DK143608B - PROCEDURE FOR THE PREPARATION OF ANTIBIOTIC A-30912 OR ITS COMPONENTS, USED A, B, C, D, E, F AND G - Google Patents

Description

(19) DENMARK

fj | Π2) PUBLICATION NOW 143608 B

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Application No. 4402/76 (51) lntC | 3 β ^ ρ 21/00 (22) Filing Day 30. s ep. 1976 C 07 0 11/00 (24) Race day 30 · Sep · 1976 (41) Aim. available Apr 3 1977 (44) Presented 14. S ep. 1981 (86) International application # - (86) International filing day - (85) Continuation day - (62) Master application no -

(30) Priority 2 Oct. 1975, 619107, US

(71) Applicant ELI LILLY AND COMPANY, Indianapolis, US.

(72) Inventor Marvin Martin Hoehn, US: Karl Heinz Michel, US.

(74) Associate Engineer Hofman-Bang & Boutard.

(54) Process for the preparation of antibiotic A-3091 2 or its components, designated A, B, C, I), E, F and G.

The present invention relates to a process for the preparation of a novel antibiotic composition called antibiotic A-30912 containing at least seven individual components A, B, C, D, E, F and G or these individual components and the process of the invention is characterized by the invention. the know-® drawing part of the claim stated.

The term antibiotic mixture as used in this specification refers to a mixture of simultaneously manufactured individual antibiotic components. As is known to those skilled in the art, the ratio of the individual components formed in an antibiotic mixture will vary, depending on the fermentation conditions used.

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The subject antibiotic mixture is extracted from the fermentation medium by polar organic solvents.

The known compound Sterigmatocystin is also produced by Aspergillus rugulosus NRRL 8113. Sterigmatocystin is either extracted separately with a non-polar organic solvent or together with the subject antibiotic mixture with polar organic solvents. put the concentrate to excess non-polar organic solvent such as diethyl ether and separate the subject antibiotic mixture as a precipitate. Sterigmatocystin is recovered from the filtrate.

The antibiotic mixture is further purified by column chromatography.

The subject antibiotic composition A-30912 and the individual components are antifungal agents.

The infrared absorption spectra of the following components of KBr tablets are shown in the accompanying drawings:

Figure 1 - Antibiotic A-30912, component A Figure 2 - antibiotic A-30912, component D, line 3 - antibiotic A-30912, component B, line 4 - antibiotic, A-30912, component C

Antibiotic A-30912 ^ Common A

This component is similar to the polypeptide antibiotic Echino-candin B recently reported by R. Benz et al., Helv.

Chim. Acta 57, 2459-2477 (1974).

Antibiotic A-30912, component A is a white amorphous solid.

An elemental analysis of component A shows the following percentage composition:

Carbon 56.52%, hydrogen 6.29%, nitrogen 8.68% and oxygen 27.09% · 3 143608

The approximate empirical formula proposed for Component A is the G-round analysis of Component A, in particular, responds well to the empirical formula C52H81N7 ° 18'H2 ° (FCS: C 56.24 - H 7.54 - N 8, 84 - 0 27.59).

The subject component A has a molecular weight of 1100, as determined by mass spectrometry and titration.

The infrared absorption spectrum of component A of a KBr tablet is shown in Figure 1 of the accompanying drawings. The following characteristic absorption maxima are observed: 2.97 (strong), 5.59 (medium), 5.47 (weak), 5.99 (strong), 6.10 (strong), 6.49 (medium), 6.56 (medium), 6.90 (medium), 8.00 (weak), 9.15 (weak) and 11.77 (weak) pm.

The ultraviolet absorption spectrum of component A in both neutral and acidic methanol shows absorption maxima at 225 nm (ε 5,000) and 284 nm (shoulder ε 2,500). The ultraviolet spectrum of component A in basic methanol shows absorption maxima at 245 nm (e 16,000) and 290 nm (ε 3,000) and also end absorption.

The 1 C NMR spectrum of component A in equine uteromethanol shows the following characteristics: δ 1760.1 δ 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.

Component A has the following specific rotations: [a] 25 = -44 ° (c 0.5, CH 2 OH) [a] | 5 = -156 ° (c 0.5, CH 2 OH).

Electrometric titration of component A in 66 ft of aqueous dimethyl-formamide indicates the presence of a titratable group having a pKa value of 12.8 (initial pH 6.9).

