Note: Descriptions are shown in the official language in which they were submitted.
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
X-3924 -2-
- 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;
X-3924 -3-
. .
: . .
io~ z~
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
X-3924 -5-
.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.
X-3924 -6-
.. . . .
3L090~
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
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
X-3924 -8-
,;,- . . ,: . . .
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
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);
X-3924 -10-
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~
. . ' , '
' ~
: ` ;
X-392~ -11- .
. -~' ' ' ,
10907Z8
_,
~_1,1 ~ o r~
_I ~ ~
~r . . . . o
,~ o o o o
.....
~,
l ~
~ ~ O _I~D
_Icocr ~ o
~r . . . . o
,~ o o o o
tn ~ . . -
~ . .
_I~r ~ u~ ~ .
I~r . . . . o
~: ~ o o o o
~I`a~ ~ o 1`
_IU~ ~ ~D ~ .
~r . . o
H 1~ O O O O . `
.~C .
,
.~ '- ' .-' .
O
~ ~.- .
0
-
. ~ ~ . '
a~ o ~ ~
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
. . '
,~
X-3924 -12-
~o~z~
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
X-3924 -13-
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.
X-3924 -14-
... .. .
10907Z8
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,
X-3924 -15-
- .. . . . .. . ..
.
,
~.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.
X-3924 -16-
'' : . ' ' .: . : '
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.
X-3924 -17-
,9 ,.
Y,~
V ~Z8
. . .
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
X-3924 -18-
28
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
X-3924 -19-
::
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
X-3924 -20-
7JZ8
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,
X-3924 -21-
~'' ' , ''' ' ' .' ' .
: . . : , ,
~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
A-7413 A-7413 A-7413
Test OrganismFactor A Factor B Factor C
. _ . _
Staphylococcus aureus 23 20 14
Bacillus subtilis 21 18 16
Sarcina lutea 22 19 16
X-3924 -22-
07ZS
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.
X-3924- -23-
.
.
~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
X-3924 -24-
~.!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:
X-3924 -25-
~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
X-3924 -27-
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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. :
X-3924 -~8-
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_I _I N ~ n ~r ~r
Sl ~ O O O O O
1:~
O
o
~ dP ~
~ ~ ~ U~' C~ ~ ~ CO
~ ~a ~
~ X O O ' ~
:'
O dP
~ ~ ~o co o a~
O~1 10 a~ o
.,1 ~a ~
X ~ ~ O O O _l O
~1
dP ~
S~ ~ ~ ~ ~ n 1` '
O N _I N O O
~I-rl
~0 o
Q ~
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o . ,.
o _
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~, o o o o U~
O O O U~ N
~r O N _i O O
''¢ _l ,. .
:
X-3924 -29-
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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 -- --
A-7413
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
X-3924 -30-
- .,: . :
~ 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
X-3924 -31-
, ~ .
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:
EXAMPLE 1
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
X-3924 -32-
.~ ' ' .
-
~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.
X-3924 -33-
- ' ' ~ ~ . . ' . ' . ' ., , :
1~07Z~
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.
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
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
X-3924 -36-
~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.
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) 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:
,'.
:
X-3924 -37-
,: ' ~ ' , ~ ' '
.
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.
EXAMPLE 6
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
X-3924 -38-
`72~3
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 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~
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 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.
EXAMPLE 19
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.
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 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.
EXAMPLES 21-25
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.
X-3924 -42-
~ 0'~
EXAMPLE 26
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.
EXAMPLES 27-29
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.
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
X-3924 -43-
. : :
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
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~
.
