CA1090728A - Antibiotic a-7413 mixture comprising factors a,b,c and d and a process for producing it - Google Patents

Abstract

Abstract The present invention relates to an antibiotic A-7413 mixture comprising factors A, B, C and D, and A-7413 factor A, A-7413 factor B, A-7413 factor C and A-7413 factor D, its production by cultivation of Antinoplanes sp. NRRL
8122 or a A-7413 producing mutant thereof, its separation from the culture media; and optionally the isolation of factors A, B, C and D. It also relates to the C1-C4-alkyl esters, C1-C5-acyl esters and thiol-C-2-C4-carboxylic acid derivatives of A-7413 factors A, B and C; and the physiologically acceptable salts of factors A, B and C and their C1-C5-acyl esters and thiol-C2-C4-carboxylic acid derivatives.

Description

1090~

The present invention relates to antibiotic mixture A-7413 comprising factors A, B, C and D, its pro-duction by eultivation of the novel Aetinoplanes sp. NRRL
8122 under submerged aerobie eonditions, its separation and the isolation of faetors A, B and C. It also relates to the Cl-C4-alkyl esters, Cl-C5-aeyl esters and thiol-C2-C4-earboxylic acid derivatives of A-7413 factors A, B and C and the physiologically acceptable salts of A-7413 faetors A, B
and C and their Cl-C5-aeyl esters and thiol-C2-C4-earboxylie aeid derivatives. The antibiotic A-7413 mixture and A-7413 compounds are useful as growth promoting agents and in the eontrol of dental earies and aene.
The A-7413 antibioties are new members of the sulfur-containing thiostrepton family of antibioties. Other members of this family include siomycin, taitomyein, thio-strepton and thiopeptin B.
The group of sulfur-containing antibiotics of this invention are designated as A-7413 antibiotics.
It is the object of this invention to provide antibiotic A-7413 mixture, A-7413 factors A, B, C and D, the Cl-C4-alkyl esters, Cl-C5-acyl esters and thiol-C2-C4-earboxylie aeid derivatives of A-7413 faetors A, B and C, and the physiologieally aeeeptable salts of A-7413 faetors A, B and C and their Cl-C5-aeyl esters and thiol-C2-C4-earboxylie aeid derivatives. It is also the objeet of this invention to provide proeesses for the production of anti-biotic A-7413 mixture by the eultivation of the novel Actinoplanes sp. NRRL 8122, the separation of antibiotic A-7413 mixture from the culture medium, the isolation of - lO~tD~ZB

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 Cl-C4-alkyl esters, Cl-C5-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 their Cl-C5-acyl esters and thiol-C2-C4-carboxylic acid ~
derivatives. : -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 their Cl-C5-acyl esters or thiol-C2-C4-carboxylic acid derivatives comprising: :
(a) cultivating Actinoplanes sp. NRRL 8122 or an A-7413 producing mutant thereof in a culture -medium containing assimilable sources of carbohydrate, nitrogen and inorganic salts under submerged aerobic fermentation con-ditions until a substantial amount of anti-biotic is produced;

. .

