GB1574023A - Antibiotic a-7413 and process for preparation thereof - Google Patents

Abstract

The antibiotic mixture A-7413, which contains the factors A, B, C and D, and the physiologically tolerated salts of factors A, B and C, are obtained by culturing the novel microorganism Actinoplanes sp. NRRL 8122, under submerged, aerobic fermentation conditions, in a culture medium containing assimilable sources of carbohydrate, nitrogen and inorganic salts until a significant quantity of the antibiotic is formed and, where appropriate, forming physiologically tolerated salts of factors A, B and C of the A-7413 antibiotic. The A-7413 antibiotic mixture obtained by the process according to the invention, and the A-7413 compounds obtained by the process, are, for example, useful agents for promoting growth and for controlling dental caries and acne.

Description

(54) ANTIBIOTIC A-7413 AND PROCESS FOR PREPARATION THEREOF (71) We, ELI LILLY AND COMPANY, a corporation of the State of Indiana, United States of America, having a principal place of business at 307 East McCarty Street, City of Indianapolis, State of Indiana, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The present invention relates to antibiotic mixture A-7413 comprising factors A, B, C and D, its production by cultivation of the novelActinoplanes sp. NRRL 8122 under submerged aerobic conditions, its separation and the isolation of factors A, B and C. It also relates to the C1-C4-alkyl esters, C1-Cs-acyl esters and thiol-C2-C4-carboxylic acid derivatives of A-7413 factors A, B and C and the physiologically acceptable salts of A-7413 factors A, B and C and the Cl-C5-acyl esters and thiol-C2-C4-carboxylic acid derivatives thereof. The antibiotic A-7413 mixture and A-7413 compounds are useful as growth promoting agents and in the control of dental caries and acne.

The A-7413 antibiotics are new members of the sulfur-containing thiostrepton family of antibiotics. Other members of this family include siomycin, taitomycin, thiostrepton and thiopeptin B.

The group of sulfur-containing antibiotics of this invention are designated as A-7413 antibiotics.

It is an object of this invention to provide antibiotic A-7413 mixture, A-7413 factors A, B, C and D, the C1-C4-alkyl esters, Cl-C5-acyl esters and thiol-C2-C4-carboxylic acid derivatives of A-7413 factors A, B and C. and the physiologically acceptable salts of A-7413 factors A, B and C and the Cl-C5-acyl esters and thiol-C2-C4-carboxylic acid derivatives thereof. It is also an object of this invention to provide processes for the production of antibiotic A-7413 mixture by the cultivation of Actinoplanes sp. NRRL 8122, the separation of antibiotic A-7413 mixture from the culture medium, the isolation of A-7413 factors A, B. C and D, and the preparation of the Cl-C4-alkyl esters, Cl-C5-acyl esters and thiol-C2-C4-carboxylic acid derivatives of A-7413 factors A. B and C and the physiologically acceptable salts of A-7413 factors A. B and C and their Cl-C5-acyl esters and thiol-C2-C4-carboxylic acid derivatives.

The present invention provides an antibiotic A-7413 mixture comprising factors A, B, C and D; A-7413 factor A. A-7413 factor B. A-7413 factor C and A-7413 D; the C1-C4-alkyl esters, C1-Cs-acyl esters and thiol-C2-C4-carboxylic acid derivatives of factors A, B and C; and the physiologically acceptable salts of factors A, B and C and the C,-C5-acyl esters and thiol-C2-C4-carboxylic acid derivatives thereof.

The present invention also provides a process for producing an antibiotic A-7413 mixture comprising factors A. B. C and D; A-7413 factor A, A-7413 factor B, A-7413 C or A-7413 D; the Cl-C4-alkyl esters, Cl-C5-acyl esters or thiol-C2-C4-carboxylic acid derivatives of factors A. B or C; or the physiologically acceptable salts of factors A. B or C or the Cl-C5-acyl esters or thiol-C2-C4-carboxylic acid derivatives thereof comprising: (a) cultivating Actitioplanes sp. NRRL 8122 or an A-7413 mixture-producing mutant thereof in a culture medium containing assimilable sources of carbohydrate, nitrogen and inorganic salts under submerged aerobic fermentation conditions; (b) optionally. separating antibiotic A-7413 mixture from the culture media; (c) optionally. isolating A-7413 factors A, B. C or D from antibiotic A-7413 mixture; (d) optionally. producing the Cl-C4-alkyl esters, Cl-C5-acyl esters or thiol- C2 C4-carboxylic acid derivatives of A-7413 factors A, B or C; and (e) optionally the producing physiologically acceptable salts of A-7413 factors A, B or C or and the Cl-C5-acyl esters or thiol-C2-C4-carboxylic acid derivatives thereof.

The present invention also provides a composition for increasing feed-utilization efficiency in ruminant animals having a developed rumen function comprising a veterinarallyacceptable carrier and antibiotic A-7413 mixture A-7413 factor A, A-7413 factor B, A-7413 factor C or A-7413 factor D; the C1-C4-alkyl ester, C,-C5-acyl ester, or thiol- C2-C4 carboxylic-acid derivatives of A-7413 factors A, B, or C; or the physiologically-acceptable salts of A-7413 factors A, B, and C or of C1-C5-acyl esters or thiol-C2-C4-carboxylic acid derivatives of A-7413 factors A, B, or C.

The present invention, also provides a method of increasing feed-utilization efficiency in ruminant animals having a developed rumen function comprising orally administering to such animals an antibiotic A-7413 mixture comprising factors A, B, C and D; A-7413 factor A, A-7413 factor B, A-7413 factor or A-7413 factor D; the C1-C4-alkyl ester, C1-C5-acyl ester, or thiol-C2-C4-carboxylic acid derivatives of A-7413 factors A,B, or C; or the physiologically-acceptable salts of A-7413 factors A, B, or C or C-C5-acyl esters or thiol C2-C4-carboxylic-acid derivatives of A-7413 factors A, B, or C.

The infrared absorption spectra of individual A-7413 factors A, B, and C in KBr disc are presented in the drawings as follows: Figure 1 - A-7413 Factor A Figure 2 - A-7413 Factor B Figure 3 - A-7413 Factor C The A-7413 mixture comprising factors A, B, C, and D is produced by cultivating under controlled conditions a strain of Actinoplanes sp. NRRL 8122.

As in the case with many antibiotic-producing cultures, fermentation of an A7413-producing strain ofActinoplanes sp. NRRL 8122 results in the production of a number of antibiotic substances. Antibiotic A-7413 factor A is the major factor produced by this culture, and factors B, C. and Dare three minor factors. Other factors are present in only very minor quantities or are relatively unstable.

The antibiotic A-7413 factors A, B, C, and D are co-produced during the fermentation and are obtained as the antibiotic A-7413 mixture. The ratio of individual factors produced in the antibiotic mixture will vary depending on the fermentation conditions and the individual antibiotic factors are separated from each other and isolated as individual compounds as hereinafter described.

A-7413 factor A is a white to light-yellow crystalline material which melts with decomposition at 205 to 212 C. A-7413 factor A crystallizes from ethanol, chloroform:ethanol, and dimethylformamide:acetone.

A-7413 factor A is soluble in methanol, chloroform. dimethylformamide. 1,2dichloroethane and dimethyl sulfoxide; is slightly soluble in ethanol and aqueous ethanol; but is insoluble in acetone, benzene. carbon tetrachloride. dichloro-methane, methyl isobutyl ketone. ethyl acetate. diethyl ether and water.

Elemental analysis of A-7413 factor A indicates the following approximate percentage composition (average): carbon, 51.92%; hydrogen. 5.25%, nitrogen, 9.85%; oxygen, 22.63%; and sulfur. 9.66%. An approximate empirical formula proposed for A-7413 factor A is C72H87N12O23S5.

The molecular weight of A-7413 factor A is approximately 1308, as determined by titration.

The infrared absorption spectrum of A-7413 factor A in KBr disc is shown in Figure 1 of the accompanying drawings. The following absorption maxima are observed: 2.93 (shoulder). 2.98 (medium). 3.24 (weak). 3.38 (shoulder). 3.44 (medium), 3.53 (weak). 5.78 (weak). 6.03 (strong). 6.56 (strong). 6.79 (medium), 7.08 (medium). 7.27 (weak). 7.49 weak 7.65 (weak). 8.08 (medium).8.41 (weak).8.62 (weak).8.81 (medium).9.03 (weak).

9.35 (medium). 9.60 (medium). 9.92 (weak). 10.20 (weak), 12.05 (weak), 12.66 (weak). and 13.51 (weak) microns.

The ultraviolet absorption spectrum of A-7413 factor A exhibits the following absorption maxima: a) in neutral, 95 vol.% aqueous ethanol: 215 nm (E1 cm 1% = 485); 260 nm (shoulder; Ei%m= 240); 300 nm (shoulder; E1 cm 1% = 170); 358 nm (shoulder; Ei - 112.5); b) in acidic ethanol: 217 nm (E1 cm 1% = 440); 265 nm (Ei Cm= 227.5); 293 nm (E1 cm 1% = 210); 358 nm (Eitcm= 95); c) in basic ethanol: 278 nm (shoulder; E1 cm 1% = 255); 408 nm (Elgcm= 80).

Electrometric titration of A-7413 factor A of 80 vol.% aqueous dimethylformamide indicates the presence of a titratable group with a pKa value of 7.9.

