MELANGE D'ANTIBIOTIQUE A-30912 EXTRAIT DE L'ASPERGILLUS RAGULOSUS

ANTIBIOTIC A-30912 MIXTURE FROM ASPERGILLUS RAGULOSAS

Abrégé anglais

ABSTRACT
This invention relates to a noval antibiotic
A-3012 mixture comprising antibiotic A-30912 factors A, B,
C, D, E, F and G. It also relates to a novel process for
the production of antibiotic A-30912 mixture by submerged
aerobic fermentation by Aspergillus rugulosus NRRL 8113,
separation of antibiotic A-30912 mixture from the culture
medium and the isolation of antibiotic A-30912 factors A, B,
C, D, E, F and G from the antibiotic A-30912 mixture. Anti-
biotic A-30912 mixture and antibiotic A-30912 factors A, B,
C, D, E, F and G are antifungal agents.

Revendications

The embodiments of the invention of which an ex-
clusive property or privilege is claimed are as follows:
1. A process for production of antibiotic
A-30912 mixture comprising, factors A, B, C, D, E, F and G
comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113
in a culture medium containing assimilable sources of carbo-
hydrate, nitrogen, and inorganic salts under submerged
aerobic fermentation conditions until a substantial amount
of antibiotic activity is produced; and
b) the separation of the antibiotic
A-30912 mixture from the culture medium.
2. An antibiotic A-30912 mixture comprising factors
A, B, C, D, E, F and G, when prepared by the process of claim
1 or by an obvious chemical equivalent thereof.
3. A process as in claim 1, and including the
additional step of isolating antibiotic A-30912 factors A, B, C,
D, E, F or G from the antibiotic A-30912 mixture.
4. A process for production of antibiotic
A-30912 factor A comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113
in a culture medium containing assimilable sources of carbo-
hydrate, nitrogen, and inorganic salts under submerged
aerobic fermentation conditions until a substantial amount
of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture
from the culture medium; and
c) the isolation of antibiotic A-30912 factor
A from the antibiotic A-30912 mixture.
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5. Antibiotic A-30912 factor A, when prepared by
the process of claim 4 or by an obvious chemical equivalent
thereof.
6. A process for production of antibiotic A-30912
factor B comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113
in a culture medium containing assimilable sources of carbo-
hydrate, nitrogen, and inorganic salts under submerged
aerobic fermentation conditions until a substantial amount
of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture
from the culture medium; and
c) the isolation of antibiotic A-30912 factor
B from the antibiotic A-30912 mixture.
7. Antibiotic A-30912 factor B, when prepared by
the process of claim 6 or by an obvious chemical equivalent
thereof.
8. A process for production of the antibiotic
A-30912 factor C comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113
in a culture medium containing assimilable sources of carbo-
hydrate, nitrogen, and inorganic salts under submerged
aerobic fermentation conditions until a substantial amount
of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture
from the culture medium; and
c) the isolation of antibiotic A-30912 factor
C from the antibiotic A-30912 mixture.
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9. Antibiotic A-30912 factor C when prepared by
the process of claim 8 or by an obvious chemical equivalent
thereof.
10. A process for production of the antibiotic
A-30912 factor D comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113
in a culture medium containing assimilable sources of carbo-
hydrate, nitrogen, and inorganic salts under submerged
aerobic fermentation conditions until a substantial amount
of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture
from the culture medium; and
c) the isolation of antibiotic A-30912 factor
D from the antibiotic A-30912 mixture.
11. Antibiotic A-30912 factor D when prepared by
the process of claim 10 or by an obvious chemical equivalent
thereof.
12. A process for production of the antibiotic
A-30912 factor E comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113
in a culture medium containing assimilable sources of carbo-
hydrate, nitrogen, and inorganic salts under submerged
aerobic fermentation conditions until a substantial amount
of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture
from the culture medium; and
c) the isolation of antibiotic A-30912 factor
E from antibiotic A-30912 mixture.
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13. Antibiotic A-30912 factor E when prepared by
the process of claim 12 or by an obvious chemical equivalent
thereof.
14. A process for production of the antibiotic
A-30912 factor F comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113
in a culture medium containing assimilable sources of carbo-
hydrate, nitrogen, and inorganic salts under submerged
aerobic fermentation conditions until a substantial amount
of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture
from the culture medium; and
c) the isolation of antibiotic A-30912 factor
F from the antibiotic A-30912 mixture.
15. Antibiotic A-30912 factor F when prepared by
the process of claim 14 or by an obvious chemical equivalent
thereof.
16. A process for production of the antibiotic
A-30912 factor G comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113
in a culture medium containing assimilable sources of carbo-
hydrate, nitrogen, and inorganic salts under submerged
aerobic fermentation conditions until a substantial amount
of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture
from the culture medium; and
c) the isolation of antibiotic A-30912 factor
G from the antibiotic A-30912 mixture.
17. Antibiotic A-30912 factor G when prepared by
the process of claim 16 or by an obvious chemical equivalent
thereof.
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Description