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thereby forming a fermentation broth containing said antibiotic;
(b) when a separated antibiotic A-7413 mixture is required, separating same from the fermentation broth;
(c) when at least one of A-7413 factor A, A-7413 factor B, A-7413 factor C and A-7413 factor D is required in isolated form, separating same from the antibiotic mixture;
(d) when Cl-C4-alkyl esters, Cl-C5-acyl esters and thiol-C2-C4-carboxylic acid derivatives of A-7413 factors A, B or C are required, reacting isolated A-7413 factor A, B or C with a correspond-ing Cl-C4-alkyl ester, Cl-C5-acyl ester or thiol-C2-C4 carboxylic acid producing reactant; ~ :
and (e) when physiologically acceptable salts of A-7413 factors A, B and C and their Cl-C5-acyl esters and thiol-C2-C4-carboxylic acid derivates are required, ~
reacting said A-7413 factor A, B or C or a Cl-C5- ~. :
acyl ester or thiol-C2-C4-carboxylic acid deriva- ;
tive thereof with a corresponding salt forming :~
reagent. . -The product of the present invention can be used to . ;
provide a composition for increasing feed-utilization efficiency in ruminant animals having a developed rumen function comprising a carrier and as active ingredient antibiotic A-7413 mixture A-7413 factor A, A-7413 factor B, A-7413 factor C or A-7413 factor D; the Cl-C4-alkyl ester, Cl-C5-acyl ester, or thiol-C2-C4-carboxylic-~3 .
"" ' i~90~Z~3 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 Cl-C5-acyl esters or thiol-C2-C4- :
carboxylic acid derivatives of A-7413 factors A, B, or C.
The product of the present invention can also be used to provide a method of increasing feed-utilization :
efficiency in ruminant animals having a developed rumen function comprising orally administering to such animals a propionate-increasing amount of an antibiotic A-7413 10 mixture comprising factors A, B, C and D; A-7413 factor A, :
A-7413 factor B, A-7413 factor or : `
.: . .

-4a-.

B

or~z~ .

A-7413 factor D; the Cl-C4-alkyl ester, Cl-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 Cl-C5-acyl esters or thiol-C2-C4-carboxylic-acid derivatives of A-7413 factors A, B, or C.
Description of the Drawings The infrared absorption spectra of individual A-7413 factor3 A, B, and C in KBr di~c 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
Detailed Description of the Invention The A-7413 m$xture comprising factors A, B, C, and D is produced by cu}tivating under controlled conditions a novel strain of Actinoplanes sp. NRRL 8122.
As is the ca~e with many antibiotic-producing cultures, fermentation of an A-7413-producing strain of A¢tinoplanee sP. NR~L 8122 results in the production of a number of antibiotic substances. Antibiotic A-7413 factor A
i8 the ma~or factor produced by this culture, and factors B, C, and D are three minor factors. Other factors are present in only very minor ~uantities or are relatively unstable.
The antibiotic A-7413 factors A, B, C, and D are co-produced durlhg the fermentation and are obtained as the antlbiotic A-7413 mixture. The ratio of individual factors produced in the antibiotic mixture will vary depending on the fermentatlon conditlon~ and the individual antibiotic .090~Z8 factor~ are separated from each other and isolated as individual compounds as hereinafter described.
A-7413 FACTOR A ;~
A-7413 factor A is a white to light-yellow crys-talline material which melts with decomposition at about 205-212 C. A-7413 factor A crystallizes from ethanol, chloroform:ethanol, and dimethylformamide:acetone.
A-7413 factor A is soluble in methanol, chloroform, dimethylformamide, dichloroethane and dimethyl sulfoxide; is ~lightly 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 apparent 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 (~houlder), 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.

.. . . .

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The ultraviolet absorption spectrum of A-7413 factor A exhibits the following absorption maxima:
a) in neutral, 95% aqueous ethanol:
215 nm (ElCm= 485);

260 nm (shoulder; ElCm= 240);

300 nm (shoulder; ElCm= 170);

358 nm (shoulder; ElCm= 112.5); -b) in acidic ethanol:
217 nm (ElCm= 440);

265 nm (El%m= ~27.5);

293 nm (ElCm= 210);

358 nm (ElCm= 95);

c) in basic methanol:
278 nm (shoulder; ElCm= 255);

408 nm (ElCm= 80).

