DE2643485A1 - ANTIBIOTICUM A-30912 AND PROCESS FOR ITS MANUFACTURING - Google Patents

Description

PFENNING MAAS

ΜΞΙΝία - LEMKE - Mockery

SCHLElSSHEiMERSTR. 299

aOOO MUNICH 40

X-4646A

Eli Lilly and Company, Indianapolis, Indiana, V.St.A. Antibiotic A-30912 and process for its preparation

The invention relates to a new antibiotic active mixture designated A-30912 and at least seven contains individual factors A, B, C, D, E, F and G. This antibiotic effective mixture is obtained by cultivating a new strain of the organism Aspergillus rugulosus NRRL 8113. the As used herein, the term "antibiotic active mixture" refers to a mixture of together formed individual antibiotic factors. As for anyone familiar with the production of antibiotics through fermentation As is readily apparent, the ratio of the individual factors produced in an antibotic mixture depends on the fermentation conditions used.

The individual antibiotic factors according to the invention are referred to as antibiotic A-30912 factors A, B, C, D, E, F and G.

The invention relates to the new antibiotic A-30912 mixture and the antibiotic A-30912 factors A, B, C, D, E, F and G.

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The subject matter of the invention also includes methods for Generation and separation of the antibiotic A-30912 mixture, which includes factors A, B, D, D, E, F, and G, and isolating these factors. This procedure is characterized in that

a) Aspergillus rugulosus NRRL 8113 in a nutrient medium that Contains assimilable substances, carbohydrate, nitrogen and inorganic salts, under submerged aerobic conditions Fermentation conditions are grown until significant antibiotic activity is produced, and

b) optionally the antibiotic A-30912 mixture is separated from the medium and

c) if applicable, the antibiotic A-30912 factors A, B, C,

D, E, F or G isolated from the Antibiotic A-30912 mixture will.

The Antibiotic A-30912 mixture is made with polar organic Solvents extracted from the fermentation medium.

The well-known compound sterigmatocystin is also used produced by Aspergillus rugulosus NRRL 8113. Sterigmatocystin is either separated with a non-polar organic Solvent or together with the antibiotic A-30912 mixture extracted with polar organic solvents. In the latter case, the antibiotic A 30912 mixture is used by the sterigmatocystin separated by the fact that the solvent extract is concentrated, the concentrate to an excess of one Non-polar organic solvent, such as diethyl ether, given and the A-30912 antibiotic mixture as a precipitate is separated. Sterigmatocystin is obtained from the filtrate. The antibiotic A-30912 mixture is represented by column chroma- • tography further cleaned.

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-γ 2843485

The antibiotic A-30912 mixture and the individual A-30912 factors are antifungal agents.

In the accompanying drawings are the infrared absorption spectra of the following A-30912 factors represented in KBr slices:

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 corresponds to the polypeptide antibiotic Echinocandin B (F. Benz et al., HeIv. Chim. Acta Volume 57, pp. 2459-2477, 1974) close.

Antibiotic A-30912 Factor A is a white amorphous solid substance, whose elemental analysis shows the following percentage composition:

C 56.52%; H 7.29%; N 8.68%; 0 27.09%.

The following empirical formula is proposed for antibiotic A-30912 factor A: C _r - ₁ _c-} H_ _q _o_N_0 ₁ __ _iq . Within this range, the elemental analysis of A-30912 factor A corresponds particularly well to the empirical formula Cc ₂ Hg ₁ N ₇ O-Ig-H ₂ O (calc .: C 56.24; H 7.54; N 8.84; 0 27.39).

Antibiotic A-30912 factor A has a molecular weight of 1100 as determined by mass spectrometry and titration.

The infrared absorption spectrum of antibiotic A-30912 Factor A in KBr disk is shown in Figure 1 of the accompanying drawings shown. There are the following characteristic ones

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Observe absorption maxima: 2.97 (strong), 3.39 (medium strong), 3.47 (weak), 5.99 (strong), 6.10 (strong), 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-3O912 Factor A in neutral and in acidic methanol shows absorption maxima at 225 nm (epsilon 18,000), 275 nm (epsilon 3000) and 284 nm (shoulder, epsilon 2500). The ultraviolet spectrum of factor A in basic methanol shows absorption maxima at 245 nm (epsilon 16,000) and 290 nm (epsilon 3000) and also final absorption.

The magnetic C nuclear resonance spectrum of antibiotic A-30912 factor A in perdeuterated methanol shows the following Characteristics:

delta 176.1, 174.3, 173.4, 172.7, 172.4, 169.8, 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 values of the specific Rotation:

/alpha/!? ⁵ -44 ° (ο 0.5, CH ₀ OH)

Electrometric titration of antibiotic A-30912 factor A. in 66 percent aqueous dimethylformamide shows the presence of a titratable group with a pKa value of 12.3 (initial pH 6.9).

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The amino acid analysis of antibiotic A-30912 factor A after its hydrolysis shows the presence of threonine, hydroxyproline and three other as yet unidentified amino acids.