Amino acid analysis of component A indicates presence after hydrolysis of threonine, hydroxyproline and three other unidentified amino acids so far.

An antibiotic component A is soluble in a wide 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. Component A is also soluble in aqueous solutions, especially those having a pH greater than 7.0.

Antibiotic A-50912, component E

A component E is a white amorphous solid. Elemental component analysis of component E gave the following percentage composition: carbon 56.37 ft, hydrogen 8.17 ft, nitrogen 8.54 ft, oxygen (by difference) 26.92 ft.

Component E has an approximate molecular weight of 1100, based on amino acid analysis and its close relationship to component A in structural terms.

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The infrared absorption spectrum of component D recorded in a KBr tablet is shown in Figure 2. The following characteristic absorption 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) pn.

The ultraviolet (UV) spectrum of component D in neutral and acidic methanol shows absorption maxima at 225 nm (ε 18,000) and 275 nm (£ 2,500). The UV spectrum of component D in basic methanol shows absorption maxima at 240 nm (ε 11,000) and 290 nm (S 3,000).

Component D has the following specific rotation: [oc] δ 5 = -50 ° (c 0.34, CH 3 OH).

Amino acid analysis of component D after hydrolysis indicates the presence of threonine, hydroxyproline, histidine and three other unidentified amino acids so far. In of the unidentified amino acids is identical to one of the unidentified amino acids in component A.

The present component D is soluble in a wide variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethylsulfoxide and ethyl acetate, but is insoluble in non-polar organic solvents such as diethyl ether and petroleum ether. The present component D is soluble in aqueous solutions, especially when the pH is greater than 7.0.

itotibiotic A 30 ^ 12, component B__

The component B in question is a white amorphous solid. Elemental analysis of component B gave the following approximate percentage composition: carbon 57.36 hydrogen 5.92 nitrogen 8.75 / 6, oxygen 26.19

The infrared absorption spectrum of component B recorded in a KBr tablet is shown in Figure 3. The following characteristic absorption maxima were observed: 6 143608 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 V ™ ·

The ultraviolet absorption spectrum of component B in both neutral and acidic methanol shows absorption maxima at 223 nm (shoulder, ε 16,000) and 278 nm (ε 2,400). The ultraviolet spectrum of component B in basic methanol shows absorption maxima at 242 nm (£ 13,900) and 292 nm (G 2,800).

Component B has the following approximate specific rotations: δ> δ 5 = -47 ° (c 0.5, CH 3 OH) [a] p 5 = -170 ° (c 0.5, OH 2 OH).

Electrometric titration of component B in 66 $ aqueous dimethyl-formamide indicates the presence of a titratable group with a pK value of 13.0 (initial pH 7.91).

Or

An amino acid analysis of component B following an acidic hydrolysis indicates the presence of threonine, hydroxyproline and several hitherto unidentified amino acids.

Component B is soluble in a wide variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide and ethyl acetate. The component is insoluble in non-polar organic solvents such as diethyl ether and petroleum ether.

Component B is also soluble in aqueous solutions, especially those having a pH greater than 7.0.

Antibiotic A-30912, component C

The component C in question is a white amorphous solid. Elemental analysis of component C gave the following approximate percentage composition: carbon $ 56.76, hydrogen $ 7.88, nitrogen $ 10.61, oxygen $ 25.09.

The infrared absorption spectrum of component C of a KBr tablet is shown in Figure 4. 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 pm.

The ultraviolet absorption spectrum of component C in both neutral and acidic methanol shows absorption maxima at 223 nm (shoulder, g 7.300) and 275 nm (g 1.350). The ultraviolet spectrum of component C in basic methanol shows absorption maxima at 240 nm (g 12,400) and 290 nm (e 5,200).

Component C has the following approximate specific rotations: [α] δ 5 = -33 ° (c 0.5, CH 3 OH) 0] ¾ = -119 ° (c 0.5, CH 3 OH).

Electrometric titration of component C in 66% aqueous dimethylformamide indicates the presence of a titratable group with a pK - cl value of about 11.08 (initial pH 7.93).

Amino acid analysis of component C following an acidic hydrolysis indicates the presence of threonine, hydroxyproline and several hitherto unidentified amino acids.

The present component C is soluble in a wide 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. Component C is also soluble in aqueous solutions, especially those having a pH greater than 7.0.

The seven individual components of antibiotic mixture A-30912 can be separated and identified using thin layer chromatography (TIC). Silica gel is a preferred adsorbent and benzene: methanol (7: 3, V: V) is a preferred solvent system.