1084~145
This invention relates to a novel antibiotic
A-30912 mixture comprising at least 7 individual factors A,
B, C, D, E, F and G, The antibiotic A-30912 mixture is
produced by culturing a novel strain of the organism
Aspergillus ru~ulosus NRRL 8113.
The term "antibiotic mixture" as used in this -;:
specification refers to a mixture of co-produced individual
antibiotic factors. As will be recognized by those familiar
with antibiotic production by fermentation, the ratio of
10 individual factors produced in an antibiotic mixture will .
vary, depending on the fermentation conditions used.
The individual antibiotic factors of the present :
invention are designated antibiotic A-30912 factors A, B, C, ..
D, E, F, and G. ;
It is the object of this invention to provide
the novel antibiotic A-30912 mixture and antibiotic A-30912
factors B, C, D, E, F and G.
It is a].so the object of this invention to provide
processes for procluction and separation o antibiotic
A-30912 mixture comprising factors A, B, C, D, E, F and G
and the isolation of factors A, B, C, D, E, F and G.
The present invention provides a novel antibiotic
A-30912 mixture comprising factors A, B, C, D, E, F and G. ;,
The present invention also provides the novel
process for production of antibiotic A-30912 mixture com-
prising factors A, B, C, D, E, F and G comprising:
a) cultivation of Asper~lllus rugulosus NRRL 8113
in a culture medium containing assimilable sources of carbo-
X-4646A -2-
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101~145
hydrate, nitrogen, and inorganic salts under submerged
aerobic fermentation conditions until a substantial amount
of antibiotic activity is produced; and
b) the separation of the antibiotic
A-30912 mixture from the culture medium; and
c) optionally, the isolation of antibiotic
A-30912 factors A, B, C, D, E, F or G from the antibiotic
A-30912 mixture.
The antibiotic A-30912 mixture is extracted from
the fermentation medium with polar organic solvents.
The known compound sterigmatocystin is also pro-
duced by Aspergillus rugulosus NRRL 8113. Sterigmatocystin
is extracted either separately with a nonpolar organic
solvent or together with the antibiotic A-30912 mixture
with polar organic solvents. In the latter case, the
antibiotic A-30912 mixture is separated from sterigmato-
cystin by concentrating the extracting solvent, adding the
concentrate to an excess of a nonpolar organic solvent such
as diethyl ether, and separating the A-30912 antibiotic mix-
ture as a precipitate. Sterigmatocystin is separated in thefiltrate. The antibiotic A-30912 mixture is further puri-
fied by column chromatography.
The antibiotic A-30912 mixture and the individual
A-30912 factors are antifungal agents.
X-4646A -3-
. :~ . . ' ' , . :

108014~i
Infrared absorption spectra of the following
A-30912 factors in KBr disc are presented in the accom-
panying drawings:
Figure 1 - antibiotic A-30912 factor A
Figure 2 - antibiotic A-30912 factor D
Figure 3 - antibiotic A-30912 factor B -
Figure 4 - antibiotic A-30912 factor C
Antibiotic A-30912 factor A
A-30912 factor A is also similar to the polypep-
tide antibiotic Echinocandin B recently reported by F. Benz
et al., Helv. Chim. Acta_57, 2459-2477 (1974).
Antibiotic A-30912 factor A is a white amorphous
solid. Elemental analysis of A-30912 factor A gave the
following percentage composition:
Carbon, 56.52%; hydrogen, 7.29%; nitrogen, 8.68%; -
oxygen, 27.09%.
The approximate empirical formula proposed for antibiotic
A-30912 factor A i8 C51_53H79-83N717-19
approximate range, the elemental analysis of A-30912 factor
A corresponds especially well with an empirical formula of
C52H81N7O18-H2O (Calcd.: C, 56.24; H, 7.54; N, 8-84;
O, 27.39).
Antibiotic A-30912 factor A has an approximate
molecular weight of 1100, as determined by mass spectrometry
and titration.
The infrared absorption spectrum of antibiotic
A-30912 factor A in KBr disc is shown in figure 1 of the
accompanying drawinqs. The following characteristic absorp-
X-4646A -4-

1~801~5
tion maxima are observed: 2.97 (strong), 3.39 (medium),
3.47 (weak), 5.99 (strong), 6.10 (Strohg), 6.49 (medium),
6.56 (medium), 6.90 (medium), 8.00 (weak), 9.13 (weak), and
11.77 (weak) microns.
The ultraviolet absorption spectra of antibiotic
A-30912 factor A in both neutral and acidic methanol exhibit
absorption maxima at 225 nm (~ 18,000), 275 nm (~ 3,000) and
284 nm (shoulder ~ 2,500). The ultraviolet spectrum of
factor A in basic methanol shows absorption maxima at
245 nm ~ 16,000) and 290 nm (~ 3,000) and also end absorp-
tion.
The 13C nuclear magnetic resonance spectrum of
antibiotic A-30912 factor A in perdeuteromethanol shows the
following characteristics:
~ 176.1, 174.3, 173.4, 172.7, 172.4, 169.~, 158.4, 132.8,
130.9, 129.6, 129.0, 116.2, 77.0, 75.7, 74.4, 71.3, 70.9,
69.6, 68.3, 62.4, 58.7, 56.9, 56.1, 52.9, 39.0, 38.5, 36.8,
35.2, 33.9, 32.9, 32.6, 30.7, 30.4, 30.2, 28.2, 27.0, 26.5,
23.6, 20.1, 19.6, 14.4, and 11.3 ppm.
Antibiotic A-30912 factor A has the following
specific rotations: [a]D ~44 (c 0.5, CH3OH)
[a]3565 -156 (c 0.5, CH30H)
Electrometric titration of antibiotic A-30912
factor A in 66% aqueous dimethylformamide indicated the
presence of a titratable group with a PKa value of 12.8
(initial pH 6.9).
Amino-acid analysis of antibiotic A-30912 factor A
indicated the presence, after hydrolysis, of threonine,
X-4646A -5-
. : . .
.

