FI72534B - FOERFARANDE FOER FRAMSTAELLNING AV ANTIBIOTIKA AKLACINOMYCIN A OCH B ELLER DERAS BLANDNINGAR. - Google Patents

Description

725 34

The present invention relates to a novel process for the preparation of aclasinomycins A and B, the anti-tumor antibiotics, and to a process for the preparation of aclacinomycins A and B, respectively. Aclasinomycin has the structural formula: r COOCK-j 0: 0 OH H ja

In aclasinomycin A, R is work 1 J-r- 1 'i / CH' \ K (C H,)., IV / i I n i j

0M

In aclasinomycin B, R is (¾ i — n 0 I 1

Ah3 \ N (CH3) 2 0 I o .a v o 1 '2 ‘72534

Aclasinomycins A and B belong to the group of anthracycline antiotics and are effective against various Gram-positive bacteria and inhibit tumor growth in animals. Aklaslnomysllnlt A and B and their methods of preparation were first described in 5 JP Patent 864851, filed July 27, 1974. The JP patent corresponds to, for example, GB patent 1491266 and U.S. patents 3988315 and 4071411.

In the above patents, Streptomyces galllaeus MA 144 M1, deposited in the American Type Culture Collection under the designation A.T.C.C., is used as the aclasmomycin-producing strain. Well. 31133 and at the Fermentation Research Institute of Japan under the designation FERM No. 2455.

Said strain has the following characteristics: poorly developed inflorescence on a solid agar medium, har-15 in color. Forms spiral-shaped spore mycelium, the spores are on the surface, oval-shaped, 0.4 to 0.8 μm in size. The colonies are corrugated and the substrate mycelium is yellowish-brown. The soluble pigment is missing or light brown. Some agar media produce a yellow-green soluble pigment.

Streptomyces galllaeus is cultured aerobically, the growth optimum is 24-30eC, at 50eC there is no growth. It hydrolyzes gelatin, peptonol milk and weakly hydrolyzes starch. Melanoid pigments are formed on organic substrates. It grows on Pridheim's and Gottlieb's medium with D-glucose, D-xylose, L-arabinose, L-rhamnose, D-galactose or inocitol, but does not grow on medium containing D-mannitol.

When the strain is grown in a nutrient solution containing soy flour, glucose, starch and various salts under aerobic conditions, 46 mg / l of aclasinomycin A and 23 mg / l of aclacinomycin B are obtained after 36 hours.

Il '3 "72534

The antibiotics are "both in the mycelium and in the culture medium. They are extracted from the mycelium with acetone and from the medium with ethyl acetate. The extracts are combined and the aclasinomycins A and B are separated by column chromatography.

A disadvantage of the above-mentioned known method is the low production capacity of the microorganism. In addition to aclasinomycins, some antibiotics belonging to the E-pyrromycin group are formed, 10 which complicates the technology of dry cleaning.

The main object of the present invention has been to find new cultures producing aclasinomycins A and B which have a higher activity in the production of aclasinomycin A than the known one and which synthesize these antibiotics as main components.

As a result of the search, a new strain 12/3-A belonging to the species Streptomyces lavendofoliae, which forms aclasinomycin A as the main component, was found in a soil sample in Central Asia.

The strain Streptomyces lavendofoliae is deposited in the strain collection 20 at the Antibiotic Institute under the designation VNIIA No. 1670 20.1.1982.

The Strain Collection of Microorganisms of this Institute (VNIIA) is registered with the International Federation of Strain Collections in 1972, No. 337.

In addition, Streptomyces lavendofoliae strain 12/3-A has been deposited in the 25 strain collection Centraalbureau voor Schirrmelcultures (CBS) on March 31, 1983, with CBS 261.83. · - 4 - 72534

Streptomycee lavendofoliae strain 12/3-A has the following morphological, physiological and antagonistic properties.

MORPHOLOGICAL CHARACTERISTICS

Streptomyces lavendofoliae strain 12/3-A forms an abundant purple-vaa-5 lean red (Ä 3) air cluster. The sporangia are spiral-shaped, with a slatted surface size of 0.7-1.4, etc. The subs-wire mycelium is belge-pink, even in the dirty evening color aetl (K1 + M2). The pigment secreted into the medium is dark brown, up to brownish-pink-purple (K1 + 10 Γ2 + Η2). the colors are defined according to the Bondarzev color scale, a publication of the SNTL Academy of Sciences from 1954.