The R ^ values of antibiotic A-30912, component A-G, using silica gel (Merck, Darmstadt), benzene: methanol (7: 3) as solvent system and Candida albicans bioautography are listed in Table I.

TABLE I

Antibiotic A-30912 component R ^ value A 0.35 B 0.45 C 0.54 B 0.59 E 0.27 E 0.18 G-0.13 R13 values for the component A in question in various papers chromatography systems using Candida albicans as a detection organism are listed in Table II.

TABLE II

A-30912 component A

R ^ value Solvent system 0.76 Butanol saturated with water.

0.69 Butanol saturated with water + 2 f> 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); 0.05M sodium citrate paper buffer at pH 5.7.

The organism useful in the preparation of the antibiotic composition in question was isolated from a soil sample from the ruins of Pompeii, Italy. Bone antibiotic A-30912-producing organism is classified as a strain of Aspergillus rugulosus belonging to the Aspergillus nidulans group. This rating 9 143808 is based on descriptions of K.B. Raper and D.I. Fenne! in "The.

Genus Aspergillus, "The Williams and Wilkins Company, Baltimore,

Mi., 1965.

The color names are given in accordance with 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., 1950.

The cultures were grown at 25 ° C unless otherwise indicated.

Cultural characteristics of Aspergillus rugulosus NRRl · 8115 Czanek's o-solution agar

The culture grows slowly, reaching a diameter of 1.5 - 2 cm in 15 days at 25 ° C. The surface of the colony is convex and velvety and becomes wrinkled with age near the center and then budded. The mycelial periphery is a 2 mm wide band of deeply submerged colorless hyphae and is also wavy. A pinkish-brown secretion forms on the marginal air peaks. In 7 to 14 days, a pale violet soluble pigment is produced in the agar that surrounds the colony. The pigment diffuses throughout the colony over 15 days.

After 5 days, the colony's surface is white to brownish yellow, and the colony's back is brownish-orange in the middle and brownish to brownish-violet in the outer regions. At 10 days, the colony has become moderately yellow to pink (ISCC-NBS 29 and Maerz and Paul 11-A-7). After 14 days, the colony has turned greyish-red (ISCC-NBS 18 and Maerz and Paul 4-G-7). The marginal areas become warty and strongly yellow (ISCC-NBS 84 and Maerz and Paul 10-L-5) due to conidia formation. Scattered dark yellow clusters of htill cells are found randomly scattered throughout the surface and along the outer edge of the colony. With age, the bright yellow spots over marginal areas turn yellowish-green. After 3 weeks, an orange-purple tint can be observed in the new air bodies in the outer area.

10 143608 Initially, the back of the colony is a bit concave. As it matures, the colony becomes flat as the surface of the agar and the back becomes somewhat wavy. On the 10th day, the back is light brown (ISCC-NBS 57 and Maerz and Paul 5-A-10). On the 15th, it is greyish red (ISCC-NBS 19 and Maerz and Paul 6-J-3).

The conidia forming stage is sparse and conidiophores are scattered over the surface, sometimes appearing as spots or as submarginal bands. The conidia heads are first spherical with radially arranged sterigms. With age, they can be developed as short cylindrical shapes that are more compact. The spherical bodies have a diameter of 70-80 µm and an average of 50 µm.

The cylindrical bodies can be up to 140 µm long and 70 µm wide.

The conidia are spherical to sub-spherical, wrinkled and yellow-green. They have a diameter of between 2.8 and 3.9 µm with an average of 3.2 µm.

Sterigmata sit next to each other and are colorless. Primary sterigmata have a length of 4.7 to 11.0 µm with an average of 7.9 µm. At their widest point, they are 2.4 pm and narrow in to 1.6 pm. Secondary sterigmata may occur singly or in pairs from the primary sterigmata, and these are flask-shaped. At their widest point they measure 3 pm and narrow to 0.4 pm where they become annular. The annular opening is 1.2 µm long. The total length is 5.5 - 12.6 µm with an average of 9.2 µm.

The vesicles are spherical to sub-spherical or hemispherical and can be flattened to a peak and become brownish with age. They have a diameter of 7.4 to 11.2 µm and an average of 9.4 µm.