Antibiotic A-30912 factor A is in a number of organic solvents, such as methanol, ethanol, dimathy1formamide, Dimethyl sulfoxide and ethyl acetate soluble, but non-polar organic solvents such as diethyl ether and petroleum ether insoluble. Antibiotic A-30912 Factor A is also in soluble in aqueous solutions, especially those with a pH value above 7.0.

A-30912 Factor D.

Antibiotic A-30912 Factor D is a white amorphous solid substance, whose elemental analysis gives the following percentage composition: C 56.37%; H 8.17%; H 8.54 I; 0 (by difference) 26.92%.

Antibiotic A-30912 factor D has a molecular weight of about 1100, due to the 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 a KBr disk is shown in Figure 2 of the accompanying drawings shown. The following characteristic absorption maxima can be 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 spectra of Antibiotic A-30912 Factor D in neutral and acidic methanol show absorption maxima at 225 nm (epsilon 18,000) and 275 nm (epsilon 2500).

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The UV spectrum of A-30912 factor D in basic methanol shows absorption maxima at 240 nm (epsilon 11,000) and 290 nm (epsilon 3000).

Antibiotic A-30912 factor D has the following value of the specific Rotation:

/ alpha / ^ ⁵ -50 ° (c 0.34, CH ₃ OH).

The amino acid analysis of antibiotic A-30912 factor D after its hydrolysis shows the presence of threonine, hydroxyproline, Histidine and three other as yet unidentified amino acids. One of the as yet unidentified amino acids ^ of A-30912 factor D is identical to one of the as yet unidentified amino acids of antibiotic A-30912 factor A.

Antibiotic A-30912 Factor D is present in a number of different organic solvents, such as methanol, ethanol, dimethylformamide, Dimethyl sulfoxide and ethyl acetate soluble, but in non-polar organic solvents such as diethyl ether and Petroleum ether insoluble. Antibiotic A-30912 factor D is in soluble in aqueous solutions, especially those with a pH value above 7.0.

A-30912 factor B

Antibiotic A-30912 Eaktor B is a white amorphous solid substance, whose elemental analysis gives the following percentage composition: C 57.36%; H 5.92%; N 8.75%; 0 26.19%,

The infrared absorption spectrum of A-30912 factor B in a KBr disk is shown in Figure 3 of the accompanying drawings

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shown. They are the following characteristic absorption maxima Observe: 2.99, 3.41, 3.49, 6.06, 6.15, 6.54, 6.61, 6.94, 7.62, 8.07, 9.26 and 9.39 Micron.

The ultraviolet absorption spectra of A-30912 factor B in neutral and in acidic methanol, absorption maxima are at 223 nm (shoulder, epsilon 16,000) and 278 nm (epsilon 2400). The ultraviolet spectrum of antibiotic A-30912 factor B in basic methanol shows absorption maxima at 242 nm (epsilon 13900) and 292 nm (epsilon 2800).

A-30912 Factor B has the following specific rotation values:

^ ^5-47 ° (c 0.5, CH ₃ OH) 170 ^O (c 0.5, "CH ₃ OH).

The electrometric titration of A-30912 factor B in 66 percent aqueous dimethylformamide results in the presence of a titratable group with a pKa value of about 13.0 (initial pH 7.91).

The amino acid analysis of A-30912 factor B after usual acid hydrolysis shows the presence of threonine, hydroxyproline and several as yet unidentified amino acids.

A-30912 Factor B is in a number of different organic solvents, such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide and methyl acetate, but soluble in non-polar organic solvents such as diethyl ether and petroleum ether insoluble. In addition, A-30912 factor B is soluble in aqueous solutions, especially those with a pH above 7.0.

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A-3O912 factor C

Antibiotic A-30912 factor C is a white amorphous solid substance, whose elemental analysis has the following percentage composition yields: C 56.76%, H 7.88%, N 10.61%, O 25.09%.

The infrared absorption spectrum of A-30912 factor C in one KBr disk is shown in Figure 4 of the accompanying drawings. There are the following characteristic absorption maxima observable: 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 neutral and in acidic methanol show absorption maxima at 223 nm (shoulder, epsilon 7300) and 275 nm (epsilon 1350). The ultraviolet spectrum of antibiotic A-30912 factor C in basic methanol shows absorption maxima at 240 nm (epsilon 12400) and 290 nm (epsilon 5200).

A-30912 Factor C has the following specific rotation values:

/ alpha / j * ⁵ - 33 ° (c 0.5, CH-.OH)

-. - JL / j

/ alpha /? cc-119 ° (c 0.5, CH ₀ OH).

The electrometric titration of A-30912 factor C in 66 percent aqueous dimethylformamide shows the presence of a titratable group with a pKa of about 13.08 (Initial pH 7.93),

The amino acid analysis of A-30912 gives factor C according to the usual Acid hydrolysis indicates the presence of threonine, hydroxyproline and several as yet unidentified amino acids.