Amino-acid analysis of A-7413 factor A, after acidic hydrolysis, indicates the presence of ammonia (1.03 CL moles/mg), glycine (0.33 CL moles/mg), threonine (0.40 moles/mg), aspartic acid (0.1 per, moles/mg), and an as-yet-unidentified amino acid )approx. 0.4 CL moles/mg).

A-7413 factor A has a specific rotation, [a 5, of +54.5 (e 2.0, CHCl3) A-7413 factor A, crystallized from chloroform:ethanol, has the following characteristic X-ray powder diffraction pattern (Cu++ radiation, 1.5405 #, nickel filter, d=interplanar spacing in angstr5ms): Relative d Intensity 12.44 100 10.77 70 7.96 100 5.71 50 5.09 80 4.53 100 4.25 80 3.88 80 3.61 10 3.44 10 3.03 5 A-7413 factor B is a white to light-yellow amorphous material which melts above 300 C.

A-7413 factor B is soluble in methanol, chloroform, dimethylformamide, 1,2dichloroethane and dimethyl sulfoxide; is slightly soluble in ethanol and aqueous ethanol; but is insoluble in acetone. benzene. carbon tetrachloride, dichloromethane, methyl isobutyl ketone. ethyl acetate. diethyl ether and water.

Elemental analysis of A-7413 factor B indicates the following approximate percentage composition: carbon. 66.34%; hydrogen, 8.73%; nitrogen, 2.98%; oxygen, 19.39%; and sulfur. 2.83%.

The infrared absorption spectrum of A-7413 factor B in KBr disc is shown in Figure 2 of the accompanying drawings. The following absorption maxima are observed: 2.97 (strong), 3.38 (strong). 3.42 (strong). 3.50 (strong , 5.78 shoulder), 5.99 (medium), 6.50 (medium), 6.80 (medium). 6.90 (shoulder). 7.00 shoulder). 7.22 (medium), 7.27 (shoulder), 7.42 (weak). 7.58 (weak). 7.78 (shoulder). 7.97 (medium), 8.33 (shoulder). 8.53 (medium), 9.00 (shoulder), 9.26 (strong). 9.71 (strong). 11.11 (weak), 11.79 (weak), 12.35 (weak) and 13.25 (weak) microns.

The ultraviolet absorption spectrum of A-7413 factor B shows the following absorption maxima: a) in neutral, 95 vol.% aqueous ethanol: 268 nm (E'qC m= 104.3); 357 nm (shoulder; E1 cm 1% = 30); b) in acidic ethanol: 268 nm (E1 cm 1% = 108.5); 357 nm shoulder; E1 - 35); c) in basic ethanol: ~~~ 268 nm (shoulder; E1 cm 1% = 178.6).

A-7413 factor B has a specific rotation, [ai5, of -26.2 (e 7.5, DMSO).

Amino-acid analysis of A-7413 factor B, after acidic hydrolysis, indicates the presence of ammonia (0.46 IL moles/mg), glycine (0.1 moles/mg), threonine (0.1 IL moles/mg), aspartic acid (0.02 moles/mg), and an as-yet-unidentified amino acid (approx. 0.11 moles/ mg).

A-7413 factor C is a white to light-yellow amorphous material which melts above 250 C.

A-7413 factor C is soluble in methanol, chloroform, dimethylformamide, 1,2dichloroethane and dimethyl sulfoxide; is slightly soluble in ethanol and aqueous ethanol; but is insoluble in acetone, benzene, carbon tetrachloride, dichloromethane, methyl isobutyl ketone, ethyl acetate, diethyl ether and water.

Elemental analysis of A-7413 factor C indicates the following approximate percentage composition: carbon, 69.38%; hydrogen. 9.92%; nitrogen, 2.34%; oxygen, 16.58! o; and sulfur, 1.73%.

The infrared absorption spectrum of A-7413 factor C in KBr dise is shown in Figure 3 of the accompanying drawings. The following absorption maxima are observed: 3.00 (medium).

3.38 (shoulder). 3.42 (strong), 3.51 (strong). 5.73 (medium). 6.02 (medium), 6.14 (shoulder), 6.52 (weak). 6.56 (weak). 6.77 (medium). 6.80 (shoulder), 6.97 (weak), 7.20 (weak), 8.25 (weak), 8.33 (weak), 8.40 (weak). 8.86 (weak), 9.39 (weak), 10.05 (weak), 10.53 (weak), 10.70 (weak). 11.77 (weak) and 13.66 (weak) microns.

Amino-acid analysis of A-7413 factor C. after acidic hydrolysis, indicates the presence of ammonia (0.24 IL moles/mg). glycine (0.05 IL moles/mg), threonine (0.04 moles/mg), aspartic acid (0.01 moles/mg). and phenylalanine (0.05 moles/mg).

The ultraviolet absorption spectrum of A-7413 factor C shows the following absorption maxima: a) in neutral. 95 vol.% aqueous ethanol: 205 nm (E'gem= 356); 235 nm (shoulder; E1 - 180); 260 nm (shoulder; E1 cm 1% = 127); 290 mm (shoulder; E1 cm 1% = 104); b) in acidic ethanol: 205 nm (E1 cm 1% = 356); 235 nm (shoulder; E1 cm 1% = 180); 260 nm (shoulder; E1 cm 1% = 127); 290 nm (shoulder; E'8Cm= 103); 355 nm (shoulder; E1 cm 1% = 40); c) in basic ethanol: 260 nm (shoulder; E1 cm 1% = 268); 325 nm (shoulder; E1 cm 1% = 189).

The Rt values of A-7413 factors A. B. C. and D in various paper-chromatographic systems.

using Bacillus subtilis ATCC 6633 as a detection organism. are given in Table I: TABLE I Rf Values Solvent System A7413-A A7413-B A7413-C A7413-D n-Butanol saturated with 0.57 0.46 0.82 0.61 water Methyl isobutyl ketone: n-butanol:water (25:21:4) 0.49 0.33 0.90 0.61 Methanol:water (1:1) 0.62 0.58 0.31 0.62 Water: methanol: acetone (12:3:1); adjusted to pH 10.5 with NH40H and then lowered to pH 7.5 with H3PO4 0.30 0.26 0.06 0.20 Methanol:0.1 N HCl (3:1) 0.71 0.71 0.42 0.71 The Ref values of A-7413 factors A, B, C, and Din two thin-layer chromatographic systems on silica gel (precoated plates, E. Merck, Darmstadt, F-254, layer thickness 0.25 nm), again using B. subtilis ATCC 6633 as a detection organism, are listed in Table II: TABLE II Solvent System A7413-A A7413-B A7413-C A7413-D Chloroform: methanol (9:1 by vol.) 0.26 0.09 0.46 0.55 Acetonitrile: water (9:1 by vol) 0.23 0.03 0.42 0.48 Each of A-74 13 factors A. B and C has an acid function capable of forming salts and esters.

A-7413 factors A. B. and C. and the Cl-C5-acyl-ester and thiol-C2-C4-carboxylic-acid derivatives thereof are capable of forming salts. The physiologically-acceptable alkali-metal.

alkaline-earth-metal and amine salts of A-7413 factors A, B. and C; and of the thiol-C2-C4- carboxvlic-acid derivatives of A-7413 factors A, B, C are also part of this invention "Physilogically-acceptable" salts are salts which are also pharmaceutically acceptable, that is. salts in which the toxicity of the compound as a whole is not increased relative to the non-salt form. Representative and suitable alkali-metal and alkaline-earth-metal salts include the sodium. potassium. lithium. cesium, rubidium, barium, calcium, and magnesium.

Suitable amine salts include the ammonium; the primary, secondary. and tertiary C1-C4- alkylammonium; and hydroxy-C2-C4alkylammonium salts. Illustrative amine salts include those formed by reaction with ammonium hydroxide. sec-butylamine, isopropylamine.

diethvlamine. diisopropylamine. ethanolamine and triethylamine.

The alkali-metal and alkaline-earth-metal cationic salts are prepared according to procedures commonly employed for the preparation of cationic salts. For example. the free acid form of An74 13 factor A is dissolved in a suitable solvent. such as methanol or ethanol; to this solution is added a solution containing the stoichiometric quantity of the desired inorganic base. The salt thus formed can be isolated by routine methods, such as filtration or evaporation of the solvent.

The salts formed with organic amines can be prepared in a similar manner. For example, the amine can be added to a solution of A-7413 factor A in a suitable solvent such as methanol; and the solvent and excess amine can be removed by evaporation.

The Cl-C4-alkyl ester derivatives of An7413 factors A, B, and C are also part of this invention. These ester derivatives are prepared by conventional means.

Each of the A-74 13 factors A. B. and C has at least one hydroxyl group capable of forming acvl-ester derivatives. The C,-C5-acyl-ester derivatives of A-7413 factors A, B, and C are prepared by standard techniques. For example, A-7413 factor A free acid, in a suitable solvent. is reacted with the appropriate acid anhydride for a suitable length of time to give the desired A-7413 factor A acyl-ester derivative.

A-7413 factors A. B. and C are also capable of forming derivatives with thiolcarboxylic acids. These derivatives are prepared according to the method of M. Ebata et al.. J. Antibiotics 22 (10)451-456 (1969). Although the character of these derivatives is not known. the derivatives retain at least one carboxyl group and are able to form salts. The thiol C2-C4-carboxylic acid derivatives of A-7413 factors A, B, and C which are a part of this invention include, for example, the derivatives prepared from mercaptoacetic acid (thioglycolic acid), 2-mercapto-propionic acid (thiolactic acid), 3-mercaptopropionic acid, mercaptosuccinic acid (thiomalic acid), and L-cysteine.

