flasks, at 32 ° C for 8-10 days, on a rotating shifter with 250 revolutions per minute and with a deflection of 50 mm. B. Fermentation of A-35512 in fermenters. In order to obtain a greater amount of inoculum, 20 ml of incubated vegetative medium, obtained as described above, is used to infect 400 ml of stage II growth medium with the same composition as the composition of the vegetative medium. The medium of the second stage is incubated in 2 liter flasks for 24 hours at 32 ° C, on a rotating jig with 250 revolutions per minute and a swing of 50 mm. 800 ml of stage II vegetative medium is used to prohibit 100 liters of sterile production medium. The banned production medium is fermented in a 165 liter fermentor for about 8-10 days at 32 ° C. The contents of the fermentor are aerated 6 (J using 0.25 air volumes per minute per volume of nutrient medium, and mixed using a stirrer usually at 200 revolutions per minute. Example II. Incubated vegetarian nutrient, prepared as indicated in point A, may be stored for later use by keeping it under liquid nitrogen vapor. For this purpose, in a test tube measuring 13 X 100 mm, quilted and fitted with a screw cap, 2 ml of a suspension of the dispersant is placed in the following composition: Ingredient Glycerin Lactose Demineralised water Amount 20% 10 70% / "2 ml of the vegetative culture incubated for 48 hours, prepared as described above, is added to the above suspension. The mixture is frozen and held under vapor in a container containing liquid nitrogen Stored in this way, the vegetative inoculum is washed and washed with a dipping vial in a water bath at 43 ° C. 1 ml of the thawed inoculum is to contaminate 30 m of veggie medium with the composition given in A. The forbidden vegetative medium is used, as described above, for fermentation in swing flasks or to obtain more inoculum for fermentation in fermenters. Example III. The fermentation process is carried out as described in example I, using the production medium for cultivation in flasks or fermenters with the following composition: Ingredient Peks, tapioca trin Molasses BQzjp (stockable meat peptone MgSO4-7H20 CaCO03 Water Amount (g / liter) 75, Q 40.0 15.0 0.5 2.0 to 1.0 liters Example IV Isolation of Antibiotic Mixture A-35512. x The total fermentation broth (946 liters) obtained as described in example I is filtered using filtration aid (Hyflo Super-cel diatomaceous earth from Johns-Manville Products Corp.). The pH value of the wort is 6.8-7.2. The clear filtrate is passed through a column containing Amberlite XAD-4 resins (manufactured by Rohm and Haas Co.), using 10 ml of bouillon and 100 ml of wort percolation. Subsequent fractions are tested for biological activity using standard disc analysis for Sarcin lute. The biologically inactive fractions are discarded. The columns are washed with water equal to (225 22 1/8 volume of wort ki, at a rate of 150 ml / minute. Inactive rinse water is discarded. The product is then eluted with 600 liters of 50% aqueous methanol at a rate of 200 ml / minute. The eluates containing the A-35512 antibiotic mixture are concentrated under reduced pressure to a volume of approximately 15 liters. One liter contains about 200 grams of A-35512 antibiotic mixture. Example 5: Isolation of individual factors from A-35512 antibiotic mixture. About 3000 grams of A-35512 antibiotic mixture dissolved in 15 liters of methanol, o-! kept in the manner described in example IV, chromatographed on a column filled with 100 liters of polyamide produced by Woelm. The column is eluted with demineralised water at a rate of about 80-120 ml / minute. The collected fractions are examined by thin-layer chromatography on cellulose or chromium - tografiL bdbujpwej, using to develop mixtures of n-butanol, pyridine, acetic acid and water Q15: lli0: 3: ll2) and to induce bioautoigpra! fie against Sercina lu'tea. The first 100 liters of eluate are discarded and fixed flow rate of about 16-200 ml / minute, 12 fractions with a volume of 12 liters each are peeled, then the eluent is changed and eluted in a * ° gradient of methanol and water, using the following procedure. The tank containing 360 liters of methanol is connected by a joint siphon with a tank containing 120 liters of water. The solvent mixture is mixed and applied to the columns. 24 fractions of 24 liters each are collected, eluting at a rate of 200-300 ml / min. Based on the results of bioautography, the individual fractions are combined into groups and evaporated under reduced pressure to give factor A-35512-B dihydrochloride. and the following enriched mixtures of other factors as shown in Table 13. Table 13 Fractions 1-10 11-24 15-31 32-44 Volume Qg and (in liters) 120 216 168 312 A + HB B + CC , E, F, G Amount 192 g 269 g 590 g 224 g 55 Example VI. Purification of factor A-35512-B. Partially purified 400 g of factor A-35512-B dihydrochloride, prepared according to Example V, are dissolved in 1.2 liters of 50% aqueous methanol and chromatographed on an oxide column. aluminum. The column is prepared as follows. 