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A-30912 Factor C is present in a number of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide and ethyl acetate soluble but insoluble in non-polar organic solvents such as diethyl ether and petroleum ether. A-30912 Factor C is also soluble in aqueous solutions, especially those with a pH value above 7.0.

The seven individual factors of the Antibiotic A-30912 mixture can be separated and identified by thin layer chromatography (TLC). Silica gel is a preferred one Adsorbent and benzene: methanol (7: 3, v: v) is a preferred one Solvent system.

The R _f values of the antibiotic A-30912 factors A to G in TLC using silica gel (Merck, Darmstadt) and benzene: methanol (7: 3) as the solvent system and of Candida albicans for bioautography are listed in Table I below :

Tabel

Antibiotic A-30912 factor R _f value

A 0.35

B 0.45

C 0.54

D 0.59

E 0.27

F 0.18

G 0.13

The R _f values of antibiotic A-30912 factor A in various paper chromatographic systems, again using Candida albicans as the detection organism, are given in Table II.

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45 *

Table II A-30912 Pactor A.

^R f -Value O , 76 0 , 69 O , 75 O , 17th

solvent

butanol saturated with water

with water plus 2% p-toluenesulfonic acid saturated butanol

Methanol: 0.1 η HCl (3: 1)

Butanol: ethanol: water (13.5: 15: 150)

0.78 methanol: 0.05M sodium citrate of

pH 5.7 (7: 3); Paper buffered with 0.05M sodium citrate of pH 5.7

The one suitable for the preparation of the Antibiotic A-30912 mixture Organism was isolated from a soil sample from the ruins of Pompeii, Italy. The A-30912 generating Organism is classified as a strain of Aspergillus rugulosus Thorn and Raper, in the Aspergillus 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 by A. Maerz and M. R. Paul in "Dictionary of Color," McGraw-Hill Book Company, New York, N.Y., 1950.

Unless otherwise specified, the cultures are grown at 25 ⁰ C.

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Culture characteristics of A. rugulosus NRRL 8113 Czapek solution agar

The culture grows slowly and reaches a diameter of 1.5 to 2.0 cm in 15 days at 25 C. The surface of the Colony is convex and velvety, becomes wrinkled with age near the center, and then bulges. The outer rim of the mycelium consists of a 2 mm thick band of deeply submerged colorless hyphae and is curved. On the aerial hyphae on A pinkish-brown exudate forms at the edge. In the period of 7 to 14 days, the agar surrounding the colony becomes a Pale purple liquid pigment formed. After 15 days the pigment diffuses through the entire colony.

After 5 days, the color of the surface of the colony ranges from white to buff and the back of the colony is brownish-orange in the middle part and brownish to brownish purple in the outer areas. In 10 days it will the colony is moderately yellowish-pink (ISCC-NBS 2 9 and Maerz and Paul 11-A-7). After 14 days the colony is light grayish red (ISCC-NBS 18 and Maerz and Paul 4-G-7). The edge area is covered with small warts as a result of the formation of conidia and is bright yellow (ISCC-N3S 84 and Maerz and Paul 10-L-5). Scattered, blunt-yellow tufts of envelope cells appear randomly distributed on the surface and along the edge of the colony. They get strong with age yellow spots and marginal areas yellowish-green. After three weeks there will be an orange in the new air components of the edge observed purple tone. The back of the colonies is initially slightly concave and flattens out with the tire the agar surface and becomes a little wrinkled. After 10 days the reverse is pale brown (ISCC-NBS 57 and Maerz and Paul 5-A-10) and grayish-red after 15 days (ISCC-NBS 19 and Maerz and Paul 6-J-3).

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The conidiogenic state is sparse. Conidiophores are scattered over the surface and sometimes appear as spots or in a band near the edge. Cpnidial heads are initially loosely radial and spherical. With aging they can develop as short columnar shapes that are more compact. Spherical heads have a diameter from 70 to 80, u and are on average 50, u. Columnar Heads can be up to 140 »long and 70» thick.

The conidia are spherical to less than spherical, slightly wrinkled and greenish-gold in bulk. The diameter is 2.8 to 3.9, u, on average 3.2 .u.

Sterigmata are arranged in two rows and are colorless. Primary sterigmata have a length of 4.7 to 11.0 / U, im Mean 7.9, u. At their thickest point they are 2,4, u and run towards 1.6 ^ u. Secondary sterigmata can be used individually or occur in pairs, arise from the primary and are bottle-shaped. They measure at their thickest point 3.0 yU and taper apical to 0.4 .u where they are tubular v / earth. The tubular tip is 1.2 u long. The total length is from 5.5 to 12.6, u, on average 9.2, u.

Vesicles are spherical to sub-spherical or hemispherical and can be flattened at the top, turning brownish with age. The diameter ranges from 7.4 to 11.2, u and averages 9.4 .u.