The newly-found and hitherto undescribed micro-organism which produces the A-7413 antibiotic complex has been characterized taxonomically as a species of the Actinoplanes genus.

The genus Actinoplanes is a member of the family Actinoplanaceae. The Actinoplanaceae are a family of microorganisms of the order Actinomycetales,. having been first described by Couch [J. Elisha Mitchell Sci. Soc., 65, 315-318 (1949); 66, 87-92 (1950); Trans. New York Acad. Sci.. 16, 315-318 (1954); J. Elisha Mitchell Sci. Soc., 71, 148-155 and 269 (1955); "Bergey's Manual of Determinative Bacteriology," 8th Edition, 706-711 (1974); J. Elisha Mitchell Sci. Soc., 79, 53-70 (1963)].

A culture of the A-7413-producing microorganism has been deposited with the permanent culture collection of the Northern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois 61604, where it has been accorded the accession number NRRL 8122.

The characteristics ofActinoplanes sp. NRRL 8122 are given in the following paragraphs.

The methods recommended for the International Streptomyces Project [E.B. Shirling and D.

Gottleib, Intern. Bull. Systematic Bacteriol. 16,313-340 (1966)] for the characterization of Streptomyces species have been used along with certain supplementary tests. Color names were assigned according to the I.S.C.C.-- N.B.S. method [K.L. Kelly and D.B. Judd, "The ISCC-NBS Method of Designating Colors and a Dictionary of Color Names." U.S. Dept. of Commerce circular No.553, Washington, D. C.] . The Maerz and Paul color blocks (A Maerz and M.R. Paul. "Dictionary of Color." McGraw-Hill Book Company. New York. N.Y., 1950) are enclosed in parenthesis.

Vegetative mycelia and sporangia are extensively produced on sweetgum (Liquidambar) pollen. There is no evidence of hyphae penetrating the pollen. No sporangia are produced on Pinus pollen.

Sporangia are usually 9IL to 1 14,u in diameter. varying in shape from globose to subglobose to irregular. The principal shape is irregular. Spores are spherical. multiflagellated. 1.4 CL to 1.7 IL; onlv a few become motile.

CULTURAL CHARACTERISTICS (after 21 days at 30"C.) Yeast-malt (ICP No. 2) Growth abundant. light brown (12I8); no soluble pigment; sporangia are produced.

Czapek's agar Growth abundant, moderate orange (l lJ8); no soluble pigment; no sporangia are produced.

Oatmeal agar (ICP No. 3) Growth fair. pale yellow green (lOB1); no soluble pigment: sporangia are produced Inorganic salts-starch Growth moderate. brownish orange (ICP No. 4) (13A10); slight brownish soluble pigment; sporangia are produced.

Glvcerol-asparagine Growth abundant. medium reddish (ICP No. 5) orange (lOA10); no soluble pig ment; sporangia are produced.

Bennett's medium Growth fair. pale yellow (All); neither soluble pigment nor sporangia are produced.

Tomato paste-oatmeal Growth sparse: neither soluble pigment nor sporangia are pro duced.

Tvrosine agar Growth fair, yellowish gray (13A2); neither sporangia nor soluble pigment is produced.

Yeast extract agar Growth abundant, brownish orange (13B9); neither soluble pigment nor sporangia are produced.

Glucose-asparagine Growth moderate, pale orange yellow (1 1A4); neither soluble pigment nor sporangia are produced.

Calcium malate Growth moderate, light yellowish pink (10A2); neither sporangia nor soluble pigment is produced.

Nutrient agar Growth sparse; neither soluble pigment nor aerial hyphae are produced.

Emerson's agar Growth fair, light brown (13F8); slight reddish brown pigment; no sporangia are produced.

Action on skim milk No growth.

Nitrate reduction Positive.

Gelatin liquefaction None after 21 days.

Melanin production on Positive.

peptone-iron agar (ICP No. 6) Temperature requirements Good growth from 26 to 37"C.

on glycerol-asparagine Reddish orange color most intense agar at 26"C. No growth at 43"C.

Carbon utiliration: Utilization code: + = utilization (+) = probable util.

(-) = doubtful util.

- = no utilization Rhamnose (+) Cellobiose ( ) Inositol (DL) -) Cellulose Melezitose Fructose (+) Dextrose ( + D-Xvlose ( + D-Mannitol Raffinose Sucrose + Maltose -) L-Arabinose -) Lactose (+) Minus Carton As in the case with other organisms. the characteristics of the A-7413-producing culture.

Actinoplanes sp. NRRL 8122. are subject to variation. For example. artificial variants and mutants of the NRRL 8122 strain may be obtained by treatment with various known mutagens such as ultraviolet rays. X-rays. high-frequency waves, radioactive rays and chemicals. All natural and artificial variants and mutants which belong to this Actinoplanes species and produce the A-7413 antibiotics may be used in this invention.

The culture medium employed to grow Actinoplanes sp. NRRL 8122 can be any one of a number of media. For economy in production. optimal antibiotic yield. and ease of product isolation. however. certain culture media are preferred. Thus, for example. a preferred carbohydrate source in large-scale fermentation is dextrin. although glucose. fructose, maltose and sucrose may also be used. Although not essential for growth. an oil such as corn oil improves antibiotic production. Other useful sources of carbon include peanut oil, soybean oil and fish oil. A preferred nitrogen source is soybean flour, although soybean grits, peptones, oatmeal, peanut meal, soybean meal, cotton-seed meal and amino acids are also useful. Among the inorganic salts which may be incorporated in the culture media are the customary soluble salts capable of yielding sodium, potassium, iron, zinc, cobalt, magnesium, calcium, ammonium, chloride, carbonate, sulfate and nitrate ions.

Essential trace elements necessary for the growth and development of the organism should also be included in the culture medium. Such trace elements commonly occur as impurities in other constituents of the medium in amounts sufficient to meet the growth requirements of the organism.

It may be necessary to add small amounts (i.e. 0.2 ml/l.) of an antifoam agent such as polypropylene glycol to large-scale fermentation media if foaming becomes a problem.

For production of substantial quantities of the A-7413 antibiotics, submerged aerobic fermentation in tanks is preferred. Small quantities of the A-7413 antibiotics may be obtained by shake-flask culture. Because of the time lag in antibiotic production commonly associated with inoculation of large tanks with the spore form of the organism, it is preferable to use a vegetative inoculum. The vegetative inoculum is prepared by inoculating a small volume of culture medium with the spore form or mycelial fragments of the organism to obtain a fresh, actively growing culture of the organism. The vegetative inoculum is then transferred to a larger tank. The medium used for the growth of the vegetative inoculum can be the same as that employed for larger fermentations, but other media can also be employed.

The A-7413-producing organism may be grown at temperatures between 20 and 37"C.

Optimum A-7413 production appears to occur at temperatures of 25 to 30"C.

As is the customary procedure in aerobic submerged culture processes, sterile air is blown through the culture medium. For efficient growth of the organism, the volume of air employed in the tank production is preferably sufficient to maintain a dissolved oxygen saturation of greater than 20 percent.

The initial pH of the uninoculated culture medium varies with the medium used. Preferably. the pH should be in the range of 6.5 to 7.5. At the end of the fermentation, the harvest pH is usually slightly lower. in the range of 6.0 to 7.0.

During the fermentation, antibiotic production can be followed by testing samples of the broth or of extracts of the mycelial solids for antibiotic activity. Organisms known to be sensitive to the A-7413 antibiotics are useful for this purpose. One especially useful assay organism is Bacillus subtilis ATCC 6633. The bioassay is conveniently performed by paperdisc assay on agar plates.

Generally. antibiotic activity is detectable on the second day of the fermentation. Maximum production of antibiotic acivity usually occurs between the third and the tenth days.

Following their production under submerged aerobic fermentation conditions. the A-7413 antibiotics previously described can be recovered from the fermentation medium by methods used in the fermentation art. The antibiotics produced during fermentation of the A7413-producing organism are found mainly in the mycelial mass. A preferred method of recovering the A-7413 antibiotics is. therefore. by extraction of the separated mycelia.

Extraction of the mycelial mass is best accomplished with methanol, but other alcohols and chloroform are also suitable. The A-7413 antibiotics are recovered from the extracting solvent by routine procedures to give a mixture of the A-7413 antibiotics. the A-7413 mixture.

The A-7413 mixture may be further purified. and the individual A-7413 factors may be separated by a variety of recognized methods such as. for example, extraction and adsorption procedures. Adsorptive materials such as alumina. silica gel. ion exchange A, B, and C were tested for antimicrobial activity at 1 mg/ml on 6.35-mm discs. The results of these tests, given as the diameter in millimeters of the observed zones of inhibition, are summarized in Table III.

TABLE III A-7413 A-7413 A7413 Test Organism Factor A Factor B Factor C Staphlococcus aureus 23 20 14 Bacillus subtilis 21 18 16 Sarcina lutea 22 19 6 Furthermore, A-7413 factor A, when given by subcutaneous injection to mice, has in vivo antimicrobial activity. Two doses of A-7413 factor A were administered to mice in illustrative infections. The protection afforded is measured as an ED50 value [effective dose to protect 50 percent of the test animals; see Warren Wicket al., J. Bacteriol. 81, 233-235 (1961)]. The ED50 values for A74 13 factor A against these infections are given in Table IV: TABLE IV ED50 * Challenge (mg/kg x 2) LD50 Streptococcus pyogenes 0.42 161 Diplococcus pneumoniae 0.39 387 Staphylococcus aureus 31.00 4,000 * Therapy at one and five hours post-infection.