Electrometric titration of A-7413 factor A in 80%

aqueous dimethylformamide indicates the presence of a titratable group with a PXa value of 7.9.
Amino-acid analysis of A-7413 factor A, after acldic hydxolysis, indicates thé presence of ammonia (1.03 les/mg), glycine (0.33 ~ moles/mg), threonine (0.40 ~
moles/mg), aspartic acid (0.1 ~ moles/mg), and an as-yet-unidentified amino acid (approx. 0.4 ~ mQles/mg).
A-7413 factor A has a specific rotation, la]25, of +54.5 (c 2.0, CHC13) A-7413 factor A, crystallized from chloroform:ethanol, ha~ the following characteristic X-ray powder diffraction X-3924 _7_ ~ zs :::
:

pattern (Cu++ radiation, 1. 5405 ~, nickel filter, d=inter-planar spacing in angstroms):

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 300C.
A-7413 factor B is soluble in methanol, chloro-20 form, dimethylformamide, dichloroethane and dimethyl sul- .
foxidei 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, 8.7~; 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 ,;,- . . ,: . . .

109072~3 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 neutraI, 95% aqueous ethanol:
268 nm (El%m= 104.3);
357 nm (shoulder; ElCm- 30);

b) in acidic ethanol:
268 nm (ElCm= 108.5);
357 nm (shoulder; ElCm= 35);

c) in basic ethanol:
268 nm (shoulder; ElCm= 178.6).
A-7413 factor B has a specific rotation, [a]DRT, of -26.2 (c 7.5, DMSO).
Amino-acid analysis of A-7413 factor B, after acidic hydrolysis, indicates the presence of ammonia (0.46 moles/mg), glycine (0.1 ~ moIes/mg), threonine (0.1 ~
les/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 250C.

X-3924 _9-~0~07Z~

A-7413 factor C is soluble in methanol, chloro-form, dimethylformamide, dichloroethane and dimethyl sul-foxide; is slightly soluble in ethanol and aqueous ethanol;
but i~ 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%;
10 and ~ulfur, 1.73%. -The infrared absorption spectrum of A-7413 factor C in KBr disc i8 3hown 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 analy~is of A-7413 factor C, after acidic hydrolysis, indicates the presence of ammonia (0.24 moles/mg), glycine (0.05 ~ moles/mg), threonine (0.04 ~
mole~/mg), aspartic acid (0.01 ~ moles/mg), and phenylalanine (0.05 ~ mole~/mg).
The ultraviolet absorption spectrum of A-7413 factor C shows the following absorption maxima:
a) in neutral, 95% aqueous ethanol: -205 nm (ElCm- 356);
235 nm (~houlder; ElCm- 180);

i~ ~2~

260 nm (shoulder; ElCm= 127); ~ .
290 nm (shoulder; ElCm= 104);
bJ in acidic ethanol:

: : . m (Elcm 356);
235.nm (shoulder; El% = 180);
260 nm (shoulder: BlCm- 127);
290 nm (shoulder; El%m- 103);

355 nm (shoulder; ElCm= 40);
c) in basic ethanol: . :
260 nm (shoulder; El%m- 268);
325 nm (shoulder; ElCms 189).
The Rf values of A-7413 factors A, B, C, and D in variou~ paper-chromatographic ~ystem~, using Bacillus subtilis.ATCC 6633 a~ a detection organism, are given in T~bl- I~

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X-392~ -11- .

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_I ~ ~
~r . . . . o ,~ o o o o .....
~, l ~
~ ~ O _I~D
_Icocr ~ o ~r . . . . o ,~ o o o o tn ~ . . -~ . .

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I~r . . . . o ~: ~ o o o o ~I`a~ ~ o 1`
_IU~ ~ ~D ~ .
~r . . o H 1~ O O O O . `
.~C .
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O _i ~0 ~ -- :
3 ~
,a ~ ~ o :c ~ 3~ h ~ o ~ ~ Z . ~ .
u~ ~ A ~ ~a a ~d ,C
:~ ,oo a3 ~C :C ~ O
U~ mU~ 3 ~ ` O rl _I--I O~ _I ~ O ' '~
~ O_I O ~ Z
P ~~C ~ ~~ ~ ~ I` ~ ,'-. "
o m ;~; R æ 3--3 Ql æ