Conidiophores are smooth, relatively thick-walled and initially glassy and transparent and then turn light cinnamon brown. The vesicles are slightly thicker and they can taper slightly near the foot cell. The average Thickness is 5.9, u. Conidiophores have a length of 47.7 to 166.6 .u, on average 106, u. You go from submerged hyphae or laterally from aerial hyphae threads.

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The ascogenic state occurs up to 20 days. The small yellowish tufts of envelope cells that appear on the surface can also occur anywhere within the mycelium. They consist of envelope cells that contain one or more Wrap the cloistothecia. Envelope cells are spherical to sub-spherical or oval to elongated, are thick-walled and transparent. Spherical envelope cells have a diameter of 18 to 24, u, on average 21.8 .u.

Kleistothecia are spherical to sub-spherical, thick-walled, relatively tough and fibrous. At first it will be relatively colorless they are reddish-purple and dark with age. The diameter measures from 165 to 470 u, on average 275 u.

Malt extract agar

Colonies grown at 25 C expand rapidly, reaching 4 to 5 cm in 10 to 12 days. At first greyish white the colonies become olive green in 4 days (ISCC-NBS 90 and Maerz and Paul 23-E-6). The indented weakly lobed one outer edge consists of tightly packed short white aerial hyphae. Small yellowish clusters of sheath cells appear as points at the edge and are scattered at random over the felt-like agar surface. After 20 days these tend to Clusters of sheath cells to encrust a large part of the surface. The underside of the colony is greyish-yellow (ISCC-NBS 90 and Maerz and Paul 11-B-1).

The ascogenic state appears in 15 days. The small yellowish tufts of sheath cells that appear on the surface occur also occur anywhere within the mycelium. They consist of envelope cells that contain one or more Wrap the cloistothecia. Envelope cells can crust over large areas over the heads of the conidia. They are spherical to sub-spherical or oval to elongated, are thick-walled and transparent. Spherical envelope cells have a diameter from 18 to 24, u, on average 21.8, u.

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Kleistothecia are spherical to sub-spherical and dark reddish brown. They have a diameter of 389 to 700, u, on average 506 .u.

Asci are fragile, transparent, and sub-spherical to oval; sub-spherical asci have a diameter of 8.7 to 11.9, u, on average 10.3, u. Oval asci have dimensions from 10.3 to 14.2 .u χ 8.7 to 10.3, u, on average 12.2 9.1, u.

Ascospores are red-orange, slightly wrinkled, with two parallel, finely folded equatorial elevations, the 0.8 u broad and unbroken. The ascospores appear lens-shaped along the long axis. If the elevation is peripheral, the ascospores are spherical. In the spherical shape they have a diameter of 4.9 to 6.3 / U, on average 5.4 .u.

Two characteristics of the strain producing antibiotic A-30912 of Aspergillus rugulosus are different from the characteristics of A. rugulosus described by Raper and Fennel (loc. cit.). The strain producing A-30912 has larger conidial heads and ascospores.

An Aspergillus rugulosus culture which is suitable for producing the antibiotic A-30912 mixture is at Northern Regional Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Peoria, Illinois 61604 and has been included in its culture collection, where it is available to the general public under the number NRRL 8113 is accessible.

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For the cultivation of Aspergillus rugulosus NRRL 8113 you can several different culture media can be used. With a view to economical production, optimal yields however, and ease of product isolation, certain nutrient media are preferred. For example, glucose is preferred Substance that provides carbohydrates in large-scale fermentations, but molasses, starch, lactose, Sucrose, maltose, glycerin and the like can be used. Preferred nitrogen sources are enzyme hydrolyzed Casein and soluble meat peptone, but grain distillate residues, monosodium glutamate and similar substances can also be used be used. Inorganic salts can be incorporated into the nutrient medium; this includes the usual soluble ones Salts that provide sodium, magnesium, calcium, ammonium, chlorine, carbonate, sulfate, nitrate and the like ions capital.

The nutrient medium should also contain essential trace elements that are necessary for the growth and development of the organism are. Such trace elements are usually present as impurities in the other components of the medium in quantities that which meet the growth requirements of the organism.

For large-scale fermentations, it may be necessary to use small amounts (for example 0.2 ml / 1) of a defoamer, such as Polypropylene glycol, to be added if foaming is difficult.

A submerged aerobic fermentation in Tanks preferred. Small amounts of the Antibiotic A-30912 mixture can be obtained by shake flask culture. Because of the delay in the start of antibiotic production, what usually occurs when inoculating large tanks with the spore shape of the organism is the use a vegetative inoculum preferred. The vegetative inoculum

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is prepared by inoculating a small volume of suturing medium with the spore form or mycelium fragments of the organism, whereby a fresh, well-growing culture of the organism is obtained. The vegetative inoculum is then transferred to a large tank. The medium used to grow the vegetative inoculum can be the same as that used for larger fermentations, but other media can also be used. The antibiotic A-30912-producing organism can be grown at temperatures between about 20 and 43 ⁰ C, and it grows well at temperatures of about 25 to 30 ⁰ C. An optimal production of the antibiotic A-30912 mixture is carried out at a temperature of apparent about 25 ⁰ C.