A special advantage of the A-7413 compounds is their ability to inhibit organisms which are resistant to other antibiotics. In Table V are summarized the results of standard agardilution tests (using the ICS method) wherein A-7413 factor A was tested against a variety of Staphylococcus aureus strains. Results are given as the minimal inhibitory concentration (MIC) at which inhibition of the S. aureus strain occurred. The results obtained with the known antibiotic vancomycin in the same test are included for comparison.

TABLE V MIC (mcg/ml) S. aureusStrain A-7413 factor A Vancomycin 3055* 0.125 1.0 3123* 0.125 1.0 H290* 0.125 1.0 3074** 0.125 1.0 H43** 0.125 1.0 H144** 0.125 1.0 H541 ** 0.062 1.0 3125*** 0.125 1.0 3130*** 0.062 1.0 3131 *** 0.062 1.0 3139*** 0.062 1.0 3133*** 0.062 1.0 3134*** 0.062 1.0 3l35*** 0.062 0.5 3136*** 0.125 0.5 3137*** 0.125 1.0 3138*** 0.062 1.0 3139*** 0.125 1.0 3140*** 0.125 0.5 *Penicillin G susceptible **Penicillin G resistant; methicillin susceptible ***Penicillin G and methicillin resistant [The strain numbers are laboratory-assigned reference numbers] In Table VI are summarized the results of agardilution tests wherein A-7413 factor A was tested against a variety of Streptococcus species. These tests employed trypticase-soy agar plus blood, 10-2 dilution of an overnight broth culture in 0.3% by wt. agar as an inoculum, giving approximately 5,000 bacteria per 7.5-mm of agar surface. Again, the results for vancomycin in the same test are reported for comparison. All strains tested are penicillin-Gresistant, Group-D-Streptococcus strains.

TABLE VI MIC (mcg/ml) Streptococcus sp. A- 7413 factor A Vancomycin 238 0.25 2.0 282 0.25 2.0 9901 0.125 4.0 9913 0.25 2.0 9933 0.25 8.0 9960 0.25 4.0 12253F 0.125 2.0 Shrigley 0.125 4.0 Mitis 0.125 4.0 55992 0.125 4.0 8043 0.125 4.0 In addition. A-7413 factor A is effective against Neisseria meningitides. In agar-dilution tests using trypticase-soy agar with 5% by vol. rabbit blood and 1% by vol. isovitalex. and a 1:100 by vol. dilution of an overnight broth culture as inoculum. A-7413 factor A had the following MIC values: N. meningitides Cultures MIC (mcg/ml) Os 4.0 Sabderlin 2.0 The A-7413 compounds are relatively nontoxic. For example. the acute toxicity (LD50) of A-7413 factor A. when administered by intraperitoneal injection to mice. was greater than 400 mg per kg.

Another advantageous property of the A-7413 complex and the A-7413 compounds is their ability to inhibit Propionibacterium acnes. a pathogen associated with acne. Representative A-7413 factor A was tested against P. acnes by the following procedure: Two-fold serial dilutions of test compounds are made in Actinomyces Broth (Baltimore Biological Laboratories). Each tube is inoculated with P. acnes to contain 104 organisms per ml. After four-days incubation at 37O C. the tubes are observed. The lowest concentration of test compound which prevents growth is recorded as the minimal inhibitory concentration (MIC).

The results of this test are summarized in Table VII.

TABLE VII A-7413 Factor A P. acnes Culture MIC (mcg/ml) ATCC 6919 s 1.25 Clinical Isolate 1 2.50 Clinical Isolate 2 S 1.25 The A-7413 mixture and the A-7413 compounds also inhibit the growth of microorganisms which contribute to the development of periodontal disease. In Table VIII is summarized the activity of A-7413 factor A against representative oral bacteria. Activity was measured using the standard agar-dilution method and recording the minimal inhibitory concentrations (MIC) after an incubation of 48 hours.

TABLE VIII Organism MIC (mcg/ml) Streptococcus mutant* < 0.25 Lactobacillus casei** < 0.25 Neisseria perfiava ** 32.0 * tested on Mitis Salivarius agar with tellurite and thioglycolic acid added.

**tested on Brain Heart Infusion Agar.

In addition. the tests using an artificial S. mutans plaque system, the A-7413 mixture and A-7413 factor A inhibited plaque formation at levels as low as 0.01 vol. percent.

Another important property of the A-7413 mixture and of the A-7413 compounds is the ability to improve feed-utilization efficiency in animals. For example, the A-7413 antibiotics improve feed-utilization efficiency in ruminants which have a developed rumen function.

It is known that the efficiency of carbohydrate utilization in ruminants is increased by treatments which stimulate the animals' rumen flora to produce propionate compounds rather than acetate or butyrate compounds (for a more complete discussion see Church et al.

in "Digestive Physiology and Nutrition of Ruminants," Vol. 2, 1971, pp. 622 and 625).

The efficiency of feed use can be monitored by observing the production and concentration of propionate compounds in the rumen using the method described by Arthur P. Raun in U.S.

Patent 3,794.732 (see especially Example 5). Table IX shows the ratio of volatile-fatty-acid (VFA) concentrations in A-7413-factor-A-treated flasks to concentrations in control flasks in this test.

Carbohydrate-utilization efficiency is further measured by in vivo tests performed in animals which have a fistula installed in their rumen, making it possible to withdraw specimens of the contents of the rumen.

The procedure used in testing cattle is also described in Raun's U.S. Patent 3,794,732 (see Example 7).

Table X summarizes the results of such a test with A-7413 factor A wherein the mean percent increases in ruminal propionic acid concentration were averaged over six analyses in a 14-day treatment period.

TABLE X Increase Increase Molar % Propionic over Relative Treatment Acid Conc. Control to Control Control 20.8 -- - A-7413 factor A 100 mg/day 25.4 4.6 22.1% In similar test in sheep. using fistulated wethers, A-7413 mixture also increased feed efficiency. The results of this test are summarized in Table XI.

TABLE XI In crease In crease Molar% over Relative Treatment propionate control to control Control 24.2 -- -- An7413 mixture 30 mg/day 26.2 2.0 8.3% *Sampled 6 days over a 17-day treatment period The An74 13 mixture and An74 13 compounds are typically effective in increasing propionates and, thereby. the efficiency of feed utilization when administered to ruminants orally preferably at rates of from 0.05 mg/kg/day to 10 mg/kg/day. Most beneficial results are achieved at a rate of about one mg/kg/day. A preferred method of administration of the A-7413 mixture or An74 13 compound is by mixing it with the animals' feed; however. it can be administered in other ways. for example. tablets, drenches, boluses or capsules. Formulation of these various dosage forms can be accomplished by methods well known in the veterinary pharmaceutical art. Each individual dosage unit should contain a quantity of A-7413 compound or mixture directly related to the proper daily dose for the animal to be treated.

An example of the useful growth-promoting property of the A-7413 mixture and A-7413 compounds is found in poultry. In floor-pen tests using broiler chicks, A-7413 factor A added to the feed at a rate of 10 grams per ton of feed significantly improved weight gains and feed utilization efficiency. The A-7413 mixture and A-7413 compounds are typically effective in promoting growth in poultry when administered with the animals' feed at rates of from 0.5 to 50 grams of A-7413 mixture or compound per ton of animal feed. Most beneficial results are seen when the A-7413 mixture or compound is administered at rates of from 2.5 to 10 grams of A-7413 mixture or compound per ton of animal feed.

The culture solids, including medium constituents and mycelia, can be used without extraction or separation, but preferably after removal of water, as a source of the A-7413 mixture. For example, after production of A-7413 antibiotic activity, the culture medium can be dried by lyophilization; the lyophilized medium can then be mixed directly into a feed premix.

In order to illustrate more fully the operation of this invention. the following examples are provided: EXAMPLE 1 A. Shake-flask Fermentation ofA-7413 A culture of Actinoplanes sp. NRRL 8122 was prepared by growing the organism on an 18 by 150 mm agar slant having the following composition: Ingredient Amount Sucrose 30 g Peptone 5 g K2HPO4 I g Czapek's mineral mix solution* 5 ml Agar 25 g Deionized water q.s. 1 liter *Czapek's Mineral Miv- Solution 100 g KCI 100 g MgSO4-7H20 2 g FeSo4,7H2O q.s. to 1 liter with deionized water.

The slant medium was inoculated with Actinoplanes sp. NRRL 8122. and the inoculated slant was incubated at 250C. for 10 to 14 days. The mature slant culture was covered with water. scraping with a sterile loop to loosen and fragment the mycelia and release the spores from the sporangia. One-half of the resulting suspension was used to inoculate 50 ml of a liquid vegetative medium having the following composition: Ingredient A mount Glucose 10 g Dextrin 20 g Soybean flour 25 g Yeast extract 2.5 g CaCO3 2.5 g Deionized water q.s. 1000 ml The inoculated vegative medium was incubated in a 250-mI Erlenmeyer flask at 25 C. for 72 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.