Cultures are grown at 28 ° C for 28 days.

table 1

Culture medium_Description_Features_ 1_2_3_

Teapeka agar mycelium well developed, purple-pink (* 3) substrate-belge-pink-purple mycelium colored (K1 + K2) soluble brown-pink pigment (Kl)

Gause I agar mycelium abundant, pink (K3 + A3) substrate gray-1-age-old (Γ2 + Η2) soluble brown-pink pigment (K1) / li 1_2_3__

Krasilnikov llmar mycelium abundant, purple-pale medium 1 red (13) substrate-beige-dirty purple mycelium (K1 + H2) soluble brown-pink pigment purple (Κ1 + Γ2 + Η2) Starchy-agar substrate stripe beige-light (Kl) mast red soluble beige-light (Kl) pigment red

Glucose aerial mycelium growth abundant, light asparagine gray (K 6) substrate sand brown mycelium brown (J17-K7) soluble sand brown pigment (J17- + K7)

Waxman agar mycelium growth abundant, reddish gray (A3) substrate- brownearn Chestnut mycelium from brown to dirty purple (B2-H2) soluble brownish-brown pigment (B2) r - 6 - 72534 1_2_3 _

Yeast and soluble atmospheric mycelium growth abundant, red-starch-agar van gray (A3) substrate brown (B 7) mycelium soluble sandy brown pigment to brown in color (B7-K7)

Glucose-yeast aerial mycelium growth abundant, lighter agar (D1) substrate dark brown (JI5) mycelium soluble brown (K 7) pigment

Oat-agar mycelium growth good, reddish gray (A3) substrate-turquoise-brown (®2) mycelium soluble light brown (B7) pigment

Malt agar Mycelium growth abundant, reddish gray (A3) substrate-brown-maroon mycelium colored (K7 + K4) soluble dark brown (B2-H <7) pigment brown t li 1_2_3_ - 7 - 72534

Gauee 2 agar atmospheric growth satisfactory, light gray (A6) substrate brown (K 7) mycelium soluble brown (K 7) pigment MPA aerial mycelium growth weak substrate beige-sand mycelium (5 6) soluble missing pigment

Tyrosine-agar mycelium growth abundant, purple-light red () K3) substrate plum black (01) mycelium soluble Umbra-colored (07) pigment

Tresner agar mycelium growth good, purple-light iron citrate-red (K 3) with solution substrate dark umbrella mycelium (Π2) soluble dark umbrella pigment (Π2) - 8 - 72534 1_2_3_

Potato pellets aerial mycelium growth good, white to pink (D3-K3) substrate — tuna-brown mycelium (Kl-Bl) soluble turquoise pigment (Kl-Bl)

PHYSIOLOGICAL SIGNS

Aerobic, the optimum growth temperature is 24-30 ° C. At 50eC there is no growth. Dissolves gelatin moderately, peptones milk, which darkens, hydrolyzes starch moderately. Generates 5 melanoids (Tresner's medium with iron citrate, (tyrosine agar).

Streptomyces lavendofoliae strain 12/3-A makes good use of the following carbon sources: D-glucose, D-xylose, L-arabinose, D-fructose, D-galactose, inositol. It uses little or no of the following substances: L-rhamnose, sucrose, raffinose and D-mannitol. Streptomyces lavendofoliae strain 12/3-A is maintained on an agar slant under a layer of vaseline oil at a temperature of + 5 ° C for six months.

ANTAGONISTIC SIGNS

15 Streptomyces lavendofoliae 12/3-A is cultured in liquid medium in a shaking vessel. After three days, aklaslnomysil A produces about 56-60 mg / l, which has the following antlmlkroblnee effect, cytotoxic activity and antitumor effect when studied in experimental animals (Tables 2-4) · f li 72534 - 9 -

Table 2

From the antimicrobial spectrum of aklaslnoroyellnl A produced by Streptomyces lavendofoliae 12/3-A and the reference substance.

TEST ORGANISMS _MIC pg / ml 1) _ S tr. lavendofoliae Produced by Japanese (Tokyo) 12/3-A Zaidanhoji Biseiaclasinomycin A but su Manufactured by Kagaku Kenkyukai

__ '_aklasinomysllnl A

_1_2_3_

Staphylococcus aureus 209P 15.56 7.8

Staph, aureus 209P, resistant to streptomycin 15.56 15.56 res. for penicillin 15.56 15.56 res. for gentamicin 15.56 15.56 res. kanamysUnille 11.6 11.6 res. for oleandomycline 15.56 11.6 res. for llnomomycin 15.56 23.3 res. for violarin 23.3 19.0 res. for erythromycin 31.2 23.2 res. for dactinomycin 31.2 70.0 res. for novobiocin> 250.0> 250.0 res. for rifampicin 125.0 125.0 res. vancomysilnllle 125.0 125.0 E.coli 675> 250.0> 250.0

Pseudomonas aeruginosa> 250.0 ^ 250.0

Klebsiella pneumoniae 444> 250.0 ^ 250.0

Providence of ATCC 7240> 250.0 -> 250.0

Candida albicans> 250.0 ^ 250.0

) The MIC, the minimum anti-growth drug concentration, is determined by increasing in two replicate experiments from a standard microbial inoculum in a peptone-broth medium with an increasing drug concentration of 10 per ml for 20 hours at 37 ° C.

and at pH 7.0.

- 10 - τ. , 72534

Table 3

Akia sinomys Unin A tumors estSvB effect in animal experiments.

Strain Shelf life Optimal dose Life expectancy Tumor animal (day) (mg / kg) kimaMrainen growth retardation () prolongation (Z) dosing (%)

La CB ^ 1- * 9 2.5 92

LLC

CBF1 1-> 9 5 67 S-37 unprocessed 1- ^ 5 2.5 80 tomato mice

Table A

Cytostatic effect of aclasinomycin A and its DNA complex in suspended culture of tumor cell P-388.

x)

Formulation ED50 * Pg / ®1

Aklasinomysilnl A 0.01

Aclasmnomycin A-DNA complex 0.01 x) Dose that slows down the production of nucleic acids (DNA + RNA) 50 Z.