As is common with submerged aerobic cultivation methods, it becomes sterile Air blown through the nutrient medium. In the interests of effective antibiotic production, this is due to tank production Applied air volume preferably over 0.4 volumes of air per volume of the nutrient medium and minute (V / V / M).

The formation of the antibiotic A-30912 mixture can occur during the fermentation can be followed in such a way that samples of alcoholic extracts of the fermentation mash against a Organism whose sensitivity to the A-30912 antibiotics is known to be tested for antibiotic activity. A sample organism that is suitable for testing suitable if the antibiotic A-30912 mixture is present, is Candida albicans. The bio-sample is expediently carried out using paper disk samples on seeded agar plates.

In general, antibiotic activity can be detected on the second day of fermentation. The maximum of the Formation of antibiotic activity usually occurs between about the third and sixth day.

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The Antibiotic A-30912 factors are antifungal agents. The antifungal effects of antibiotic A-30912 factors is determined by in vitro tests with antibiotic A-30912 factor A. and D illustrates. These tests are summarized in Table III. The antifungal effect was enhanced by the conventional one Disk diffusion method determined (6 mm pads are immersed in solutions containing the compound to be tested contain. The pads are placed on agar plates inseminated with the test organism). The results are as the minimum inhibiting concentration (MIC) per disc in which the tested compound inhibits the test organism, specified.

T a b e 1 1 e

III

Test organism MIC (mcg / disc)

Candida albicans Trichophyton mentagrophytes

Antibiotic

A-30912 factor A

0.625 0.078

Antibiotic

A-30912 Factor D.

0.5 0.07

A-30912 Factor A is in in vitro disc diffusion tests very effective against dermatophytes. The results of these tests are shown in Table IV.

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-VS-

Table IV

A-30912 Factor A against Dermatophytes

Dermatophytes

Trichophyton mentagrophytes Trichophyton gallinae Trichophyton meginini Trichophyton quinckeanum Trichophyton rubrum Trichophyton schoenceinii Trichophyton terrestre Trichophyton tonsurans Microsporiura gypseum, Microsporium audouinii Microsporium canis Microsporium cookei Nannizzia incurvata Phalaphere jean salemi Epidermatophyton floccosum Geotrichum candidum Keratinomyces ajellio

number of
Isolates MIC (mcg / disc) 13th 1.25-0.039 1 > 1.25 1 0.0195 1 > 1.25 1 ^ 0.0098 1 0.0195 1 0.0195 9 > 1.25-0.156 5 0.156-0.038 4th 1.25-0.156 6th ⁿ 1.25-0.0098 2 1.25-0.0195 1 0.312 1 > 1.25 1 1.25 4th ^ 1.25-0.156 1 0.156

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The antifungal effect of Antibiotic A-30912 factors is also demonstrated by in vivo tests. These in vivo tests are carried out in the following manner: Three doses of the test compound are administered to Candida albicans infected mice at 0, 4 and 24 hours after infection. The protection achieved with the test compound is determined as the EDj-Q value (the effective dose in mg / kg which leads to protection of 50% of the mice; see W. Wick et al., J. Bacteriol. Vol. 81 , pp 233-235, (1961). The ED ₅ values for antibiotic A-30912 factor A against Candida albicans in mice is 30 mg / kg (intraperitoneal) and 50 mg / kg (subcutaneous). The ED ₅ value for antibiotic A-30912 factor D against Candida albicans in mice is 33 mg / kg (subcutaneous).

There are no signs of acute toxicity when the antibiotic A-30912 factor A intraperitoneally (ip) or subcutaneously to mice at a dose of 100 mg / kg twice daily for three days (total 600 mg / kg). Even then, no signs of acute toxicity are observed when antibiotic A-30912 factor A administered ip to mice three times a day at a dose of 200 mg / kg (total dose 600 mg / kg).

-1

With sc administration of antibiotic A-30912 factor D on Mice three times a day at a dose of 14 mg / kg each (total dose 42 mg / kg) no signs of acute toxicity can be observed.

When used as antifungal agents, Antibiotic A-30912 factors become parenteral and common administered in conjunction with a pharmaceutically acceptable carrier or diluent. The dosage of the antibiotic A-30912 factor depends on several conditions for Example of the type and severity of each infection.

The invention is further illustrated by the following examples.

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Example 1 A. Shake flask fermentation

A culture of Aspergillus rugulosus NRRL 8113 is on Prepare and receive agar slants of the following composition:

Ingredient amount (%)

Dextrin 1.0000

Enzymatic hydrolyzate of casein 0.2000

Yeast extract 0.1000

Meat extract 0.1000

KCl. 0.0200

MgSO ₄ .7H ₂ O 0.0200

FeSO ₄ .7H ₂ O 0.0004

Water 98.5596

N-Z-Amines A, Sheffield Chemical Co., Norwich, N.Y.