This incubated vegative medium may be used directly to inoculate the second-stage vegetative medium. Alternatively and preferably. it can be stored for later use by maintaining the culture in the vapor phase of liquid nitrogen. The culture is prepared for such storage in multiple small vials as follows: In each vial is placed 2 ml of incubated vegetative medium and 2 ml of a glycerol-lactose solution having the following composition: Ingredient Amount Glycerol 20% Lactose 10% Deionized water 70% The prepared suspensions are stored in the vapor phase of liquid nitrogen.

A stored suspension (1 ml) thus prepared was used to inoculate 50 ml of a first-stage vegetative medium having the same composition earlier described for the vegetative medium. The inoculated first-stage vegetative medium was incubated in a 250-ml widemouth Erlenmeyer flask at 25 C for 72 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.

B. Tank Fermentation ofA-7413 In order to provide a larger volume of inoculum, 40 ml of the above-described incubated vegetative medium was used to inoculate 400 ml of a second-stage vegetative medium having the same composition as that of the first-stage vegetative medium, This inoculated secondstage vegetative medium, in a 2-liter flask, was incubated at 25"C. for about 48 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.

One liter of the second-stage vegetative inoculum thus prepared was used to inoculate 100 liters of sterile production medium of the following composition: Ingredient Amount Soybean flour 35 g Corn oil 40 g MgSO4-7H20 2 g CaCO3 2g FeCl2-4H20 0.06 g Deionized water q.s. 1 liter After sterilization by heating at 1200C. for 30 minutes, the pH of the medium was 70.0.

The inoculated production medium. in a 165-liter fermentation tank, was allowed to ferment for about 7 days at a temperature of 25 C. The fermentation medium was aerated with sterile air at the rate of approximately 0.5 to 1.0 volume of air per volume of culture medium per minute. The medium was stirred with conventional agitators at 250 RPM.

EXAMPLE 2 The A-7413 antibiotics were produced as described in Example 1, but a slant medium having the following composition was used to provide spores or mycelium for the initial inoculum: Ingredient Amount Na2S203 0.5 g Yeast extract 2.0 g CaCO3 3.0 g Vegetable juice* 200 ml Deionized water 800 ml pH adjusted to 7.2 by the addition of dilute sodium hydroxide *V-8 Juice. Campbell Soup Company. Camden, N. J. 08101. U.S.A.

EXAMPLE 3 The A-7413 antibiotics were produced as described in Example 1, but using a vegetative medium and a second-stage vegetative medium of the following composition: Ingredient Amount Glucose 10.0 g Dextrin 20.0 g Soybean flour 150.0 g Yeast extract 2.5 g Soybean oil (refined) 5.0 g CaCO3 2.5 g Deionized water q.s. 1 liter EXAMPLE 4 Separation of the A-7413 Mixture Whole fermentation broth (200 liters), prepared as described in Example 1, was made acidic (pH 3.5) by the addition of dilute sulfuric acid. The resulting acidic broth was filtered using a filter aid (Hyflo Super cell, (Registered Trade Mark) a diatomaceous earth, Johns Manville Products Crop.). Methanol (100 liters) was added to the separated mycelial cake; this methanol suspension was stirred for 30 min. and then was separated by filtration.

Methanol (100) liters) was again added to the separated mycelial cake, again stirred for 30 min. and separating by filtration. The two methanol extracts were concentrated under vacuum, removing the methanol to give an aqueous concentrate (10.5 liters). This aqueous concentrate was cooled (5"C.) for 24 hours. The oily upper layer which formed was separated and discarded. The aqueous lower layer (2 liters) was adjusted to pH 4.3 by the addition of dilute sulfuric acid. The resulting solution was extracted twice with one-half volumes of a chloroform:methanol (4:1 by vol.) solution. These extracts were combined and evaporated to dryness under vacuum. The residue thus obtained was dissolved in chloroform (150 ml); this solution was added to n-pentane (1500 ml). The resulting precipitate was separated by centrifugation and was dried under vacuum to give 15.8 g of the A-7413 mixture as a tan powder.

EXAMPLE 5 Isolation of the A-7413 Factors A-7413 mixture (26.4 g), obtained as described in Example 4, was dissolved in 200 ml of a chloroform:methanol (19:1 by vol.) solution. The resulting solution was applied to a 5.8- x 94.0-cm column of silica gel (Matheson. Grade 62, equilibrated with 5% waster), prepared in chloroform:methanol (19:1 by vol.). The column was developed using chloroform:methanol (19:1 by vol.). collecting 150-ml fractions. Elution of the column was monitored by assaying fractions against Staphylococcus aureus, Bacillus subtilis, and Sarcina lutea and by thin-layer chromatography bionutography, using S. lutea as the detecting organism. Fractions were combined according to factor content and activity exhibited. The combined fractions were each evaporated to dryness under vacuum. Each of the residues thus obtained was dissolved in chloroform (50 ml); each chloroform solution was added to n-pentane (500 ml) to precipitate the desired factor. The results of the column were as follows: Factor Approximate Obtained Fractions Yield Purity A 18-23 10.153 g pure A 24-30 597.6 mg 60% B 31-43 278.8 mg 80% C 49-56 218.4 mg 60% The factors which were impure were subjected to further chromatography on silica gel columns. using the above-described procedure. to obtain purified factors B and C and an additional amount of purified factor A.

EXAMPLE 6 Crvstallizan'on of A-7413 Factor A Purified factor A (1 g). obtained as described in Example 5, was dissolved in chloroform (10 ml). Absolute ethanol (10 ml; absolute ethanol contains 0.5% by vol. benzene) was added. The resulting solution was allowed to stand for two hours at room temperature and then was cooled to 5 C. overnight. The crystals which formed were separated by centrifugation. washed with ethanol and dried to give 513 mg of crystalline A-7413 factor A.

An7413 factor A crystallized in a similar manner using the following solvents: chloroform:sec-butanol chloroform:n-propanol chloroform: isopropanol dimethylformamide:acetone acetone: ethanol aqueous ethanol EXAMPLE 7 A-7413 Factor A Ammonium Salt A-7413 factor A (200 mg). prepared as described in Example 6. was added to 0.01 N ammonium hydroxide (10 ml). This suspension was stirred for 20 minutes. using a Virtis blender. The insoluble material was then separated by centrifugation and was discarded. The supernatant solution was freeze dried to give 158.7 mg of A-7413 factor A ammonium salt as a yellow. water-soluble powder.

EXAMPLE 8 A-7413 Factor A Potassium Salt A-7413 factor A (3 g), prepared as described in Example 6, was suspended in water (150 ml). The pH of the resulting suspension was 4.3 and was adjusted to pH 9.45 by the addition of 0.05 N potassium hydroxide (41 ml). This solution was stirred, using a blender, for 30 minutes. The insoluble material was then separated by centrifugation. The supernatant solution was freeze-dried to give 2.61 g of A-7413 factor A potassium salt as a yellow, water-soluble powder.

The potassium salt was further purified and crystallized by dissolving this powder (200 mg) in methanol (8 ml), centrifuging off insoluble impurities, adding diethyl ether to the separated supernatant solution and cooling (5 C.) for three days. The crystals which formed were separated by centrifugation and were dried to give 141.8 mg of crystalline A-7413 factor A potassium salt.

EXAMPLE 9 A-7413 Factor A Calcium Salt A .7413 factor A (200 mg), prepared as described in Example 6, was dissolved in methanol (20 ml). and 0.1 N calcium hydroxide was slowly added to the methanol solution with stirring until the solution had a pH of 9.1. Diethyl ether (6 volumes) was added to the resulting solution to precipitate the salt. The precipitate was separated by centrifugation and was dried to give 80.1 mg of A-7413 factor A calcium salt. The product contained 1.81 % calcium when analyzed by atomic-absorption analysis.

EXAMPLE 10 A-7413 Factor A Triethylamine Salt A-7413 factor A (200 mg) was treated according to the method of Example 7, but using 0.01 N triethylamine. to give 122.2 mg of the triethylammonium salt of A-7413 factor A.

EXAMPLE 11 A-7413 Factor A Disodium Salt A-7413 factor A (300 mg) was treated according to the method of Example 7, but using 0.01 N sodium hydroxide (30 ml). to give 260 mg of the disodium salt of A-7413 factor A as a yellow. water-soluble compound (2.67% Na by atomic-absorption analysis).

EXAMPLE 12 A- 7413 Factor A Monosodium Salt A-7413 factor A (200 mg) was treated according to the method of Example 9, but using 0.1 N sodium hydroxide to adjust the pH of the solution to pH 8.6, to give 151.6 mg of the monosodium salt of A-7413 factor A as a water-soluble compound (1.43 % Na by atomic absorption analysis).

EXAMPLES 13-18 13. A-7413 factor B disodium sat. prepared according to the method of Example 8. but using A-7413 factor B and 0.01 N sodium hydroxide.

14. A-7413 factor B ammonium salt. prepared according to the method of Example 7, but using A-7413 factor B.

15. A-7413 factor B barium salt. prepared according to the method of Example 9, but using A-7413 factor B and 0.1 N barium hydroxide.

16. A-7413 factor C monosodium salt. prepared according to the method of Example 9, but using A-7413 factor C and 0.1 N sodium hydroxide.

17. A-7413 factor C isopropylamine salt. prepared according to the method of Example 10, but using A-7413 factor C and 0.01 N isopropylamine.

18. An7413 factor C magnesium salt. prepared according to the method of Example 9, but using A-7413 factor C and 0.1 magnesium hydroxide.