108~45
hydroxyproline and three other as-yet-unidentified amino
acids.
Antibiotic A-30912 factor A is soluble in a
variety of organic solvents such as methanol, ethanol,
dimethylformamide, dimethyl sulfoxide, and ethyl acetate;
but is insoluble in nonpolar organic solvents such as
diethyl ether and petroleum ether. Antibiotic A-30912
factor A is also soluble in aqueous solutions, especially
those having a pH greater than 7Ø
A-3091? Factor D
Antibiotic A-30912 factor D is a white amorphous
solid. Elemental analysis of A-30912 factor D gave the
following percentage composition: carbon, 56.37 percent;
hydrogen, 8.17 percent; nitrogen, 8.54 percent; oxygen (by
difference), 26.92 percent.
Antibiotic A-3091Z factor D has an approximate
molecular weight of 1100, based upon amino-acid analysis and
its close structural relationship to antibiotic A-30912
factor A.
The infrared absorption spectrum of antibiotic
A-30912 factor D in KBr disc is shown in figure 2 of the
accompanying drawings. The following characteristic absorp-
tion maxima are observed: 2.98 (strong), 3.31 (weak), 3.36
(shoulder), 3.40 (medium), 3.48 (weak), 5.76 (weak), 6.01
(strong), 6.10 (shoulder), 6.49 (medium), 6.57 (medium),
6.90 (medium), 7.81 (weak), 8.07 (weak), and 9.16 (weak)
microns.
The ultraviolet absorption (UV) spectra of anti-
biotic A-30912 factor D in neutral and acidic methanol
X-4~46~ -6-

8V14~
exhibit absorption maxima at 225 nm (~ 18,000) and 275 nm
( 2,500). The UV spectrum of A-30912 factor D in basic
methanol exhibits absorption maxima at 240 nm ( 11,000) and
290 nm (~ 3,000).
Antibiotic A~30912 factor D has the following
specific rotation: [~]D ~50 (c 0.34, CH30H).
Amino-acid analysis of antibiotic A-30912 factor
D, after hydrolysis, indicated the presence of threonine,
hydroxyproline, histidine and three other as-yet-unidenti-
fied amino acids. One of the unidentified antibiotic
A-30912-factor-D amino acids is identical to one of the
unidentified antibiotic A-30912-factor-A amino acids. ~;
Antibiotic A-30912 factor D is soluble in a
variety of organic solvents such as methanol, ethanol,
dimethylformamide, dimethyl sulfoxide, and ethyl acetate;
but is insoluble in non-polar organic solvents such as
diethyl ether and petroleum ether. Antibiotic A-30912
factor D is soluble in aqueous solutions, especially those
having a pH greater than 7Ø
A-30912 Factor B
Antibiotic A-30912 factor B is a white amorphous
solid. Elemental analysis of A-30912 factor B gave the
following approximate percentage composition: carbon,
57.36 percent; hydrogen, 5.92 percent; nitrogen, 8.75
percent; oxygen, 26.19 percent.
The infrared absorption spectrum of A-30912
ractor B in KBr disc is shown in figure 3 of the accom-
~)any;ng drawings. The following characteristic absorption
maxima are observed: 2.99, 3.41, 3.49, 6.06, 6.15, 6.54,
30 6.61, 6.94, 7.62, 8.07, 9.26, and 9.39 microns.
X-4646A -7-

lasv~s
The ultraviolet absorption spectra of A-30912
factor B in both neutral and acidic methanol exhibit ab-
sorption maxima at 223 nm (shoulder, 16,000) and 278 nm (~ ;~
2,400). The ultraviolet spectrum of antibiotic A-30912
factor B in basic methanol shows absorption maxima at 242 nm
(~ 13,900) and 292 nm (~ 2,800).
A-30912 factor B has the following approximate
specific rotations: [a]D -47 (c 0.5, CH30H)
[a]365 -170 (c 0.5, CH30H).
Electrometric titration of A-30912 factor B in 66
aqueous dimethylformamide indicated the presence of a
titratable group with a PKa value of about 13.0 (initial pH
7.91).
Amino-acid analysis of A-30912 factor B indicated
the presence, after standard acid hydrolysis, of threonine,
hydroxyproline and several as-yet-unidentified amino acids.
A-30912 factor B is soluble in a variety of organic
solvents such as methanol, ethanol, dimethylformamide,
dimethyl sulfoxide, and ethyl acetate; but is insoluble in
nonpolar organic solvents such as diethyl ether and petroleum
ether. A-30912 factor B is also soluble in aqueous solutions,
especially those having a pH greater than 7Ø
A-30912 Factor C
Antibiotic A-30912 factor C is a white amorphous
solid. Elemental analysis of A-30912 factor C gave the
following approximate percentage composition: carbon, 56.76
percent; hydrogen, 7.88 percent; nitrogen, 10.61 percent;
oxygen, 25.09 percent.
X-4646A -8-