The slope is inoculated with Aspergillus rugulosus NRRL 8113 and then incubated at 25 ° C. for about 7 days. The mature slant is covered with bovine serum and allowed to dissolve the spores are scraped off with a sterile snare. Half of the suspension obtained is used for inoculating 50 ml of a vegetative medium of the following composition is used:

Component ■ amount (%)

Sucrose 2.5

Molasses 3.6

Corn steep water 0.6

Enzymatic hydrolyzate of casein 1.0

K ₂ HPO ₄ 0.2

Water 92.1

N-Z Case, Sheffield Chemical Co., Norwich, N.Y.

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The inoculated vegetative medium is incubated in a 25O ml Erlenmeyer Wide-necked flask at 25 ⁰ C for 24 hours on a shaker rotating at 250 revolutions / minute by a sheet of 5 cm diameter.

This incubated vegetative medium can be used directly to inoculate the vegetative medium of the second stage will. Instead, it can be saved for later use, which is preferred by keeping the culture holds in the vapor phase of liquid nitrogen. For such storage, the culture is as follows in many small vials are placed: 2 ml of incubated vegetative medium and 2 ml of a glycerol-lactose solution are placed in each vial the following composition:

Component quantity

Glycerine 20%

Lactose 10%

deionized water 70%

The suspensions produced in this way are stored in the vapor phase of liquid nitrogen.

1 ml of a suspension prepared and stored in this way is used to inoculate 50 ml of vegetative medium of the first Stage used with the composition given above. The inoculated medium is then placed in a 250 ml wide-necked Erlenmeyer flask Incubated for 22 hours at 25 ° C on a shaker that rotates through an arc at 250 revolutions / minute rotates with a diameter of 5 cm.

B. Tank fermentation

To achieve a large volume of inoculum, 10 ml of the incubated vegetative medium described above first study of inoculating 400 ml of vegetative medium

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the second stage with the same composition as the vegetative medium is used. The medium of the second stage is incubated in a 2 liter Erlenmeyer wide-necked flask for 25 hours at 25 ^° C. on a shaker which rotates at 250 revolutions / minute through an arc with a diameter of 5 cm.

800 ml of the second stage incubated vegetative medium prepared as described above are added to the Inoculate 100 liters of sterile production medium using the following composition:

Inventory quantity

Glucose 25 g / liter

Starch 10 g / liter

Peptone 10 g / liter

Blackstrap molasses 5 g / liter

Enzymatic hydrolyzate of casein 4 g / liter

MgSO ₄ .7H ₉ O 0.5 g / liter

+++
Czapek mineral mixture 2.0 ml / liter

CaCO ₃ 2.0 g / liter

deionized water, to 1 liter

W.P. No. 159, Inolex Biomedical Corp., Glenwood, 111. N-Z Amine A, Sheffield Chemical Co., Norwich, N.Y. Czapek mineral mixture has the following composition:

Component quantity

FeSO ₄ .7H ₉ O (dissolved in

2 ml conc. HCl) 2 g

KCl 100 g

MgSO ₄ .7H ₂ O 100 g

deionized water, to 1 liter

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After sterilization by treatment for 30 minutes in an autoclave at 121 ° C. and a pressure of about 1.1 to 1.25 atmospheres, the pH of the medium is 6.8. The inoculated production medium is allowed to ferment in a 165 liter fermentation tank at a temperature of 25 ^° C. for 4 days. The fermentation medium is aerated with sterile air in a ratio of 0.5 V / V / M and stirred with a conventional stirrer at 300 revolutions / minute.

Example 2 Separation of the Antibiotic A-30912 mixture

200 ml of the total fermentation mash obtained as described in Example 1 are stirred thoroughly with 200 ml of methanol for one hour and then using a filter aid (Hyflo Super-Cel, Diatomaceous Earth, Johns-Manville Products Corp.) filtered. The pH of the filtrate is adjusted to 4.0 by adding 5η HCl. The acidified filtrate will extracted twice with equal volumes of chloroform. The chloroform extracts are combined and reduced to volume in vacuo concentrated by about 4 liters. The concentrate is added to about 60 liters of diethyl ether, causing the A-30912 mixture to precipitate. The precipitate is collected by filtration and dried, and 38 g of the antibiotic A-30912 mixture are obtained as a gray powder. Concentrating the filtrate in vacuo gives an oil which is dissolved in 500 ml of methanol will. The methanol solution is added to 7.5 liters of diethyl ether, creating another antibiotic A-30912 mixture fails. This precipitate is also filtered off and dried, leaving another 3.5 g of the antibiotic A-30912 mixture can be obtained.

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Example 3 Isolation of Antibiotic A-30912 Factor A

20 g of the antibiotics A-30912 mixture obtained as described in Example 2 are poured onto a silica gel column (4 χ 107 cm, Woelm) in acetonitrile: water (95: 5). the Column is filled with acetonitrile: water (95: 5) at one flow rate from 1 to 2 ml / minute, collecting fractions of approximately 20 ml each. the Fractions are determined by thin layer chromatography using silica gel and the acetonitrile: water (95: 5) solvent system and bioautography checked with Candica albicans.