EXAMPLE 19 A-7413 Factor A Acetvl Ester Derivative An74 13 factor A (500 mg) was dissolved in dimethyl sulfoxide (20 ml). Acetic anhydride (8 ml) was added to this solution. and the mixture was allowed to stand at room temperature for 22 hours. The mixture was concentrated under vacuum to a volume of about 15 ml. Methanol (15 ml) was added to the concentrate. and the resulting mixture was added to diethyl ether (240 ml). The precipitate which formed was separated by filtration and dried under vacuum to give 216 mg of the acetyl ester derivative of A-7413 factor A.

EXAMPLE 20 A-7413 Factor A Acetyl Ester Derivative A-7413 factor A (500 mg) was dissolved in pyridine (20 ml). Acetic anhydride (8 ml) was added to this solution. and the mixture was allowed to stand at room temperature for 22 hours. The mixture was then evaporated to dryness under vacuum. The residue was dissolved in chloroform (4 ml). and this solution was added to n-pentane (60 ml). The precipitate which formed was separated by centrifugation and dried under vacuum to give 421 mg of the acetyl ester derivative of A-7413 factor A.

EXAMPLES 21-25 21. A-7413 factor A propionyl ester derivative, prepared according to the method of Example 20, but using propionic anhydride.

22. A-7413 factor B n-butyryl ester derivative, prepared according to the method of Example 20, but using A-7413 factor B and n-butyric anhydride.

23. A-7413 factor C n-valeryl ester derivative, prepared according to the method of Example 20, but using A-7413 factor C and n-valeric anhydride.

24. A-7413 factor A succinyl ester derivative, prepared according to the method of Example 20, but using succinic anhydride.

25. A-7413 factor B formyl ester derivative, prepared according to the method of Example 20, but using formic anhydride.

EXAMPLE 26 An 7413 Factor A Methyl Ester A-7413 factor A (100 mg) was dissolved in a solution of methanol (5 ml) and chloroform (0.6 ml). An ethereal solution of diazomethane (4 ml) was added to the A-7413 factor-A solution. The resulting solution was stirred for 30 minutes and then was allowed to stand at room temperature for 4.5 hours. This solution was evaporated to dryness under vacuum. The residue obtained was dissolved in methanol (4 ml), and this solution was evaporated to dryness under vacuum. The residue obtained was dissolved in chloroform (3 ml), and the chloroform solution was added to n-pentane (30 ml). The precipitate which formed was separated by centrifugation and dried to give 87 mg of A-7413 methyl ester.

EXAMPLES 27-29 27 A-7413 factor B ethyl ester, prepared according to the method of Example 26, but using A-7413 factor B and diazoethane.

28. A-7413 factor C 2-propyl ester, prepared according to the method of Example 26. but using A-7413 factor C and diazo-2-propane.

29. A-7413 factor A n-butyl ester. prepared by reaction of A-7413 factor A with n-butanol by standard procedures. using dicyclohexylcarbodiimide as a dehydrating agent.

EXAMPLE 30 A-7413 Factor A Bis(Mercaptoacetic Acid) Derivative A-7413 factor A (free acid; 200 mg) was dissolved in N.N-dimethylformamide (2.8 ml).

Mercaptoacetic acid (200 mg) was added to this solution. The resulting solution was saturated with nitrogen by bubbling the gas through the solution for 30 minutes, and then was allowed to stand at room temperature for 20 hours. The solution was concentrated to a small volume; the concentrated solution was added to diethyl ether (25 volumes). The precipitate which formed was separated by filtration and dried to give 179 mg of the bis(mercaptoacetic acid)derivative of A-7413 factor A.

EXAMPLES 31-35 31. An7413 factor B bis(2-mercaptopropionic acid) derivative. prepared according to the method of Example 30. but using A-7413 factor B and 2-mercaptopropionic acid.

32. A-7413 factor C bis (3-mercaptopropionic acid) derivative. prepared according to the method Example 30. but using A-7413 factor C and 3-mercaptopropionic acid.

33. A-7413 factor A bis(mercaptosuccinic acid) derivative. prepared according to the method of Example 30. but using mercaptosuccinic acid.

34. A-7413 factor A mono-mercaptosuccinic acid derivative. prepared according to the method of Example 33. but allowing the solution to stand for only 6 hours.

35. A-7413 factor A L-cysteine derivative. prepared according to the method of Example 30. but using a,, L-cysteine and purifying the product by chromatography.

WHAT WE CLAIM IS: 1. An antibiotic A-7413 factor A. factor B. factor C or factor D; mixtures thereof; the C1-C4-alkyl esters. C1-Cs-acyl esters and thiol-C2-C4-carboxylic acid derivatives of factors A.

B and C; and the physiologically acceptable salts of factors A. B and C and the C,-C5-acyl esters and thiol-C2-C4-carboxylic acid derivatives thereof. factor A being a white to lightyellow crystalline material when crystallized from ethanol; which is soluble in methanol.

chloroform. dimethylformamide. 1 .2-dichloroethane and dimethyl sulfoxide; is slightly solu ble in ethanol and aqueous ethanol; but is insoluble in acetone. benzene. carbon tetrach loride. dichloromethane methyl isobutyl ketone, ethyl acetate. diethyl ether and water; which melts with decomposition at 205 to 212"C. and which has: A) a molecular weight of approximately 1308. ad determined by titration; B) an approximate elemental composition of 51.92 percent carbon. 5.25 percent hydrogen, 9.85 percent nitrogen. 22.63 percent oxygen. and 9.66 percent sulfur; C) a specific rotation. [0g}d5. of +54.5 (c 2.0. CHCl3); D) an infrared absorption spectrum in KBr disc with the following observable absorption maxima: 2.93 (shoulder). 2.98 (medium). 3.24 (weak). 3.38 (shoulder). 3.44 (medium).

3.53 (weak). 5.78 (weak). 6.03 (strong). 6.56 (strong). 6.79 (medium). 7.08 (medium). 7.27

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (1)

1. **WARNING** start of CLMS field may overlap end of DESC **.

   EXAMPLES 21-25 21. A-7413 factor A propionyl ester derivative, prepared according to the method of Example 20, but using propionic anhydride.

   22. A-7413 factor B n-butyryl ester derivative, prepared according to the method of Example 20, but using A-7413 factor B and n-butyric anhydride.

   23. A-7413 factor C n-valeryl ester derivative, prepared according to the method of Example 20, but using A-7413 factor C and n-valeric anhydride.

   24. A-7413 factor A succinyl ester derivative, prepared according to the method of Example 20, but using succinic anhydride.

   25. A-7413 factor B formyl ester derivative, prepared according to the method of Example 20, but using formic anhydride.

   EXAMPLE 26 An 7413 Factor A Methyl Ester A-7413 factor A (100 mg) was dissolved in a solution of methanol (5 ml) and chloroform (0.6 ml). An ethereal solution of diazomethane (4 ml) was added to the A-7413 factor-A solution. The resulting solution was stirred for 30 minutes and then was allowed to stand at room temperature for 4.5 hours. This solution was evaporated to dryness under vacuum. The residue obtained was dissolved in methanol (4 ml), and this solution was evaporated to dryness under vacuum. The residue obtained was dissolved in chloroform (3 ml), and the chloroform solution was added to n-pentane (30 ml). The precipitate which formed was separated by centrifugation and dried to give 87 mg of A-7413 methyl ester.

   EXAMPLES 27-29 27 A-7413 factor B ethyl ester, prepared according to the method of Example 26, but using A-7413 factor B and diazoethane.

   28. A-7413 factor C 2-propyl ester, prepared according to the method of Example 26. but using A-7413 factor C and diazo-2-propane.

   29. A-7413 factor A n-butyl ester. prepared by reaction of A-7413 factor A with n-butanol by standard procedures. using dicyclohexylcarbodiimide as a dehydrating agent.

   EXAMPLE 30 A-7413 Factor A Bis(Mercaptoacetic Acid) Derivative A-7413 factor A (free acid; 200 mg) was dissolved in N.N-dimethylformamide (2.8 ml).

   Mercaptoacetic acid (200 mg) was added to this solution. The resulting solution was saturated with nitrogen by bubbling the gas through the solution for 30 minutes, and then was allowed to stand at room temperature for 20 hours. The solution was concentrated to a small volume; the concentrated solution was added to diethyl ether (25 volumes). The precipitate which formed was separated by filtration and dried to give 179 mg of the bis(mercaptoacetic acid)derivative of A-7413 factor A.

   EXAMPLES 31-35 31. An7413 factor B bis(2-mercaptopropionic acid) derivative. prepared according to the method of Example 30. but using A-7413 factor B and 2-mercaptopropionic acid.

   32. A-7413 factor C bis (3-mercaptopropionic acid) derivative. prepared according to the method Example 30. but using A-7413 factor C and 3-mercaptopropionic acid.

   33. A-7413 factor A bis(mercaptosuccinic acid) derivative. prepared according to the method of Example 30. but using mercaptosuccinic acid.

   34. A-7413 factor A mono-mercaptosuccinic acid derivative. prepared according to the method of Example 33. but allowing the solution to stand for only 6 hours.

   35. A-7413 factor A L-cysteine derivative. prepared according to the method of Example 30. but using a,, L-cysteine and purifying the product by chromatography.

   WHAT WE CLAIM IS:

   1. An antibiotic A-7413 factor A. factor B. factor C or factor D; mixtures thereof; the C1-C4-alkyl esters. C1-Cs-acyl esters and thiol-C2-C4-carboxylic acid derivatives of factors A.