10 kg of acidic alumina (from Woelm) is mixed with 50% of an aqueous solution of methanol. The mixture is allowed to stand, the Su-23 12S 225 24 pennatant is decanted and discarded. The alumina is remixed with 50% aqueous methanol and charged to a column 13.5 cm in diameter, then washed with 50% aqueous methanol until a clear eluate is obtained. The product is eluted with a 50% aqueous methanol solution at a rate of about 8 - 10 ml / minute, collecting fractions of about 240-300 ml. Subsequent fractions are examined by means of thin layer chromatography, using a bio-autograph as described in Example 5 for development. From the results, the individual fractions are combined and the purified factor A-35512-B dihydrochloride is obtained. The results are shown in Table 14. Table 14 Fractions 17-21 22-29 30-37 Amount 9.6 g 72.0 g 117.0 g Each of the above fractions was crystallized at 4 ° C. from a concentrated 50% aqueous methanol solution. The resulting A-35512-B dihydrochloride contains about 4.6% chlorine. The pH value of a solution of factor A -3 / 5512-B dihydrochloride in a 66% aqueous solution of dimethylformamide is about 6.5. Example VII. Preparation of A-35S12-B aglycone. 5.0 g of factor A-35512-B dihydrochloride, measured in accordance with Example VI, is dissolved in 200 ml of water. The solution obtained is acidified with 14 ml of 4N hydrochloric acid and heated to reflux for two hours, then cooled and evaporated to 3/4 of the original volume under reduced pressure. Then 6N hydrochloric acid is dripped in until the precipitate is completely settled. The precipitate is filtered off and dried to give 3.56 g of crude aglycone A-35512-B hydrochloride. After concentration and analysis of the effluent, glucose, fucose, mannose and rhamnose are present. The raw aglycone A-35512-B is purified. by chromatography on acid-washed alumina (Woelm, type I), eluting with a 1: 9 mixture of water and methanol. The successive fractions are analyzed by means of thin-layer chromatography on cellulose. The fractions containing the A-35512-B sail are pooled and concentrated under reduced pressure. 398 mg of partially purified product are obtained. The results of thin layer chromatography using ninhydrin development indicate the presence of biologically inactive impurities. Portions (100 mg) of the above partially purified factor A-35512-B aglycone are further purified by MN-SC polyamide chromatography. -6 (manufacturer Machery, Nogel and Co., seller Brinkman Instruments Co., size 0.07 mm) when used for eluting water. The fractions are analyzed by thin layer chromatography as previously described. The eluate fractions containing the factor A-35512-B aglycone are pooled and lyophilized. There are obtained 64 mg of purified aglicon hydrochloride of factor A-35512-B. The overall yield of the starting A-35512-B factor is 5.08%. Example VIII. Preparation of factor A-35512-B aglycone free base. 50 mg of factor A-35512-B aglycone hydrochloride obtained according to Example VII are dissolved in 10 ml of a 1: 1 mixture of methanol and water. The solution is neutralized with 3.5 ml of Bio-Rad AC-3-X ion exchanger in hydroxide form. The mixture is stirred for 15 minutes at room temperature, then the exchanger is drained off, and the permeate is concentrated under reduced pressure to half its volume, at a temperature not exceeding 60 ° C, and then lyophilized. 68 mg of factor A-35512-B aglycon free base is obtained. Patent claim A method of producing an aglycon derivative of factor B of the A-35512 antibiotic mixture, characterized in that Streptomyces candidus NRRL 8156 is grown in a medium containing an available carbohydrate source. and nitrogen and inorganic salts, under the conditions of sub-surface aerobic fermentation, to obtain significant antibiotic activity, then the A-35512 antibiotic mixture is separated from the culture medium, from this mixture the factor B of the A-antibiotic mixture is separated. 35512 and the refrigerant is hydrolyzed with a moderately strong acid. 15 20 25 30 35 40 45 0128 225 HOOC NH. 0H Cl H ^ N COOH DO 80 60 40 20 \ O e, 5 Formula i * i "iii i '¦ i — i — i — i— ~ ii 1CW JSPO 3PÓO 25ÓO 2000 1800 1500 1400 1200 1000 800 600 400 250 ( OM-0 # 6 / i.? 1M 80 £ 0 40 20 0 40 S ¦ 00 3 - '' t '5500 3000 A \ E50O 5 i 2ooo 4600 A ^ 7 & 3 10 12 14 16lW0tfJ0 / i0 - IWito. / ^ ¦ 16 00 lAOtf 4 * 00 400 © 80O 60 (7 W 15o FIG. 2. PL

Claims (2)

Claim 1. A method of producing an aglycon derivative of factor B of the A-35512 antibiotic mixture, characterized in that the microorganism Streptomyces candidus NRRL 8156 is grown in a medium containing a source of assimilable carbohydrate and nitrogen and inorganic salts under the conditions of sub-surface aerobic fermentation, to obtain significant antibiotic activity, and the A-35512 antibiotic mixture is separated from the culture medium, the B-factor of the A-35512 antibiotic mixture is separated from this mixture and this factor is hydrolyzed with a moderately strong acid . 15 20 25 30 35 40 45 0128 225 HOOC NH. 0H Cl H ^ N COOH DO 80 60 40 20 \ O e, 5 Formula i * i "iii i '¦ i — i — i — i— ~ ii 1CW JSPO 3PÓO 25ÓO 2000 1800 1500 1400 1200 1000 800 600 400 250 ( OM-0 # 6 / i.? 1M 80 £ 0 40 20 0 40 S ¦ 00 3 - '' t '5500 3000 A \ E50O 5 i 2ooo 4600 A ^ 7 & 3 10 12 14 16lW0tfJ0 / i0 - IWito. / ^ ¦ 16 00 lAOtf 4 * 00 400 © 80O 60 (7 W 15o FIG. 2. PL