Conidiophores are smooth, relatively thick-walled and are translucent at first, after which they develop a color that is light cinnamon brown. They are slightly wider at the vesicles and can narrow a little near the foot cell. The average width is 5.9 pm. Conidiophores have a length of 47.7 - 166.6 µm with an average of 106 µm. They come from submerged hyphae or laterally from aerial filaments.

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The ascogenic state comes around by the 20th day. Small yellowish blisters of Mile cells appearing on the surface can now be found anywhere in the mycelium. They “consist of Mile cells that envelop one or more cleistothecia. Httlle cells are spherical to suh spherical or oval to oblong and are thick-walled and transparent. Spherical httle cells have a diameter of 18 - 24 µm and an average of 21.8 µm.

Cleistothecia are spherical to sub-spherical, thick-walled and relatively tough and fibrous. At first they are colorless, after which they become reddish-violet and darken with age. The. has a diameter of 165 - 470 µm and an average of 275 µm.

Colonies grown at 25 ° C grow rapidly and reach a diameter of 4-5 cm in 10-12 days. First they are greyish white, then the colonies become moderately olive green (ISCC-NBS 90 and Maerz and Paul 23-E-6) in 4 days. The corrugated periphery consists of tightly packed, short, white air vents. Small yellowish clusters of Mile cells sit as small dots on the outer region and are randomly distributed over the agar surface. After 20 days, these ML cell clusters tend to envelop most of the surface. The back of the colony is greyish yellow (ISCC-NBS 90 and Maerz and Paul 11-B-1).

The ascgenic condition appears on the 15th day. Small yellowish clusters of httle cells can now be found throughout the mycelium. They consist of Mile cells that encapsulate one or more cleistothecia. Httlle cells can cover large areas above the conidia heads. The femoral cells are spherical to sub-conical or oval to oblong, and are thick-walled and transparent. Spherical httle cells have a diameter of 18-24 µm with an average of 21.8 µm.

Clestothecia are spherical to sub-spherical and are dark reddish brown. They range from 389 - 700 µm in diameter with an average of 506 µm.

Asci are brittle, transparent and sub-spherical to oval.

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Sub-spherical asci are 8.7 - 11.9 µm in diameter with an average of 10.5 µm. Oval asci are 10.3 - 14.2 µm x 8.7 - 10.3 µm with an average of 12.2 x 9.1 µm.

The ascospores are red-orange, wrinkled with two parallel, gently folded equatorial strands up to 0.8 µm wide and unbroken. The ascospores appear lens-shaped through the long axis. When the thread is encircling, the ascospor is spherical.

One spherical "body" is 4.9 - 6.3 µm in diameter with an average of 5.4 µm.

Two characteristics of the antibiotic A-30912-producing strain of Aspergillus rugulosus differ from those of Aspergillus rugulosus described by Raper and Fennel. The A-30912-producing strain has larger conidial bodies and ascospores.

The Aspergillus rugulosus culture useful in the preparation of the antibiotic in question is deposited and made part of the stem culture collection in the Northern Regional Research laboratory, U.S. Department of Agriculture, Agricultural Research Service, Peoria, Illinois 61604, from which it is available to the public under NRRI 8113.

A wide variety of culture media can be used to grow Aspergillus rugulosus NRRI 8113. However, in order to achieve economical production, optimal yield and easy product isolation, certain culture media are preferred. For example, a carbohydrate source such as glucose is preferred for large-scale fermentation, although molasses, starch, lactose, sucrose, maltose, glycerol and others may be used. A preferred source of nitrogen is enzyme hydrolyzed casein and soluble meat peptone, although distillery waste, monosodium glutamate and others may be used. Inorganic nutrient salts can also be incorporated into the culture medium. These usually include soluble salts which can provide sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate, nitrate and other ions.

Essential trace elements useful for the growth and evolution of the organism must also be present in the culture medium.

Such trace elements are usually found as impurities in the other constituents in amounts sufficient to provide the required growth of the organism.

It may be necessary to add small amounts (i.e. 0.2 ml / g) of an antifoaming agent, such as polypropylene glycol, if foaming becomes a problem.

For preparing a substantial amount of antibiotic A-30912, submerged aerobic fermentation in tanks is preferred. Small amounts of antibiotic can be produced by shaking bottle culture. Because of the time lag in the antibiotic production, usually associated with grafting large tanks with the spore form of the organism, it is preferred to use a vegetative grafting medium. This is accomplished by grafting a small volume of culture medium with spores from the mycelial fragments or the organism to obtain a fresh, actively growing culture of the organism. The vegetative seed medium is transferred to a larger container. The medium used for growing the vegetative seed medium may be the same as that used for larger scale fermentation, but other media may also be used. The antibiotic A-30912 producing organism can be grown at temperatures between 20 and 43 ° C, preferably 25-30 ° C. Optimal production of the subject antibiotic mixture appears to occur at a temperature of about 25 ° C.