1080145
The infrared absorption spectrum of A-30912 factor
C in KBr disc is shown in figure 4 of the accompanying
drawings. The following characteristic absorption maxima -
are observed: 2.98, 3.39, 3.43, 3.51, 6.01, 6 12, 6.47,
6.90, 7.04, 7.22, 7.38, 8.00, 8.30, and 9.13 microns.
The ultraviolet absorption spectra of A-30912
factor C in both neutral and acidic methanol exhibit ab-
sorption maxima at 223 nm (shoulder, 7,300) and 275 nm (E
1,350). The ultraviolet spectrum of antibiotic A-30912
factor C in basic methanol shows absorption maxima at 240 nm
(~ 12,400) and 290 nm ( 5,200).
A-30912 factor C has the following approximate
specific rotations: []D5 ~33 (c 0.5, CH30H)
[a]365 -119 (c 0.5, CH30H).
Electrometric titration of A-30912 factor C in 66%
aqueous dimethylformamide indicated the presence of a
titratable group with a PKa value of about 13.08 (initial pH
7.93).
Amino-acid analysis of A-30912 factor C indicated
the presence, after standard acid hydrolysis, of threonine,
hydroxyproline and several as-yet-unidentified amino acids.
A-30912 factor C is soluble in a variety of
organic solvents such as methanol, ethanol, dimethylformamide,
dimethyl sulfoxide, and ethyl acetate; but is insoluble in
nonpolar organic solvents such as diethyl ether and petroleum
ether. A-30912 factor C is also soluble in aqueous solutions,
especially those having a pH greater than 7Ø
X-4646A -9-

1~80145
The seven individual factors of the antibiotic
A-30912 mixture can be separated and identifièd by the use
of thin-layer chromatography (TLC). Silica gel is a pre-
ferred adsorbent; and benzene:methanol (7:3, V:V) is a
preferred solvent system.
The Rf values of antibiotic A-30912 factors A-G,
using silica gel (Merck, Darmstadt) TLC, the benzene:meth-
anol (7:3) solvent system, and Candida albicans bioauto- .
graphy are given in Table I.
10TABLE I
Antibiotic A-30912 Factor Rf Value
,~
A 0.35
B 0,45
C 0.54
D 0.59 ~ :
E 0.27
F 0.18
G 0.13
The Rf values of antibiotic A-30912 factor A in
various paper-chromatographic systems, again using Candida
albicans as a detection organism, are given in Table II.
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1080145
TABLE II
A-30912 Factor A
Rf Value Solvent system
0.76 sutanol saturated with water
0.69 Butanol saturated with water
plus 2% p-toluenesulfonic acid
0.75 Methanol:0.1 N HCl (3:1)
0.17 Butanol:ethanol:water
(13.5:15:150)
0.78 Methanol:0.05 M sodium citrate
at pH 5.7 (7:3); paper buff-
ered with 0.05 M sodium ci-
trate at pH 5.7 ~ '
The organism useful for the preparation of the
; antibiotic A-30912 mixture was isolated from a soil sample
from the ruins of Pompeii, Italy. The A-30912 producing
organism is classified as a strain of Aspergillus rugulosus
Thom and Raper, which is in the Asper~illus nidulans
form group. This classification is based on the description
of K. B. Raper and D. I. Fennel in "The Genus Aspergillus,"
The Williams and Wilkins Company, Baltimore, Md., 1965.
Color names were assigned according to the ISCC-
NBS method (K. L. Kelly and D. B. Judd, "The ISCC-NBS Method
of Designating Color and a Dictionary of Color Names," U. S.
Dept. of Commerce, Circ. 553, Washington, D. C., 1955). The
Maerz and Paul color blocks are described by A. Maerz and M.
R. Paul in "Dictionary of Color," McGraw-Hill Book Company,
New York, N.Y., I950.
Cultures were grown at 25 C. unless otherwise
specified.
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iO80145
Culture Characteristics of A. rugulosus NRRL 8113
-
Cza~ek's Solution ~ r ~ -
~ . .
The culture grows slowly, attaining 1.5 to 2.0 cm
in diameter in 15 days at 25C. The colony surface is -`?
convex and velutinous, becoming wrinkled with age near the
center and then umbonate. The mycelium periphery is a-2-mm
wide band of deeply submerged colorless hyphae and is
sinuate. A pinkish-brown exudate forms on the marginal
aerial hyphae. In from 7 to 14 days a pale purple soluble
pigment is produced in the agar surrounding the colony. The
pigment diffuses throughout the colony by 15 days.
After 5 days the colony surface ranges from white
to buff, and the colony reverse is brownish orange centrally
and brownish to brownish purple in the peripheral regions.
In 10 days the colony is moderate yellowish pink (ISCC-NBS
29 and Maerz and Paul ll-A-7). After 14 days the colony is
light grayish red (ISCC-NBS 18 and Maera and Paul 4-G-7).
The marginal area becomes verruculose and is strong yellow
(ISCC-NBS 84 and Maerz and Paul 10-L-5) due to conidiation.
Scattered dull yellowish clusters of hulle cells occur
randomly over the surface and along the margin of the
colony. With age, the strong yellow patches and marginal
area become yellowish green. After 3 weeks, an orange-
purple tone is observed in the new aerial components of the
margin. Initially, the colony reverse is slightly concave.
~s it matures, the colony flattens to the agar surface, and
~he reverse becomes slightly wrinkled. By 10 days the
reverse is light brown (ISCC-NBS 57 and Maerz and Paul
5-A-10). In 15 days it is grayish red (ISCC-NBS 19 and
Maerz and Paul 6-J-3).
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.