Fractions 74 to 125 are combined and concentrated. When the concentrated solution is left to stand, crystals precipitate and 124 mg of sterigmatocystine are obtained by separating them off. Fractions 212-273 are combined and concentrated in vacuo until an oil is obtained. This oil is dissolved in a small volume of methanol and then added to 15 volumes of diethyl ether. The precipitate formed is separated off and dried and yields 23 mg of antibiotic A-30912 factor D. Fractions 274 to 437 contain antibiotic A-30912 factors A, B _f C and D. Fractions 482 to 900 contain antibiotic A-30912 Factors A, E, F and G. Fractions 438 to 481 are combined and concentrated in vacuo to an oil. This oil is dissolved in a small volume of methanol and then added to 15 volumes of diethyl ether. The precipitate formed is separated off and dried, yielding 2.17 g of Antibiocitum A-30912 factor A.

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3o

Example 4 Isolation of Antibiotic A-30912 Factor D

A partially purified Antibiotic A-30912 mixture that the antibiotic A-30912 contains factors B, C and D will as described in Example 3 in connection with fractions 274 to 437. 750 mg of this material are attached to a silica gel column (2.2 χ 51 cm, Woelm) chromatographed, collecting fractions with a volume of about 15 ml. Elution takes place with following solvents:

Fractions solvent

1-25 acetonitrile

26-62 acetonitrl + 1% water

63-700 acetonitrile + 1.5% water

The fractions emerging from the column are analyzed by thin layer chromatography using silica gel and acetonitrile: water (95: 5) and benzene: methanol (7: 3) checked as a solvent system and bioautography with Candida albicans. The fractions 535 to 685 included Antibiocitum A -30912-Factor D, and are combined and concentrated in vacuo to an oil. This oil comes in a Dissolved a small amount of methanol and then added to 15 volumes of diethyl ether. The precipitate formed is filtered off and dried and yields 69 mg antibiotic A-30912 factor D.

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Example 5 Isolation of A-3Q912 Factors B and C

A partially purified A-30912 antibiotic mixture containing the A-30912 factors A ₇ B, C and D is obtained as described in Example 3 for fractions 212 to 437. 18 g of this material are dissolved in the smallest possible volume of acetonitrile: water (4: 1) and chromatographed on an aluminum oxide column (3.8 χ 56 cm, Woelm), fractions with a volume of about 20 ml being collected. The column is eluted with the following solvents:

Fractions solvent

1-300 acetonitrile: water (4: 1)

301: 509 Acetonitrol: Water (7: 3)

The fractions obtained are monitored by the thin layer chromatography described in Example 4. On the Based on the results obtained, the fractions are combined and concentrated to an oil. The oily ones Residues are dissolved in small volumes of methanol, whereupon 10 to 15 volumes of diethyl ether are precipitated. The type of combination of the fractions, the weight received in each case and the factor content of the combined fractions are summarized below:

Factions Weight (g) factor 6-28 O, 23 ⁺ - 6-28 5.80 A-30912 C, D 34-114 2.90 A-3O912 B 115-164 1.20 A-30912 A, B 165-509 1.90 A-30912 A

⁺ Insoluble material "before ether precipitation.

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To obtain purified A-30912 factor C, 2 g of the Solid matter of fractions 6 to 28 dissolved in methanol, adsorbed on a sufficient amount of silica gel (type 62), and, after drying, placed on a silica gel column (1.9 80 cm, type 62, in acetonitrite). The column is made with acetonitrile eluted at a flow rate of 20 ml / minute, with fractions having a volume of about 10 ml be caught. In fraction 117 the solvent is changed and acetonitrile: water (99: 1) is used. the Fractions emerging from the column are again monitored by thin layer chromatography. On the basis of The results of the thin-layer chromatography are combined and the fractions are concentrated to an oily residue. Of the oily residue is dissolved in a small volume of methanol, whereupon 10 to 15 volumes of diethyl ether are precipitated will. The weight amounts and the factor content of the fractions of interest are summarized below:

Factions Weight, g factor 341-479 0.250 D. 480-540 0.015 D. 541-899 0.391 C, D 900-1675 0.340 C.