   B and C; and the physiologically acceptable salts of factors A. B and C and the C,-C5-acyl esters and thiol-C2-C4-carboxylic acid derivatives thereof. factor A being a white to lightyellow crystalline material when crystallized from ethanol; which is soluble in methanol.

   chloroform. dimethylformamide. 1 .2-dichloroethane and dimethyl sulfoxide; is slightly solu ble in ethanol and aqueous ethanol; but is insoluble in acetone. benzene. carbon tetrach loride. dichloromethane methyl isobutyl ketone, ethyl acetate. diethyl ether and water; which melts with decomposition at 205 to 212"C. and which has: A) a molecular weight of approximately 1308. ad determined by titration; B) an approximate elemental composition of 51.92 percent carbon. 5.25 percent hydrogen,

   9.85 percent nitrogen. 22.63 percent oxygen. and 9.66 percent sulfur; C) a specific rotation. [0g}d5. of +54.5 (c 2.0. CHCl3); D) an infrared absorption spectrum in KBr disc with the following observable absorption maxima: 2.93 (shoulder). 2.98 (medium). 3.24 (weak). 3.38 (shoulder). 3.44 (medium).

   3.53 (weak). 5.78 (weak). 6.03 (strong). 6.56 (strong). 6.79 (medium). 7.08 (medium). 7.27

   (weak), 7.49 (weak), 7.65 (weak), 8.08 (medium), 8.41 (weak), 8.62 (weak), 8.81 (medium), 9.03 (weak), 9.35 (medium), 9.60 (medium), 9.92 (weak), 10.20 (weak), 12.05 (weak), 12.66 (weak), and 13.51 (weak) microns; E) an ultraviolet absorption spectrum with the following absorption maxima: a) in neutral, 95% by vol. aqueous ethanol: 215 nm (El6Xcom=485); 260 nm (shoulder' E1 cm 1% = 240); 300 nm (shoulder; Ei Cm= 170); 358 nm (shoulder' E1 cm 1% = 112.5); b) in acidic ethanol: 217 nm (E1 cm 1% = 440); 265 nm (E1 cm 1% = 227.5); 293 nm (E1 cm 1% = 210); 358 nm (E' m= 95); c) in basic ethanol: 278 nm (shoulder; E1 cm 1% = 255); 408 nm (Ei 'Cm= 80); F) an electrometric titratable group with a pKa value of 7.9 in 80% by vol. aqueous dimethylformamide; G) an amino-acid analysis. after acidic hydrolysis, which indicates the presence of ammonia, glycine, threonine and aspartic acid; H) a characteristic X-ray powder diffraction pattern (Cu++ radiation, 1.54505 #, nickel filter) having the following interplanar spacings in angströms (d): Relative d Intensity

   12.44 100

   10.77 70

   7.96 100

   5.71 50

   5.09 80

   4.53 100

   4.25 80

   3.88 80

   3.61 10

   3.44 10

   3.03 5 I) the following Rf values in the paper-chromatographic systems indicated below, using Bacillus subtilis ATCC 6633 as a detection organism: Rf Value Solvent System 0.57 n-Butanol saturated with water 0.49 Methyl isobutyl ketone: n-butanol:water (25:21:4 by vol.) 0.62 Methanol:water (1:1 by vol) 0.30 Water:methanol:acetone (12:3:1 by vol.); adjusted to pH

   10.5 with NH40H and then lowered to pH 7.5 with H3PO4 0.71 Methanol: 0.1 N HCI (3:1 by vol.) J) the following R values in the silica-gel thin-layer-chromatographic systems indicated below. using Bacillus subtilis ATCC 6633 as a detection organism: Rj Value Solvent System 0.26 Chloroform:methanol (9:1 by vol.) 0.23 Acetonitrile: water (9:1 by vol.) K) an acid function capable of forming salts and ester derivatives; L) at least one hydroxyl group capable of esterification; and M) the ability to form derivatives with thiolcarboxylic acids; and the C1C4-alkyl ester, C1-Cs-acyl ester, and thiol-C2-C4-carboxylic acid derivatives thereof; and the physiologically-acceptable salts of A-7413 factor A and of said Cl-C5-acyl ester and thiol-C2-C4-carboxylic acid derivatives thereof: factor B being a white to light-yellow amorphous material which melts above 300"C. and which is soluble in methanol, chloroform, dimethylformamide, 1,2-dichloroethane and dimethyl sulfoxide; is slightly soluble in ethanol and aqueous ethanol; but is insoluble in acetone, benzene, carbon tetrachloride, dichloromethane, methyl isobutyl ketone, ethyl acetate, diethyl ether and water; and which has: A) an approximate elemental composition of 66.34 percent carbon, 8.73 percent hydrogen, 2.98 percent nitrogen, 19.39 percent oxygen, and 2.83 percent sulfur; B) a specific rotation, [a 5, of -26.2 (c 7.5, DMSO); C) an infrared absorption spectrum in KBr disc with the following observable absorption maxima:

   2.97 (strong), 3.38 (strong), 3.42 (strong), 3.50 (strong), 5.78 (shoulder), 5.99 (medium), 6.50 (medium), 6.80 (medium), 6.90 (shoulder), 7.00 (shoulder), 7.22 (medium 7.27 (shoulder), 7.42 (weak). 7.58 (weak), 7.78 (shoulder), 7.97 (medium), 8.33 (shoulder 8.53 (medium), 9.00 (shoulder), 9.26 (strong), 9.71 (strong), 11.11 (weak), 11.79 (weak), 12.35 (weak) and 13.25 (weak) microns; D) an ultraviolet absorption spectrum with the following absorption maxima: a) in neutral, 95% by vol. aqueous ethanol: 268 nm (E'Cm= 104.3); 357 nm (shoulder; E' Ccm= 30); b) in acidic ethanol: 268 nm (E1 - 108.5); 357 nm (shoulder; E''cm= 35); c) in basic ethanol: 268 nm (shoulder; Ej,= 178.6); E) an amino-acid analysis. after acidic hydrolysis. which indicates the presence of ammonia, glycine. threonine and aspartic acid; F) the following R values in the paper-chromatographic systems indicated below, using Bacillus subtilis ATCC 6633 as a detection organism: Rf Value Solvent System 0.46 n-Butanol saturated with water 0.33 Methyl isobutyl ketone: n-butanol:water (25:21:4 by vol.) 0.58 Methanol: water (1:1 by vol.) 0.26 Water: methanol: acetone (12:3:1 by vol.) adjusted to pH 10.5 with NH4OH and then lowered to pH 7.5 with H3PO4 0.71 Methanol:0.1 N HCI (3:1 by vol.) G) the following R values in the silica-gel thin-layer-chromatographic systems indicated below. using Bacillus subtilis ATCC 6633 as a detection organism: R, Vahe Solvent System 0.09 Chloroform: methanol (9:1 by vol.) 0.03 Acetonitrile:water (9: 1 by vol.) H) an acid function capable of forming salts and ester derivatives; I) at least one hydroxyl group capable of esterification; and J) the ability to form derivatives with thiol carboxylic acids; and the C1-C4-alkyl ester, Cl-C5-acyl ester, and thiol-C2-C4-carboxylic acid derivatives thereof; and the physiologically-acceptable salts of A-7413 factor B and of said C1-C5-acyl ester and thiol-C2-C4-carboxylic acid derivatives thereof: factor C being a white to lightyellow amorphous material which melts above 250 C. and which is soluble in methanol, chloroform, dimethylformamide, 1 ,2-dichloroethane and dimethyl sulfoxide; is slightly soluble in ethanol and aqueous ethanol; but is insoluble in acetone, benzene, carbon tetrachloride, dichloromethane, methyl isobutyl ketone, ethyl acetate, diethyl ether and water; and which has: A) an approximate elemental composition of 69.38 percent carbon, 9.92 percent hydrogen, 2.34 percent nitrogen, 16.58 percent oxygen, and 1.73 percent sulfur; B) an infrared absorption spectrum in KBr disc with the following observable absorption maxima: 3.00 (medium), 3,38 (shoulder), 3.42 (strong), 3.51 (strong), 5.73 (medium), 6.02 (medium), 6.14 (shoulder), 6.52 (weak), 6.56 (weak), 6.77 (medium), 6.80 (shoulder), 6.97 (weak), 7.20 (weak), 8.25 (weak), 8.33 (weak), 8.40 (weak), 8.86 (weak), 9.39 (weak), 10.05 (weak), 10.53 (weak), 10.70 (weak), 11.77 (weak) and 13.66 (weak) microns; C) an ultraviolet absorption spectrum with the following absorption maxima: a) in neutral, 95% by vol. aqueous ethanol: 205 nm (E1 cm 1% = 356); 235 nm (hou1dor; Flcm= 180); 1? 260 nm (shoulder; Elco 127); 290 ntn (shoulder; E1 cm 1% = 104); b) in acidic ethanol: 205 nm (Elqm= 356); 235 nm (shoulder; E1 cm 1% = 180); 260 nm (shoulder' E,Cm= 127); 290 nm (shoulder' ElCm= 103); 355 nm (shoulder' Elecm= 40); c) in basic ethanol: 260 nm (shoulder; El%'m= 268); 325 nm (shoulder; El^;m= 189); D) an amino-acid analysis. after acidic hydrolysis, which indicates the presence of ammonia, glycine. threonine. aspartic acid, and phenylalanine; E) the following Rf values in the paper-chromatographic systems indicated below, using Bacillus subtilis ATCC 6633 as a detection organism: Rf Value Solvent System 0.82 n-Butanol saturated with water 0.90 Methyl isobutyl ketone: n-butanol:water (25:21:4 by vol.) 0.31 Methanol: water (1:1 by vol.) 0.06 Water: methanol: acetone (12:3:1 by vol.) adjusted to pH 10.5 with NH40H and then lowered to pH 7.5 with H3PO4 0.42 Methanol:0.1 N HCI (3:1 by vol.) F) the following Rf values in the silica-gel thin-layer-chromatographic systems indicated below. using Bacillus subtilis ATCC 6633 as a detection organism: Rr Value Solvent System 0.46 Chloroform:methanol (9:1 by vol.) 0.42 Acetonitrile:water (9:1 by vol.) G) an acid function capable of forming salts and ester derivatives; H) at least one hydroxyl group capable of esterification; and I) the ability to form derivatives with thiol carboxylic acids; and the Cl-C4-alkyl ester, Cl-C5-acyl ester, and thiol-C2-C4-carboxylic acid derivatives thereof; and the physiologically-acceptable salts of A-7413 factor C and of said C1-Cs-acyl ester and thiol-C2-C4-carboxylic acid derivatives thereof: factor D having A) the following Ri values in the paper-chromatographic systems indicated below, using Bacillus subtilis ATCC 6633 as a detection organism: Rt Value Solvent System 0.61 n-Butanol saturated with water 0.61 Methyl isobutyl ketone: n-butanol:water (25:21:4 by vol.) 0.62 Methanol:water (1:1 by vol.) 0.20 Water: methanol: acetone (12:3:1 by vol.) adjusted to pH 10.5 with NIl4 OH and then lowered to pH 7.5 with H3PO4 0.71 Methanol: 0.1 N HCl (3:1 by vol.) B) the following Rf values in the silica-gel thin-layer-chromatographic systems indicated below, using Bacillus subtilis ATCC 6633 as a detection organism: Rf Value Solvent System 0.55 Chloroform:methanol (9:1 by vol.) 0.48 Acetonitrile: water (9.1 by vol.)