1080145
:
The conidiogenous state is sparse; and conidio-
phores are scattered over the surface, sometimes occurring
as patches or in a submarginal band. Conidial heads are at
first loosely radiate and globose; with aging, they may
develop as short columnar forms which are more compact.
Globose heads are from 70~ to 80~ in diameter and average
50~. Columnar heads may be up to 140~ long and 70~ wide.
Conidia are globose to subglobose, rugulose, and
greenish gold en masse. They range from 2.8~ to 3.9~ in
diameter and average 3.2~.
Sterigmata are biseriate and colorless. Primary
sterigmata range in length from 4.7~ to 11.0~ and average
7.9lJ- At their widest point they are 2.4~ and taper to
1.6~. Secondary sterigmata may occur singly or in pairs,
arising from the primaries, and are flask shaped. At their
widest point they measure 3.0~ and taper apically to 0.4~ -
where they become tubular. The tubular apex is 1.2~ long.
The overall length range~ from 5.5~ to 12.6~ and averages
9.21J.
Vesicles are globose to subglobose or hemispheri-
cal and may be apically flattened, becoming brownish with
age. They range from 7.4~ to 11.2~ in diameter and average
9.4ll.
Conidiophores are smooth, relatively thick-walled,
and are at first hyaline then evolve to a light cinnamon
brown. They are slightly wider at the vesicle and may taper
slightly near the foot cell. The average width is 5.9~l.
X-464hA -13-

~801~5 ~ ~
Conidiophores range from 47.7~ to 166.6~ and average 106~ in
length. They arise from submerged hyphae or laterally from
aerial hyphal filaments.
The ascogenous state appears in up to 20 days.
The small yellowish clusters of hulle cells which occur on
the surface may be found at any level in the mycelium. They
consist of hulle cells which envelope one or more cleisto-
thecia. Hulle cells are globose to subglobose or oval to
elongate, are thick-walled and hyaline. Globose hulle cells
range from 18~ to 24~ in diameter and average 21.8~.
Cleistothecia are globose to subglobose, thick-
walled, relatively tough and fibrous. At first relatively
colorless, they become reddish purple and darken with age.
They measure from 165~ to 4701l in diameter and average 275~.
Malt Extract Agar
Colonies grown at 25 C. expand rapidly, attaining
4-5 cm in 10-12 days. At first a grayish white, colonies
become moderate olive green (ISCC-NBS ~0 and Maerz and Paul
23-E-6) in 4 days. The sinuate to weakly lobate periphery
consists of tightly packed, short, white aerial hyphae.
Small yellowish clusters of hulle cells dot the margin and
are randomly scattered over the felt-like agar surface.
After 20 days these hulle cell clusters tend to encrust much
of the surface. The colony reverse is grayish yellow
(ISCC-NBS 90 and Maerz and Paul ll-B-l).
The ascogenous state appears in 15 days. The
small yellowish clusters of hulle cells which occur on the
surface may be found at any level in the mycelium. They
consist of hulle cells which envelope one or more cleisto-
X-4646~ -14-

1080~45
thecia. Hulle cells may encrust large areas over the
conidial heads. Hulle cells are globose to subglobose or
oval to elongate, are thick-walled and hyaline. Globose
hulle cells range from 18~ to 24~ in diameter and average
21.8~. ;
Cleistothecia are globose to subglobose and are
dark reddish brown. They range from 389~ to 700~ in diam-
eter and average 506~.
Asci are fragile, hyaline, and subglobose to oval.
Subglobose asci are from 8.7~ to 11.9~ in diameter and
average 10.3~. Oval asci are from 10.3~-14.2~ x 8.7~-
10.31l and average 12.2~ x 9.1~.
Ascospores are red-orange, rugulose, with two
parallel, delicately pleated equatorial crests which are up
to 0.8~ wide and unbroken. The ascospore appears lenticular
through the long axis. When the crest is peripheral, the
ascospore is globose. In the globose view it is from 4.9
to 6.3~ in diameter and averages 5.4~.
__
Two characteristics of the antibiotic A-30912-
producing strain of Aspergillus rugulosus differ from the
characteristics of A. rugulosus described by Raper and
Fennel, supra. The A-30912-producing strain has larger
conidial heads and ascospores.
The Aspergillus rugulosus culture useful for the
prod~lction of the antibiotic A-30912 mixture has been
deposited and made a part oL the stock culture collection of
the Northern Regional Research Laboratory, U. S. Department
X-4646A -15-
.