As is customary in aerobic submerged fermentation processes, sterile air is blown through the culture medium. To achieve effective antibiotic production, preferably about 0.4 volume of air is used per day. volume of culture medium per minute.

The production of the subject antibiotic mixture can be monitored during the fermentation by analyzing samples of alcohol extracts of the fermentation liquid for antibiotic activity against an organism known to be sensitive to A-30912. One such organism is Candida albicans. The bioassay is conveniently conducted as a paper plate test on infected agar plates.

In general, the antibiotic effect can be determined on the second day of fermentation. Maximum antibiotic production usually occurs between the 3rd and 6th day.

Η 143808

The antihiotic and its components are antifungal agents. The antifungal effect of the components in question can be illustrated by in vitro experiments with components A and D. These tests are listed in tahel III below. The antifungal effect was measured by a conventional plate diffusion method (6 mm strips dipped in solutions containing the test compound. The strip was placed on agar plates infected with the test organism). The result is stated as the minimum inhibitory concentration per day. plate in which the test compound inhibits the development of the test organism.

TABLE · III

Minimum inhibitory concentration Test organism _ (µg / plate) _

Antihiotic A-30912 Component A Component D

Candida albicans 0.625 0.5

Trichophyton mentagrophytes 0.078 0.07

Component A is very active in in vitro plate diffusion experiments against dermatophytes. The results of these experiments are listed in Table IV.

MEL 17 A-50912, component A to dermatophytes

Number of Minimal Inhibitory Concern-Dermatophyte Isolates Tration (µg / plate)

Trichophyton mentagrophytes 13 1.25 - 0.039

Trichophyton gallinae 1> 1.25

Trichophyton meginini 1 0.0195

Trichophyton quinckeanum 1> 1.25

Trichophyton rubrum 1 <0.0098

Trichophyton scoenceinii 1 0.0195

Trichophyton terrestre 1 0.0195

Trichophyton tonsurans 9> 1.25-0.156

Microsporium gypsum 5 0.156 - 0.038

Microsporium audounii 4 1.25-0.156

Microsporium canis 6 1.25 - 0.0098

Microsporium cookei 2 1.25 - 0.0195

Nannizgia incurvata 1 0.312

Phalaphere ”jean salemi 1> 1.25

Epidermatophyton floccosum 1 1.25

Geotrichum candidum 4> 1.25 - 0.156

Keratinomyces ajellio 1 0.156

Furthermore, bone antifungal activity of the subject antibiotic components was demonstrated by in vivo experiments. The in vivo tests were carried out as follows: Three doses of the test compound were given to Candida albicans infected mice at the time 0, 4 and 24 hours after infection. The protection afforded by the test compound is measured as an ED 50 value (the effective dose in mg / kg that protects $ 50 by the mice, see W. Wick et al., J. Bacteriol.

81, 233-235 (1961)). The ED ^ values of component A for Candida albicans in mice were 30 mg / kg (intraperitoneal administration) 16 143608 and 50 mg / kg (subcutaneous administration). The ED1Q value of component D against Candida alMcans in mice was 53 mg / kg (subcutaneous administration).

There was no evidence of acute toxicity when the subject Component D was administered intraperitoneally or subcutaneously in mice at a rate of 100 mg / kg twice per day. day for 3 days (total 600 mg / kg).

Also, there was no evidence of acute toxicity when component A "was administered intraperitoneally in mice at an amount of 200 mg / kg three times a day (600 mg / kg in total).

There was no evidence of acute toxicity when component D "was administered subcutaneously in mice three times a day at an amount of 14 mg / kg (total 42 mg / kg).

When used as antifungal agents, the subject antibiotic components are administered parenterally and are usually administered together with a pharmaceutically acceptable carrier or diluent. The dosage of the antibiotic depends on a variety of conditions, such as the nature and severity of the infection in question.