   2. Factor A of antibiotic A-7413. as defined in Claim 1, and the physiologicallyacceptable salts of Factor A and of the C1-Cs-acyl esters and thio-C2-C4 carboxylic acid derivates thereof.

   3. Factor A of antibiotic An7413, as defined in Claim 1.

   4. Factor A of antibiotic A-7413, as defined in Claim 1, in ammonium salt form.

   5. Factor A of antibiotic A-7413, as defined in Claim 1. in potassium salt form.

   6. Factor A of antibiotic A-7413. as defined in Claim 1. in calcium salt form.

   7. Factor A of antibiotic An7413. as defined in Claim 1, in triethylamine salt form.

   8. Factor A of antibiotic An7413. as defined in Claim 1, in disodium salt form.

   9. Factor A of antibiotic A-7413, as defined in Claim 1. in monosodium salt form.

   10. Factor A of antibiotic A-7413, as defined in Claim 1. in the form of an acetyl ester derivative.

   11. Factor A of antibiotic A-7413. as defined in Claim 1, in methyl ester form.

   12. Factor A of antibiotic A-7413. as defined in Claim 1. in the form of a bis(mercaptoacetic acid) derivative.

   13. Factor B of antibiotic A-7413. as defined in Claim 1. and the physiologicallyacceptable salts of Factor B and of the C1-Cs-acyl esters and thio-C2-C4-carboxylic acid derivatives thereof.

   14. Factor B of antibiotic A-7413. as defined in Claim 1.

   15. Factor C of antibiotic A-7413, as defined in Claim 1. and the physiologicallyacceptable salts of Factor C and of the Cl-C5-acyl esters and thiol-C2-C4-carboxylic acid derivatives thereof.

   16. Factor C of antibiotic An7413. as defined in Claim 1.

   17. Factor D of antibiotic A-7413. as defined in Claim 1.

   18. A process for producing antibiotic A-7413 mixture comprising factors A. B. C and D; A-7413 factor A. A-7413 factor B. A-7413 factor C or A-7413 factor D; the C1-C4-alkyl esters. C1-C-acyl esters or thiol-C2C4-carboxylic acid derivatives of factors A. B or C; or the physiologically acceptable salts of factors A. B or C or the C1-Cs-acyl esters or thiol C2-C4-carboxylic acid derivatives thereof comprising: (a) cultivating Actinoplanes sp. NRRL 8122 or an A-7413 mixture-producing mutant thereof in a culture medium containing assimilable sources of carbohydrate. nitrogen and inorganic salts under submerged aerobic fermentation conditions.

   (b) optionally. separating antibiotic A-7413 mixture from the culture media; (c) optionally, isolating A-7413 factors A. B. C or D from antibiotic A-7413 mixture; (d) optionally. producing the C1-C4-alkyl esters, C1-Cs-acyl esters and thiol C2-C4-carboxylic acid derivatives of A-7413 factors A, B or C; and (e) optionally, producing physiologically acceptable salts of A-7413 factors A, B and C and the C1-Cs-acyl esters and thiol-C2-C4-carboxylic acid derivatives thereof.

   19. The process of claim 18 for producing separated antibiotic A-7413 mixutre which includes: a) cultivating Actinoplanes sp. NRRL 8122 or an A-7413 producing mutant thereof; and b) separating antibiotic A-7413 mixture from the culture medium.

   20. The process of claim 18 for producing A-7413 factor A which includes: a) cultivating Actinoplanes sp. NRRL 8122 or an A-7413 producing mutant thereof; b) separating antibiotic A-7413 mixture from the culture medium; and c) isolating A-7413 factor A from the antibiotic A-7413 mixture.

   21. The process of claim 18 for producing A-7413 factor A methyl ester which includes: a) cultivating Actinoplanes sp. NRRL 8122 or an A-7413 producing mutant thereof; b) separating antibiotic A-7413 mixture from the culture medium; c) isolating A-7413 factor A from the antibiotic A-7413 mixture; and d) producing the methyl ester of A-7413 factor A.

   22. The process of claim 18 for producing A-7413 factor A acetyl ester derivative which includes: a) cultivating Actinoplanes sp. NRRL 8122 or an A-7413 producing mutant thereof; b) separating antibiotic A-7413 mixture from the culture medium; c) isolating A-7413 factor A from the antibiotic A-7413 mixture; and d) producing the acetyl ester of A-7413 factor A.

   23. A composition comprising as an active ingredient antibiotic A-7413 mixture; A-7413 factor A. A-7413 factor B, A-7413 factor C or A-7413 factor D; a C1-C4-alkyl ester, C,-C5-acyl ester, or thiol-C2-C4-carboxylic acid derivatives of A-7413 factors A, B, or C; or a physiologically-acceptable salt of A-7413 factors A, B, or C or of said C-C5-acyl esters or thiol-C2-C4-carboxylic acid derivatives of A-7413 factors A, B, or C; associated with a veterinarally acceptable carrier therefor.

   24. A composition of claim 23 wherein the active ingredient is antibiotic A-7413 mixture.

   25. A composition of claim 23 wherein the active ingredient is A-7413 factor A or a physiologically acceptable salt thereof.

   26. A method of increasing feed-utilization efficiency in ruminant animals having a developed rumen function comprising orally administering to such animals an antibiotic A-7413 mixture; A-7413 factors A. B. C and D; and the C1-C4-alkyl ester. C1-Cs-acyl ester, and thiol-C2-C4-carboxylic acid derivatives of A-7413 factors A, B, or C; or the physiologically-acceptable salts of A-7413 factors A. B. or C or of said C1-Cs-acyl esters or thiol-C2-C4-carboxylic acid derivatives of A-7413 factors A. B, and C.

   27. The method of claim 26 wherein A-7413 mixture is administered to the ruminant animal.

   28. The method of claim 26 wherein A-7413 factor A or a physiologically acceptable salt thereof is administered to the ruminant animal.

   29. An antibiotic A-7413 mixture comprising factors A, B, C and D; factor A. A-7413 factor B. A-7413 factor C and A-7413 factor D; the C1-C4-alkyl esters. C1-CS-acyl esters and thiol-C2-C4-carboxylic acid derivatives of factors A, B and C; and the physiologically acceptable salts of factors A. B and C and the C1-C5-acyl esters and thiol-C2-C4-carboxylic acid derivatives thereof substantially as herein described.

   30. A process for producing an antibiotic A-7413 mixture comprising factors A, B, C and D; A-7413 factor A. A-7413 factor B, A-7413 factor C or A-7413 factor D; the C1-C4-alkyl esters. C1-C5-acyl esters or thiol-C2-C4-carboxylic acid derivatives of factors A, B or C; or the physiologically acceptable salts of factors A, B or C or the C1-C5-acyl esters or thiol C2-C4-carboxylic acid derivatives thereof substantially as herein described.

   31. A composition for increasing feed-ultilization efficiency in ruminant animals having a developed rumen function comprising a carrier and an active ingredient as defined in claim 1 substantially as herein described.

   32. A method as defined in claim 25 of increasing feed-utilization efficiency in ruminant animals having a developed rumen function substantially as herein described.

   33. Actinoplanes sp. NRRL 8122 in a synthetic culture medium containing assimilable sources of carbohydrate. nitrogen and inorganic salts.