~ )145
of ~griculture, Agricultural Research Service, Peoria,
Illinois 61604, from which it is available to the public
under the number NRRL 8113.
Any one of a number of culture medium can be
employed to grow Aspergillus rugulosus NRRL 8113. For
economy in production, optimal yield and ease of product
isolation, however, certain culture media are preferred.
Thus, for example, a preferred carbohydrate source in
large-scale fermentation is glucose, although molasses,
starch, lactose, sucrose, maltose, glycerol and the like may
be employed. Preferred nitrogen sources are enzyme-hydrolyzed
casein and soluble meat peptone, although distiller's grains,
monosodium glutamate and the like may be used. Nutrient
inorganic salts can be incorporated in the culture media.
These include the customary soluble salts capable of yielding
sodium, magnesium, calcium, ammonium, chloride, carbonate,
sulfate, nitrate, and the 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
c~lycol to large-scale fermentation media if foaming becomes
a ~roblem.
Ior ~roduction of a substantial quantity of the
anti~ioti( ~-~0912 mixturc, submerged aerobic fermentation
X-4646A -16-
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1~1)145
in tanks is preferred. Small quantities of the antibiotic
A-30912 mixture may be obtained by shake-flask culture.
secause of the time lag in antibiotic production commonly
associated with inoculation of large tanks with the spore
form of the organism, it is preferable to use a vegetative
inoculum. The vegetative inoculum is prepared by inocu-
lating 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 used for larger fermenf:ations, but other media
can also be employed. The antibiotic A-30912-producing
organism can be grown at temperatures between about 20 and
about 43C.; the organism grows well at temperatures of
about 25-30C. Optimum production of the antibiotic A-30912
mixture appears to occur at a temperature of about 25C.
As is customary in aerobic submerged culture
processes, sterile air is blown through the culture medium.
For efficient antibiotic production, the volume of air
employed in tank production is preferably above 0.4 volume
of air per volume of culture medium per minute (V/V/M).
Production of the antibiotic A-30912 mixture can
be followed during the fermentation by testing samples of
alcoholic extracts of the whole broth for antibiotic activity
against an organism known to be sensitive to the A-30912
antibiotics. One assay organism useful in testing for the
l)resence of the antibiotic A-30912 mixture is Candida albicans.
'rhe bioassay is conveniently performed by paper-disc assay
on seeded agar plates.
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11~80145
Generally, antibiotic activity can be detected on
the second day of fermentation. Maximum production of
antibiotic activity usually occurs between about the third
and the sixth days.
The antibiotic A-30912 factors are antifungal
agents. Illustrative of the antifungal activity of the -
antibiotic A-30912 factors are ln vitro tests with anti-
biotic A-30912 factors A and D. These tests are summarized
in Table III. Antifungal activity was measured by the
conventional disc-diffusion method (6-mm pads were dipped in
solutions containing test compound; pads were placed on agar -
plates seeded with the test organism). Results are given as
the minimal inhibitory concentration (MIC) per disc at which
the test compound inhibited the test organism.
TABLE III
Test Organism MIC (mcg/disc)
Antibiotic Antibiotic
A-30912 A-30912
Factor A Factor D
Candida albicans 0.625 0.5
-
Trichophyton mentagrophytes 0.078 0.07
A-30912 factor A is very active in in ln vitro disc-
diffusion tests against dermatophytes. The results of these
tests are summarized in Table IV.
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- 108l)145 ~:
TABLE IV
A-30912 FACTOR A VS. DERMATOPHYTES
Dermatophyte No. of Isolates MIC~mcg/disc)
TrichophrYton
mentagrophytes13 1.25 - .039
,,:
Trichophyton
gallinae 1 >1.25 :~
Trichophyton :'
me~inini 1 . 0 0195
Trichophyton
quinckeanum 1 >1 25 :
-:
10 TrichophYton
rubrum 1 <.0098
~ .
Trichophyton
schoencelnii 1 0.0195
Trichophyton
terrestre 1 0.0195
Trichophyton
tonsuranrs- ~ 9 >1.25 - 0.156
Microsporium -
gypseum 5 0.156 - 0.038
Microsporiu~
audouinii 4 1.25 - 0.156
Microsporium
canis~ ~ - 6 1.25 - 0.0098
20 Microsporium
cookel 2 1.25 - 0.0195
.
Nannizzia
incurvata 1 0.312
: . .
Phalaphere
jean SAlemi 1 >l. 25 ~
Epidermatophyton
floccosum 1 1.25
.
Geotrichum
candidum~~~ 4 ~1.25 - 0.156
Keratinomyces
ajelllo 1 0.156
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The antifungal activity of the antibiotic A-30912
factors was further demonstrated by in vivo tests. The in
vivo tests were carried out in the following manner: Three
doses of test compound are given to Candida albicans-
infected mice at 0, 4, and 24 hours post-infection. The
protection which the test compound provides is measured as
an ED50 value [the effective dose in mg/kg which protects 50
percent of the mice; see W. Wick et al., J. Bacteriol. 81,
233-235 tl961) 1. The ED50 values for antibiotic A-30912
factor A against Candida albicans in mice were 30 mg/kg
(intraperitoneal administration) and 50 mg/kg(subcutaneous
administration). The ED50 value for antibiotic A-30912
factor D against Candida albicans in mice was 33 mg/kg
(subcutaneous administration).
There were no signs of acute toxicity when anti-
biotic A-30912 factor A was administered intraperitoneally
(ip) or subcutaneously (sc) to mice at 100 mg/kg twice per
day for three days (a total of 600 mg/kg). There were also
no signs of acute toxicity when antibiotic A-30912 factor A
20 was administered ip to mice at 200 mg/kg three ti~es per day
( a total dose of 600 mg/kg).
There were no signs of acute toxicity when anti-
biotic A-30912 factor D was administered sc to mice three
times per day at 14 mg/kg (a total dose of 42 mg/kg).
When used as antifungal agents, the antibiotic
A-30912 factors are administered parenterally and are com-
m~nly administered together with a pharmaceutically-accep-
table carrier or diluent. The dosage of antibiotic A-30912
X-4646A -20-