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The Rf values of A-7413 factors A, ~, C, and D in two thin-layer chromatographic Rystems 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) 0.26 0.09 0.46 0.55 Acetonitrile:
water (9:1) 0.23 0.03 0.42 0.48 Each of A-7413 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; of the Cl-C5-acyl-ester derivatives of A-7413 factors A, B, and C; and of the thiol-C2-C4-carboxylic-acid derivatives of A-7413 factors A, B, C are also part of this invention. "Physiologically-acceptable" salts are salts which are also pharmaceutically acceptable, that is, alts 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 Cl-C4-alkylammonium; and hydroxy-C2-C4-alkylammonium salts. Illustrative amine salts include lO~V7~8 those formed by reaction with ammonium hydroxide, sec- -butylamine, isopropylamine, d~ethylamine, di-isopropylamine, ethanolamine, triethylamine, and the like.
The alkali-metal and alkaline-earth-metal cationic salts are prepared according to procedures commonly employed for the preparation of cationic salts. For example,Dthe free acid form of A-7413 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 evap-oration of the solvent.
The salts formed with organic amines can be pre-pared 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 A-7413 factors A, B, and C are also part of this invention. ~hese ester derivatives are prepared by conventional means.
Each of the A-7413 factors A, B, and C has at least one hydroxyl group capable of forming acyl-ester derivatives. The Cl-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 deri-vatives 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 Actinom;ycetales, having bèen first described by Couch [J. Elisha Mitchell Sci. Soc., 65, 315-318 (1949); 66, 87-92 (1950); Trans. New York Acad.
Scl., 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., _ , 53-70 (1963)1.
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, - .. . . . .. . ..

.
, ~.09(~7Z8 Illinois 61604, where it has been accorded the accession number NRRL 8122.
The characteristics of Actinoplanes 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.5. 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.
MORPHOLOGY
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 9 ~ to 14 ~ in diameter, varying in shape from globose to subglobose to irregular.
The principal shape is irregular. Spores are spherical, multiflagellated, 1.4 ~ to 1.7 ~; only a few become motile.

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

'' : . ' ' .: . : ' 10~07Z~3 ~, Czapek's agar Growth abundant, moderate orange (llJ8); no soluble pigment; no sporangia are produced.
Oatmeal agar tICP No. 3) Growth fair, pale yellow green (lOBl); no soluble pigment;
sporangia are produced.
Inorganic salts-starch Growth moderate, brownish orange (ICP No. 4) (13A10); slight brownish soluble pigment; sporangia are produced.
Glycerol-asparagine Growth abundant, medium reddish (ICP No. 5) orange (lOA10); no soluble pig-ment; sporangia are produced.
Bennett's medium Growth fair, pale yellow (llCl);
neither soluble pigment nor sporangia are produced.
Tomato paste-oatmeal Growth sparse; neither soluble pigment nor sporangia are pra-duced.
Tyrosine agar Growth fair, yellowish gray (12A2); 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 (llA4); neither soluble pigment nor sporangia are pro-duced.
Calcium malate Growth moderate, Iight yellowish pink (lOA2); 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.
Gela~in liquefaction None after 21 days.

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Melanin production on Positive.
peptone-iron agar (ICP No. 6) Temperature requirements Good growth from 26 to 37C.
on glycerol-asparagine Reddish orange color most intense agar at 26C. No growth at 43C.

Carbon utilization:

Utilization code: + = utilization -(+) = probable util.
(-) = doubtful util.
- = no utilization .
Rhamnose (+) Cellobiose (-) 10 i-Inositol (-) Cellulose - Melezitose - ~; Fructose (+) Dextrose (+) D-Xylose (+) D-Mannitol (+) Raffinose Sucrose +
Maltose (-) L-Arabinose (-) - Lactose (+) Minus Carbon 1. .,~,;., As in the case with other organisms, the char-,.~
acteristics of the A-7413-producing culture, Actinoplanes .; ~ f 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 i~olation, however, certain culture media are preferred. Thus, for example, a preferred carbohydrate source in large-scale fermentation is dextrin, although glucose, fructose, maltose, sucrose and the like can 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, fish oil, and the like. A preferred nitrogen source is soybean flour, although soybean grits, peptones, oatmeal, peanut meal, soybean meal, cotton-seed meal, amino acids and the like are also useful. Among the inorganic salts which can be incorporated in the culture media are the customary soluble salts capable of yielding sodium, potassium, iron, zinc, cobalt, magnesium, calcium, ammonium, c~loride, carbonate, sulfate, nitrate, and like 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 ::

10~07Z8 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 can be grown at temperatures between about 20 and about 37~C. Optimum A-7413 production appears to occur at temperatures of about 25-30C.
As is the ~ustomary 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. In general, the pH should be in the range of 6.5-7.5. At the end of the fermentation, the harvest pH is usually slightly lower, in the range of 6.0-7Ø
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 paper-disc assay on agar plates.
Generally, antibiotic activity is detectable on the second day of the fermentation. Maximum production of antibiotic activity usually occurs between about 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 A-7413-producing orga-nism 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 lower 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 B alumina, silica gel, ion exchange resin, cellulose, Sephadex, and the like can be advantageously used. For example, preparative-thin-layer chromatography over silica gel, using a chloroform:methanol (9:1) solvent system can be used to separate factors A, B, C, and D, recovering each factor by elution with methanol. For large-scale separation of factors, ~'' ' , ''' ' ' .' ' .
: . . : , , ~09V7;Z~

column chromatography is preferred. In such column separations, a preferred absorbent is silica gel, and a preferred solvent system is chloroform:methanol (19:1). Factor A, the major factor, is readily separated using this method. Purification of minor factors B, C, and D, however, requires subsequent column separations of enriched fractions. Again, silica gel is a preferred adsorbent, and chloroform:methanol (19:1) is a preferred solvent system.
For simplicity in discussions of utility, the term "A-7413 compound" is used herein to refer to a compound selected from the group consisting of A-7413 factors A, B, and C; the alkyl ester, acyl ester, and thiolcarboxylic acid derivatives of A-7413 factors A, B, and C; and the physiologically-acceptable salts of factors A, B, and C and of the alkyl acyl-ester and thiolcarboxylic acid derivatives of factors A, B, and C.
The A-7413 compounds are antimicrobial agents and are especially active against gram-positive microorganisms.
Using the standard disc-plate screening procedure, A-7413 factors 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

Test OrganismFactor A Factor B Factor C
. _ . _ Staphylococcus aureus 23 20 14 Bacillus subtilis 21 18 16 Sarcina lutea 22 19 16 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 ED~o value [effective dose to protect 50 percent of the test animals; see Warren Wick et al., J. Bacteriol. 81, 233-235 (1961)]. The ED50 values for A-7413 factor A against these infections are given in Table IV:

TABLE IV

50Challenge (mg/k 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 agar-dilution 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.

.

.

~LV90728 TABLE V
MIC (mcg/ml) S. aureus Strain 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 H114** 0.125 1.0 H541** 0.062 1.0 3125*** 0.125 1.0 3130*** 0.062 1.0 3131*** 0.062 1.0 -3132*** 0.062 1.0 3133*** 0.062 1.0 3134*** 0.062 1.0 3135*** 0.062 0.5 3136*** 0.125 0.5 203137*** 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 ~.!o~7~s In Table VI are summarized the results of agar-dilution 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 over- ~ -night broth culture in 0.3% 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 peni-cillin-G-resistant, 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 20Shrigley 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% rabbit blood and 1% isovitalex, and a 1:100 dilution of an overnight broth culture as inoculum, A-7413 factor A had the following MIC values:

~n~07zs 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 Proeionibacterium 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 Bio-logical Laboratories). Each tube is inoculated with P.
acnes to contain 104 organisms per ml. After four-days incubation at 37 C, the tubes are observed. The lowest concentration of test compound which prevents growth is recorded as the minimal inhibitory concentration (MIC). The result~ of this test are summarized in Table VII. -TABLE VII

P. acnes CultureA-7413 Factor A
MIC (mcg/ml) ATCC 6919 < 1.25 Clinical Isolate 1 2.50 Clinical Isolate 2 < 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 repre- ~

.:
X-3924 ~26-: . . - , . . . . . .