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Claims (8)

Claims V. Antibiotic A-30912 mixture containing the factors A, B, C, D, E, F and G. 2. Antibiotic A-30912 factor B ₇ a white amorphous solid substance which is soluble in methanol, ethanol, dimethylformamide, dimethyl sulfoxide and ethyl acetate and in aqueous solutions with a pH value above 7.0, but insoluble in diethyl ether and petroleum ether and the following Parameter has: a) an elemental composition of C 57.36%; H 5.92%; N 8.75%; 0 26.19%; b) an infrared absorption spectrum in a KBr disk with the following characteristic absorption maxima: 2.99, 3.41, 3.49; 6.06, 6.15, 6.54, 6.61, 6.94, 7.62, 8.07, 9.26 and 9.39 microns; c) Ultraviolet absorption spectra in neutral and in acidic methanol with absorption maxima at 223 nm (shoulder, epsilon 16,000) and 278 nm (epsilon 2400) and in basic methanol with absorption maxima at 242 nm (epsilon 13900) and 292 nm (epsilon 2800); d) the following values of the specific rotation: /alpha/;? ⁵ - 47 ° (c 0.5, CH-OH) - - L) J / _alpha / 3g ₅ -17O ° (c 0.5, CH ₃ OH); e) a titratable group having a pKa of about 13.0 in aqueous dimethylformamide; 709815 / 1US ORIGINAL fNSPECTED FRG y f) an R _f value of 0.45 in thin layer chromatography using benzene: methanol (7: 3) and Candida albicans for bioautographic determination, and g) an amino acid analysis, after conventional acid hydrolysis, the presence of threonine, hydroxyproline and several indicates as yet unidentified amino acids. 3. Antibiotic A-30912 factor C, a white amorphous solid substance, those in methanol, ethanol, dimethylformamide, dimethyl sulfoxide and ethyl acetate and soluble in aqueous solutions with a pH of over 7.0 but in diethyl ether and petroleum ether is insoluble and has the following parameters: a) an elemental composition of C 56.76%; H 7.88%; N 10.61%, 0.25.09%; b) an infrared absorption spectrum ir, a KBr disk with the following characteristic absorption maxima: 2.98, 3.39, 3.43, 3.51, 6.01, 6.12, 6.47, 6.9O, 7.04, 7.22, 7.38, 8.00, 8.30 and 9.13 microns; c) Ultraviolet absorption spectra in neutral and in acidic methanol with absorption maxima at 223 im (shoulder, epsilon 7300) and 275 nm (epsilon 1350) and in basic methanol with absorption maxima at 240 nm (epsilon 12400) and 290 nm (epsion 5200); d) the following values of the specific rotation: / alpha / p ⁵ - 33 ° (c 0.5, CH ₃ OH) / alpha / ^ - 119 ⁰ (c 0.5, CH ₃ OH); 709815/1 US e) a titratable group having a pKa of about 13.08 in 66 percent aqueous dimethylformamide; f) an R_ value of 0.54 in the case of silica gel thin layer chromatography using benzene: methanol (7: 3) and Candida albicans for bioautographic determination and g) an amino acid analysis, after usual acid hydrolysis, that the presence of threonine, hydroxyproline and several more indicates unidentified amino acids. 4. Antibiotic A-30912 factor D, a white amorphous solid substance, those in methanol, ethanol, dimethylformamide, dimethyl sulfoxide and ethyl acetate and in aqueous solutions with a pH value above 7.0 is soluble but insoluble in diethyl ether and petroleum ether and has the following parameters: a) a molecular weight of about 1100; b) an elemental composition of C 56.37%; H 8.17%; N 8.54%; 0 (by difference) 26.92%; c) the following value of the specific rotation: /alpha/;? ⁵ -50 ° (c 0.34, CH-OH); d) an infrared absorption spectrum in a KBr disk with the following characteristic absorption maxima: 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; 70981 5 / 1U5 e) Ultraviolet absorption spectra in neutral and acidic Methanol with absorption maxima at 225 nm (epsilon 18,000) and 275 nm (epsilon 2500) and in basic methanol with absorption maxima at 240 nm (epsilon 11,000) and 290 nm (epsilon 3000); f) an amino acid analysis after their hydrolysis, which indicates the presence of threonine, hydroxyproline, histidine and three other as yet unidentified amino acids, and g) an R _f value of 0.59 in silica gel thin-layer chromatography using benzene, methanol (7: 3) and Candida albicans for bioautographic determination. 5. Antibiotic A-30912 factor E, characterized by an R _f value of 0.27 in thin-layer chromatography on silica gel using benzene: methanol (7: 3) as the solvent system. 6. Antibiotic A-30912 factor F, characterized by an R _f value of 0.18 in thin-layer chromatography on silica gel using benzene: methanol (7: 3) as the solvent system. 7. Antibiotic A-30912 factor G, characterized by an R_ value of 0.13 in thin layer chromatography on silica gel using benzene: methanol (7: 3) as the solvent system. 709815/1 US FRG. - yi - 8. Process for the preparation of the Antibiotic A-30912 mixture according to claim 1, characterized in that one a) Aspergillus rugulosus NRRL 8113 in a nutrient medium, which contains assimilable substances that provide carbohydrate, nitrogen and inorganic salts, under aerobic submerged form Fermentation conditions grow until significant antiblotic activity is produced, and b) optionally the antibiotic A-30912 mixture of the fermentation mash separates and (c) if applicable, the antibiotic A-30912 factors A, B, C, D, E, F or G from Antibiotic A-30912 mixture isolated. 709815/1145