1080145
factor will depend upon a variety of conditions, such as the
nature and severity of the particular infection involved.
In order to illustrate more fully the operation of
this invention, the following examples are provided.
EXAMPLE 1
A. Shake-flask Fermentation
A culture of Aspergillus rugulosus NRRL 8113 was
prepared and maintained on an 18- x 150- ml agar slant
having the following composition:
Amount
Ingredient (percent)
Dextrin 1.0000
Enzymatic hydrolysate of casein* 0.2000
Yeast extract 0.1000
Beef extract 0.1000 ~;
KCl 0.0200
MgSO ~7H2O 0.0200
FeS04 7H20
Water 98.5596
*"N-Z-Amine A,"l Sheffield Chemical Co.,
Norwich, N.Y.
The slant was inoculated with Aspergillus rugu-
losus NRRL 8113, and the inoculated slant was incubated at
25C. for about 7 days. The mature slant culture was
covered with beef serum and scraped with a sterile loop to
loosen the spores. One-half of the resulting suspension was
used to inoculate 50 ml of a vegetative medium having the
following composition:
1 Trademark
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108()145
Amount
Ingredient (Percent)
Sucrose 2.5
Molasses 3.6
Corn steep liquor 0.6
Enzymatic hydrolysate of casein* 1.0
K2HPO4 0.2
Water 92.1
*"N-Z-Case"2,Sheffield Chemical Co.,
Norwich, N.Y.
The inoculated vegetative medium was incubated in ;~
a 250-ml wide-mouth Erlenmeyer flask at 25~ C. for 24 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 incubclted vegetative medium and 2 ml of a glycerol-
lactose solution having the following composition: ,
Ingredient Amount
Glycerol 20%
Lactose 10%
Deionized water 70~
Thc prcpared suspensions are stored in the vapor phase of
liquid nitrogen.
2. Trademark
X-4646A -22-
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.