~ 0728 sentative 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 mutans* < 0.25 Lactobacillus casei** < 0.25 Neisseria perflava** 32.0 * tested on Mitis Salivarius agar with tellurite and thioglycolic acid added.
**tested on Brain Heart Infusion Agar.
In addition, in 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 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 lV~o~z~

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.

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07zs Carbohydrate-utilization efficiency is further measured by in vivo tests performed in animals which have ~ ~
had 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 % Propionic over Relative Treatment Acid Conc. Control to Control Control 20.8 -- --factor A 25.4 4.6 22.1%
100 mg/day In a 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

Increase Increase Molar % over Relative Treatment prop onate control to control Control 24.2 -- --A-7413 mix-ture 30 mg/day 26.2 2.0 8.3%
*Sampled 6 days over a 17-day treatment period The A-7413 mixture and A-7413 compounds are typically effective in increasing propionates and, thereby, the efficiency of feed utilization when administered to - .,: . :

~ g~r~Z8 ruminants orally at rates of from about 0.05 mg/kg/day to about 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 A-7413 compound is by mixing it with the animals' feed; however, it can be adminis*ered 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 ~ains and feed utili-zation 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 , ~ .

72 ~

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:

A. Shake-flask Fermentation of A-7413 A culture of Actinoplanes sp. NRRL 8122 was pre-pared by growing the organism on an 18- x 150-mm agar slant having the following composition:
IngredientAmount Sucrose 30 g Peptone 5 g K2HP04 1 g Czapek's mineral mix soluti~n* 5 ml Agar 25 g Deionized waterq.s. 1 liter *Czapek's Mineral Mix Solution ~ -~
100 g KCl 100 g MgS04-7H20 -~-2 g FeS4-7H2 q.s. to 1 liter with deionized water The slant medium was inoculated with Act ~ es sp. NRRL 8122, and the inoculated slant was incubated at 25C. for 10 to 14 days. The mature slant culture was covered with water, scraping with a ~terile loop to loosen and fragment the mycelia and release the spores from the sporangia. One-half of the resulting ~uspension was used to .~ ' ' .

-~O~ZB

inoculate 50 ml of a liquid vegetative medium having the following composition:
Ingredient Amount 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 vegetative medium was incubated in a 250-ml Erlenmeyer flask at 25C. for 72 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.
This incubated vegetative 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.

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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 wide-mouth Erlenmeyer flask at 25 C
for 72 hour~ on a shaker rotating through an arc two inches in diameter at 250 RPM.
B. Tank_Fermentation of A-7413 In order to provide a larger volume of inoculum, 40 ml of the above-de~cribed incubated vegetative medium was uAed to inoculate 400 ml of a second-stage vegetative medium having the same composition as that of the first-stage vegetative medlum. This inoculated second-stage vegetative medlum, in a 2-liter flask, was incubated at 25~C. for about 48 hour~ on a shaker rotating through an arc two inches in dlameter 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 gSO4 7~2 2 g CaCO3 2 g FeC12-4H2o 0.06 g Deionized waterq.s. 1 liter X-3924 _34_ .

1t)~072~

After sterilization by heating at 120C. for 30 minutes, the pH of the medium was 7Ø The inoculated production medium, in a 165-liter fermentation tank, was allowed to ferment for about 7 days at a temperature of 25C. 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.