The following examples further illustrate the invention: EXAMPLE · 1 A. Shake flask fermentation

A culture of Aspergillus rugulosus NRB.L · 8113 was prepared and maintained on an 18 x 150 ml agar slab having the following composition:

Ingredient Quantity (fa)

Dextrin 1,000

Enzymatic hydrolyzate of casein * 0.2000 Yeast extract 0.1000 Meat extract 0.1000 KOI 0.0200 17 143608

Ingredient Quantity ($)

MgSO4 * 7H20 0.0200: FeS04'7H20 0.0004

Water 98.5596 * N-Z-Amin A, Sheffield Chemical Co., Norwich, N.Y.

The oblique surface was seeded with Aspergillus rugulosus NKRL 8113 and the grafted oblique surface incubated at 25 ° C for approximately 7 days. The mature oblique culture covered the bovine serum and scraped with sterile loops to loosen the spores. Half of the resulting suspension was used to seed 50 ml of a vegetative medium of the following composition:

Ingredient Quantity ($)

Sucrose 2.5

Molasses 3.6

Corn mold water 0.6

Enzymatic hydrolyzate of casein 1.0 E2HPO4 0.2

Water 92.1 * N-Z-Case, Sheffield Chemical Co., Norwich, N.Y.

The inoculated vegetative medium was incubated in a 250 ml Erlenmeyer flask at 25 ° C for 24 hours on a rotary shaker with a stroke length of 50 mm and at a rotational speed of 250 rpm. minute.

This vegetative medium can be used directly to seed the second stage medium. Alternatively and preferably, it can be stored for later use by keeping the culture under a nitrogen blanket. The culture is prepared for such storage in many small samples as follows: In each sample are placed 2 ml of grafted vegetative medium and 18 ml of a glycerol-lactose solution of the following composition:

Quantity of Ingredients

Glycerol 20 $ lactose 10 $

Deionized water $ 70

The prepared suspensions are stored in a nitrogen atmosphere,

A stored suspension (1 ml) prepared as above was used to seed 50 ml of a first stage vegetative medium of the same composition as described above. The inoculated first stage vegetative medium was incubated in a 250 ml wide-necked Erlenmeyer flask at 25 ° C for 22 hours in a shaker as described above.

B, long-fermentation

To prepare a large volume of seed medium, 10 ml of the above-described incubated first stage vegetative medium was used to seed 400 ml of a second stage vegetative growth medium of the same composition as the vegetative medium. The second stage medium was incubated in a 2 liter Erlenmeyer flask at 25 ° C for 25 hours on a shaker as above.

The incubated second stage vegetative medium (800 ml) prepared above was used to seed 100 liters of sterile production medium of the following composition:

Quantity of Ingredients

Glucose '25 g / liter

Starch 10 g / liter

Pepton * · 10 g / liter

Molasses 5 g / liter

Enzymatic hydrolyzate of casein ## 4 g / liter

MgSO ^ * 7H₂O 0.5 g / liter 19 143608

Quantity of Ingredients

Czapek's mineral stock solution 2.0 ml / liter

CaC05 2.0 g / liter of water-exchanged water up to 1 liter of P. 159, Index Biomedical Corp., Glenwood, 111.

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

** # Czapek's mineral stock solution has the following composition:

Quantity of Ingredients

EeSO 2 * 7H 2 O (dissolved in 2 ml of wife. HCl) 2 g EC1 100 g

MgSO4-7H2O 100 g

Ion-exchanged water equivalent to 1 liter

The pH of the medium was 6.8 after sterilization by autoclaving at 121 ° C for 30 minutes at a pressure of 35-40 kg. The inoculated production medium was allowed to ferment in a 165 liter fermentation tank at a temperature of 250 ° C for 4 days. The fermentation medium was aerated with sterile air at a rate of 0.5 volume / volume / minute. The fermentation medium was stirred with conventional stirrer at 300 rpm. minute.

M§ ^ 411else_af_den_antibiotiske_blanding_A-30912

The whole fermentation medium (200 liters) obtained by the procedure described in Example 1 was thoroughly stirred with 200 liters of methanol for one hour and then filtered using filter aid (Hyflo Super-cell, a diatomaceous earth, Johns-Manville Products Corp.). The pH of the filtrate was adjusted to 4.0 by the addition of 5N hydrochloric acid. The acidic filtrate was extracted twice with equal amounts of chloroform. The chloroform extracts were mixed and concentrated in vacuo to a volume of about 4 liters. This concentrate was added to 60 liters of diethyl ether to precipitate the mixture of the antibiotic components. The precipitate was separated by filtration and dried to give 38 g of the subject antibiotic mixture as a gray powder. The filtrate was concentrated in vacuo to give an oil. This was dissolved in 500 ml of methanol. The methanol solution was added to the diethyl ether (7.5 liters) to precipitate an additional amount of antibiotic mixture. This precipitate was also separated by filtration and dried to give an additional 3.5 g of the antibiotic mixture.