- 1080~45
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 22 hours on a shaker rotating through an arc two inches
in diameter at 250 RPM.
B. Tank Fermentation
In order to provide a larger volume of inoculum,
10 ml of the above-described incubated first-stage vege-
tative medium was used to inoculate 400 ml of a second-
stage vegetative growth medium having the same composition
as that of the vegetative medium. The second-stage medium
was incubated in a 2-liter wide-mouth Erlenmeyer flask at
25 C for 25 hours on a shaker rotating through an arc two `
inches in diameter at 250 RPM. ~;
Incubated second-stage vegetative medium (800 ml),
prepared as above-described, was used to inoculate 100
liters of sterile production medium having the following
composition:
Ingredient Amount
Glucose 25 g/liter
Starch 10 g/liter
Peptone* 10 g/liter
Blackstrap molasses 5 g/liter
Enzymatic hydrolysate of casein** 4 g/liter
g O4 712O 0.5 g/liter
Czapek's mineral stock*** 2.0 ml/liter
CaCO3 2.0 g/liter
Deionized water q.s. 1 liter
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1081)145
*W.P. No. 159, Inolex Biomedical Corp.,
Glenwood. Ill.
**"N-Z Amine A", Sheffield Chemical Co.,
Norwich, N.Y.
***Czapek's mineral stock has the following composition: `
Ingredient Amount
FeSO4.7H2O (dissolved in
2 ml conc HCl) 2 g
KCl 100 g
MgSO4.7H2O 100 g
Deionized water q.s. to 1 liter
The pH of the medium was 6.8 after sterilization
by autoclaving at 121C for 30 min at about 16-18 pounds
pressure. The inoculated production medium was allowed to
ferment in a 165-liter fermentation tank at a temperature of
25C for four days. The fermentation medium was aerated with
sterile air at the rate of 0.5 V/V/M. The fermentation med-
ium was stirred with conventional agitators at 300 RPM.
EXAMPLE 2
Separation of the Antibiotic A-30912 Mixture
Whole fermentation broth (200 1.), obtained by the
method described in Example 1, was stirred thoroughly with
methanol (200 1.) for one hour and then was filtered, using `
a filter aid ("Hyflo Super-cel"3, a diatomaceous earth,
Johns-Manville Products Corp.). The pH of the filtrate was ad- `
justed to pH 4.0 by the addition of 5 N HCl. The acidified
filtrate was extracted twice with equal volumes of chloro- i~
form. The chloroform extracts were combined and concentrated
3. Trademark
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108~145
under vacuum to a volume of about four liters. This con-
centrate was added to about 60 liters of diethyl ether to
precipitate the A-30912 mixture. The precipitate was -~
separated by filtration and dried to give 38 g of the
antibiotic A-30912 mixture as a gray powder. The filtrate
was concentrated under vacuum to give an oil; this oil was
dissolved in methanol (500 ml). The methanol solution was
added to diethyl ether (7.5 1.) to precipitate additional
antibiotic A-30912 mixture. This precipitate was also
separated by filtration and dried to give and additional
3.5 g of the antibiotic A-30912 mixture.
EXAMPLE 3
Isolation of Antibiotic A-30912 Factor A
, ._ - - :
Antibiotic A-30912 mixture (20 g), obtained as
described in Example 2, was placed on a silica-gel column
(4- x 107-cm, Woelm) in acetonitrile:water (95:5). The
column was eluted with acetonitrile:water (95:5) at a flow
rate of 1 to 2 ml per minute, collecting fractions having a
volume of approximately 20 ml. Fractions were checked by
thin-layer silica-gel chromatography, using the acetoni-
trile:water (95:5) solvent system and Candida albicans
bioautography.
Fractions 74 through 125 were combined and con-
centrated. The concentrated solution crystallized upon
standing to give an additional 124 mg of sterigmatocystin.
Fractions 212 through 273 were combined and concentrated
under vacuum to give an oil. This oil was dissolved in a
small volume of methanol. The methanol solution was added
X-4646A -25-
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10~ 45
.,
to diethyl ether (15 volumes). The precipitate which formed ;
was separated and dried to give 23 mg of antibiotic A-30912
factor D. Fractions 274 through 437 contained antibiotic
A-30912 factors A, B, C and D. Fractions 482 through 900
contained antibiotic A-30912 factors A, E, F and G. Frac-
tions 438 through 481 were combined and concentrated under
vacuum to give an oil. This oil was dissolved in a s~all
volume of metllanol; and the methanol solution was added to
diethyl ether (15 volumes). The precipitate which formed
10 was separated and dried to give 2.17 g of antibiotic A-30912
factor A.
EXAMPLE 4
Isolation of Antibiotic A-3091~ Factor D
A partially purified antibiotic A-30912 mixture
containing antibiotic A-30912 factors B, C, and D was
obtained as described in Example 3 for fractions 274-437.
This material (750 mg) was chromatographed on a silica-gel
column (2.2 x 51 cm, Woelm silica gel), collecting fractions
having a volume of approximately 15 ml and eluting with the
following solvents:
Fractions Solvent
.. ..
1-25 acetonitrile
26-62 acetonitrile + 1% water
63-700 acetonitrile + 1.5~ water
The column fractions were monitored by silica-
ge~l thin-layer chromatography, using acetonitrile:water
(95:5) and benzene:methanol (7:3) solvent systems and
X-4646A -26-
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- 1080145
Candida albicans bioautography. Fractions 535-685, which
contained antibiotic A-30912 factor D, were combined and
concentrated under vacuum to give an oil. ThiS oil was
dissolved in a small amount of methanol and added to diethyl
ether (15 volumes~. The precipitate which formed was - ~-
separated by filtration and dried to give 69 mg of anti-
biotic A-30912 factor D. ;,
EXAMPLE 5
Isolation of A-30912 factors E~ and C
Partially purified A-30912 antibiotic complex
containing A-30912 factors A, B, C, and D was obtained as
described in Example 3 for fractions 212-437. This material
(18 g) was dissolved in a minimal volume of acetonitrile:
water (4:1) and chromatographed on an aluminum oxide column
(3.8 x 56 cm, Woelm), collecting fractions having a volume
of approximately 20 ml. The column was eluted with the
following solvents:
Fractions Solvent
.. . .
1-300 acetonitrile:water (4:1)
301-509 acetonitrile:water (7:3)
The column fractions were monitored by silica-
gel thin-layer chromatography as described in Example 4. On
the basis of these results, fractions were combined and
concentrated to oils; the oily residues were dissolved into
small volumes of methanol; the methanol solutions were
~recipitated with 10-15 volumes of diethyl ether. The
manner in which the fractions were combined, the weight of
material obtained, and the factor content of the combined
fractions are summarized below.
X-4646A -27-
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108~)14~;
Fraction Weight (g) Factors
6-28 0.23* --
6-28 5.80 A-30912 C, D
34-114 2.90 A-30912 B
115-164 1.20 A-30912 A, B
165-509 1.90 A-30912 A
*insoluble material obtained before ether pre-
cipitation
In order to obtain purified A-30912 factor C, a
portion of fractions 6-28 (2 g) was dissolved in methanol,
adsorbed onto a sufficient quantity of silica gel (grade 62),
dried, and added on top of a silica-gel column (1.9 x 80 cm,
grade 62), packed in acetonitrile. The column was eluted
with acetonitrile at a flow rate of 2 ml/minute, collecting
factions having a volume of about 10 ml. At fraction 117,
the solvent was changed to acetonitrile:water (99:1). The
column fractions were again monitored by thin-layer chroma-
tography. On the basis of the TLC results, fractions were
combined and concentrated to give oily residues; the oily
residues were dis601ved into a small volumes of methanol;
the methanol solutions were precipitated with 10-15 volumes
of diethyl ether. The factor content and weight of the
fractions of interest are summarized below:
Fractions Weight (g) Factors
341-479 0.250 D
480-540 0.015 D
541-899 0.391 C, D
900-1675 0.340 C
X-4646A -28-
: ; ~ , . , , : . . .