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 Na2S2O3 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 additi~n of dilute B ~ 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:

X-3924 _35_ . .- . ~ : ' . :
.: . - , -)7~3 Ingredient Amount Glucose 10.0 g Dextrin 20.0 g Soybean flour 15.0 g Yeast extract 2.5 g Soybean oil (refined) 5.0 g CaCO3 2.5 g Deionized waterq.s. l liter EXAMPLE 4 --~
10 Separation of the A-7413 Mixture Whole fermentation broth (200 liters), prepared as described in Example l, was made acidic (pH 3.5) by the addition of dilute sulfuric acid. The resulting acidic B broth was filtered using a filter aid (Hyflo Super-cel~
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 chloro-form:methanol (4:1) solution. These two extracts were ~07Z8 combined and evaporated to dryness under vacuum. The residue thus obtained was dissolved in chloroform (150 ml);
~his 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.

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) solution. The resulting solution was applied to a 5.8- x 94.0-cm column of cilica gel (Matheson, Grade 62, equilibrated with 5% water), prepared in chloroform:methanol (19:1). The column was developed using chloroform:methanol (19:1), 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 bioautography, 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 chloro-form (50 ml); each chlo~oform solution was added to _-pentane (500 ml) to precipitate the desired factor. The results of the column were as follows:
,'.

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o~zs "

Factor Approximate ObtainedFractions Yield Purity ~-.. _ A 18-23 10.153 gpure 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.

Crystallization 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% benzene) was added. The resulting solution was allowed to stand for two hours at room temperature an~ 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.
A-7413 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 `72~3 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.

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 mi~utes. The , insoluble material was then separated by centrifu~ation.
,l The supernatant solution was freeze-dried to give ~.61 g of :
A-7413 factor A potassium salt as a yellow, water-sc)luble powder.
The potassium salt was further purified and crys-tallized 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 sep- ~ -arated by centrifugation and were dried to give 141.8 mg of crystalline A-7413 factor A potassium salt.

'I ' ' X-3924 -39_ - , . . . . . .
.

10~

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.

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.

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 o~ A-7413 factor A as a yellow, water-soluble compound (2.67% Na by atomic-absorption analysis).
EXAMæLE 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 X-3924 -40_ . . .

Z~

the monosodium salt of A-7413 factor A as a water-soluble compound (1.43% Na by atomic-absorption analysis).
EXAMPLES 1~-18 A-7413 factor B disodium salt, prepared according to the method of Example 8, but using A-7413 factor B and 0.01 N sodium hydroxide.
A-7413 factor B ammonium salt, prepared according to the method of Example 7, but using A-7413 factor B.
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.
A-7413 factor C monosodium salt, prepared according to the method of Example ~, but using A-7413 factor C and 0.1 N sodium hydroxide.
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.
A-7413 factor C magnesium salt, prepared according to the method of Example 9, but using A-7413 factor C and -~
0.1 magnesium hydroxide.

A-7413 Factor A Acetyl Ester Derivative A-7413 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 -temperaturé 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 .

X-3924 -41- ~

10~ 2~3 formed was separated by filtration and dried under vacuum to give 216 mg of the acetyl ester derivative of A-7413 factor A.

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 precip-itate which formed was separated by centrifugation and dried under vacuum to give 421 mg of the acetyl ester derivative of A-7413 factor A.

A-7413 factor A propionyl ester derivative, pre-pared according to the method of Example 20, but using propionic anhydride.
A-7413 factor B n-butyryl ester derivative, pre-pared according to the method of Example 20, but usingA-7413 factor B and n-butyric anhydride.
A-7413 factor C n-valeryl ester derivative, pre-pared according to the method of Example 20, but using A-7413 factor C and n-valeric anhydride.
A-7413 factor A succinyl ester derivative, pre-pared according to the method of Example 20, but using succinic anhydride.-A-7413 factor B formyl ester derivative, prepared according to the method of Example 20, but using acetic formic anhydride.