EXAMPLE 2

Isolation of the antihiotic_component_A

The antibiotic mixture A-30912 (20 g) obtained as described in Example 1 was placed in a silica gel column (4 x 107 cm) in acetonitrile: water (95 * 5). The column was eluted with acetonitrile: water (95: 5) at a rate of 1-2 ml per ml. minutes and fractions were collected. with a volume of about 20 ml. The reactions were analyzed by thin-layer chromatography on silica gel using acetonitrile: water (95: 5) and Candida albi-Cans bioautography as the solvent.

Fractions 74 - 125 were combined and concentrated. The concentrated solution crystallized on standing to give an additional 124 mg of sterigmatocystin. Fractions 212-273 were mixed and centrifuged in vacuo to give an oil. This was dissolved in a small volume of methanol. The methanol solution was added to 15 volumes of diethyl ether. The precipitate formed was separated and dried to give 23 mg of antibiotic A-30912, component D. Fractions 274-437 contained components A, B, C and D. Fractions 482-900 contained components A, E, F and G ·. Fractions 438-481 were combined and concentrated in vacuo to give an oil. This was dissolved in a small amount of methanol and the methanol solution was added to 15 volumes of diethyl ether. The precipitate which formed was separated and dried to give 2.17 g of the component A.

21 143608

EXAMPLE

iS2lS £ iSS_S £ -! ilii5i2ii £ HS _ ^: z222i2i componentJD

A particularly purified antibiotic mixture containing the components B, C and B obtained as described in Example 2 from fractions 274-437 <> 750 mg of this material was chromatographed on a silica gel column (2.2 x 51 cm, Woelm silica gel). , and fractions having a volume of ca. 15 ml, eluting with the following solvents:

Solvents Solvent 1-25 acetonitrile 26-62 acetonitrile + 1 $ water 65-700 acetonitrile + 1.5 1 ° water

The aliquots were measured by silica thin layer chromatography using acetonitrile: water (95: 5) and benzene: methanol (7: 5) as solvent systems and Candida albicans bioautography. Fractions 535 - 685 * containing the component D in question were mixed and concentrated in vacuo to give an oil. This was dissolved in a small amount of methanol and added to diethyl ether (15 volume) and the precipitate formed was separated by filtration and dried to give 69 mg of the antibiotic component D.

EXAMPLE 4

Insulation of A-30912, components B and C

A partially purified antibiotic mixture containing components A, B, C and D was obtained as described in Example 2 from fractions 212-457. 18 grams of this material was dissolved in a minimal amount of acetonitrile: water (4: 1) and chromatographed on an alumina column (5.8 x 56 cm) and fractions were collected with a volume of about 20 ml. The column was eluted with the following solvents:

22 1A36OB

Solvents 1-300 acetonitrile: water (4: 1) 301-509 acetonitrile: water (7: 3).

The column fractions were analyzed by thin-layer chromatography on silica gel as described in Example 3 · On the basis of these results, the fractions were combined and concentrated to oils. The oily residues were dissolved in a small amount of methanol and the methanol solutions precipitated with 10-15 volumes of diester.

The manner in which the fractions were formed, the weight of the fabric prepared, and the content of components are given below.

Praction 7weight (g) Component 6-28 0.23 *

6-28 5.80 A-30912, 0, D

34-114 2.90 A-30912, B

115-164 1.20 A-30912, A, B

165-509 1.90 A-30912, A

w insoluble material obtained prior to ether precipitation.

To purify component C, 2 grams of the 6-28 L methanol fractions were dissolved, adsorbed on a sufficient amount of silica gel (grade 62) which was dried and the product added to the 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 / min, collecting fractions with a volume of about 10 ml. From fraction 117, the solvent was changed to acetonitrile: water (99: 1). The column fractions were again measured by thin layer chromatography. On the basis of the TIO results, the fractions were combined and concentrated to obtain oily residues. These were dissolved in a small volume of methanol and the methanol solutions precipitated with 10-15 volumes of diethyl ether. The component content and weight of the fractions of interest are given below;