~ 0'~

A-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 d,ssolved in chloroform (3 ml), and the chloroform solution was added to _-pentane (30 ml). The precipitate which formed was separated by centrifugation and dried to give 87 mg of A-7413 methyl ester.

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

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.

A-7413 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.
-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.
A-7413 factor A bis(mercaptosuccinic acid) derivative, prepared according to the method of Example 30, but using mercaptosuccinic acid.
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.
A-7413 factor A L-cysteine derivative, prepared according to the method of Example 30, but using L-cysteine and purifying the product by chromatography.

X-3924 ~44~

.

Claims (16)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. 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 their C1-C5-acyl esters or thiol-C2-C4-carboxylic acid derivatives comprising:
(a) cultivating Actinoplanes sp. NRRL 8122 or an A-7413 producing mutant thereof in a culture medium containing assimilable sources of carbohydrate, nitrogen and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic is produced thereby forming a fermentation broth containing said antibiotic;
(b) when a separated antibiotic A-7413 mixture is required, separating same from the fermentation broth;
(c) when at least one of A-7413 factor A, A-7413 factor B, A-7413 factor C and A-7413 factor D is required in isolated form, separating same from the antibiotic mixture;
(d) when C1-C4-alkyl esters, C1-C5-acyl esters and thiol-C2-C4-carboxylic acid derivatives of A-7413 factors A, B or C are required, reacting isolated A-7413 factor A, B or C with a corresponding C1-C4-alkyl ester, C1-C5-acyl ester or thiol-C2-C4 carboxylic acid producing reactant;

and (e) when physiologically acceptable salts of A-7413 factors A, B and C and their C1-C5-acyl esters and thiol-C2-C4-carboxylic acid derivates are required, reacting said A-7413 factor A, B or C or a C1-C5-acyl ester or thiol-C2-C4-carboxylic acid derivative thereof with a corresponding salt forming reagent.

2. 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 factor D; the C1-C4-alkyl esters, C1-C5-acyl esters and thiol-C2-C4-carboxylic acid deriva-tives of factors A, B and C; and the physiologically ac-ceptable salts of factors A, B and C and their C1-C5-acyl esters and thiol-C2-C4-carboxylic acid derivatives when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.

3. A process for producing antibiotic A-7413 mixture comprising factors A, B, C and D comprising:
(a) cultivating Actinoplanes sp. NRRL 8122 or an A-7413 producing mutant thereof in a culture medium containing assimilable sources of carbohydrate, nitrogen and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic is produced; and (b) separating antibiotic A-7413 mixture from the culture media.

4. Antibiotic A-7413 mixture comprising factors A, B, C and D when prepared by the process of claim 3 or by an obvious chemical equivalent thereof.

5. The process 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 mixutre.

6. A-7413 factor A when prepared by the process of claim 5 or by an obvious chemical equivalent thereof.

7. The process for producing A-7413 factor B
which includes:
a) cultivating Acetinoplanes 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 B from the antibiotic A-7413 mixture.

8. A-7413 factor B when prepared by the process of claim 7 or by an obvious chemical equivalent thereof.

9. The process for producing A-7413 factor C
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 C from the antibiotic A-7413 mixture.

10. A-7413 factor C when prepared by the process of claim 9 or by an obvious chemical equivalent thereof.

11. The process 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.

12. Methyl ester of A-7413 factor A when prepared by the process of claim 11 or by an obvious chemical equivalent thereof.

13. The process for producing A-7413 factor A
acetyl ester derivative which invludes:
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 derivative of A-7413 factor A.

14. A-7413 factor A acetyl ester derivative when prepared by the process of claim 13 or by an obvious chemical equivalent thereof.

15. The process for producing A-7413 factor A
bis(mercaptoacetic acid) which includes:
a) cultivating Acetinoplanes 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 A-7413 factor A bis(mercaptoacetic acid).

16. A-7413 factor A bis(mercaptoacetic acid) when prepared by the process of claim 15 or by an obvious chemical quivalent thereof.