The results of the comparison of the characteristics of the strain 12/3-A according to the invention with the characteristics of Streptomyces galilaeus MA 144M1, a known producer of aklaslnomllnle A and B, confirm that the morphological and physiological characteristics of their cultures differ. r

II

72534

On the other hand, the strain according to the invention corresponds in its characteristics to the properties of Streptomyces lavendofollae as defined by Bergey (Table 5) ·

Table 5

Comparison of the morphological and physiological characteristics of the culture of strain 12/3-A according to the invention with the characteristics of the prototype Str. Galilaeus MA 144M1 and Str. Lavendofoliae.

Descriptive Strain Str. Galilaeue Str. Lavendo- 12/3-A MA 144M1 foliae _ (prototype) _ 1_2_3_4_

Spore straps in spiral shape with spiral shape. splraallm.

ItiOt smooth, oval smooth, oval smooth, shaped oval m.

Synthetic media:

Air mycelium growth good, even growth moderate growth good, abundant, purplish-red, light-purple-light (M3) from gray to red to earthy

Substrate belgep reddish yellow-brown yellow-yellow mycelium dirty lilac in some growth brown to color (Π 7-M2) substrates yellow-brown to green-brown

Soluble TVIllium brown missing or yellow pigment turnrian brownish brown or spawn purple tunic brown (Κ1 + Γ2 + Η2) 1_2___3_A_ - 12 - 72534

Organic media:

Air mycelium growth good, red growth moderate growth good, normal gray (A3), light purple-light gray from red

Substrate light brown to yellowish brown dark brown or mycelium to brown (J17-H37-K7) very dark brown

Soluble tan brown brown brown or pigment (J17-B2-H <7) very brown

Potato slices:

Air mycelium growth good, white growth good, horizontal growth good, reddish lean gray dark brown (D3- Ä 3) reddish

Substrate- tunman- (K 1 + 81) brown-yellow-tan-brown mycelium brown nen

Soluble turnnan- ^ i + gi) Chestnut tan brown pigment brown brown

Melanoldlt formed form formed

Growth in cellulose good moderate goodB

Milk peptonolate peptonolate peptonolate brown moderately brown Starch hydrolyses hydrolyses hydrolyses hydrolysis moderately weakly moderately

Moderate weak moderate hydrolysis of gelatin 1_2_3_4_ - 13 - 72534

Cane sugar - + - D-glucose + + + D-xylose + + + L-arabinose + + + L-rhamnose - + - D-fructose + + - D-galactose + + +

Raffinose - + - D-mannitol - - -

Inositol - + - +

Taxonomic determination of culture 12/3-A was performed on the basis of the literature: Krasiljnikov H.A. "Rays", "Nauka" in 1970, pages 172, 328; Bergey’s Manual of Determinative Bacteriology, Editors: Buchanan R.E., Gibbons N.E.

5 et.al., pp. 808-810.

The culture medium can be any medium used by a strain of aclasinomycin belonging to the species Str. Lavendofoliae.

Fructose, glucose, arablnose, xylose, galactose, starch and the like are recommended for the control sources of the culture medium.

The nitrogen source may be meat extract, peptone, maleic extract, yeast extract, casein hydrolyzate, corn flour, soybean meal, cotton meal, etc., as well as organic and inorganic compounds still containing nitrogen compounds, such as ammonium salts (nitrates, sulfates, phosphates), phosphates. urea and others.

Mineral salts such as calcium carbonate, sodium and potassium phosphates, sodium or potassium chloride, magnesium salt, copper and the like may be added to the medium.

S

- 14 - 72534 ^ To produce larger amounts of glass glasses A and B, the culture is performed in a two-stage aerated deep culture. Vegetative mycelium is used to inoculate the fermentor. The medium growing the vegetative mycelium may be the same as that used in the biosynthesis of aclasinomycins A and 6, but may also be different.

Of the revised media alternatives, it was found that media B-2 and B-3 were the most preferred:

Raw material _Culture media_ _________ B-2 (Z) _B-3 (Z) 1. Ammonium sulphate 0,1 1,5 2. Soya flour 1,1 1,5 3. Potato starch 2,6 3,0 4. Calcium carbonate 0,8 1 .4 5. Sodium chloride 0.3 0.3 6. Copper sulphate 0.007 0.007 7. Defoamer AC-60 0.5 0.5

The media are sterilized at 124-126 ° C for 30-40 min.

10, after which the pH of the medium is neutral.

Str. Lavendofollae, strain 12/3-A when grown Under aerated conditions, 1 l of air / 1 l of medium per minute at a temperature of 20-30 ° C, a pH of about 6-8.5, preferably 6.9-7.9, gives 48 After -96 hours, up to about 60 mg / l of aclasinomycin A and 4-10 mg / l of aclasinomycin B.

Table 6

Comparison of physicochemical properties of aclasinomycin A from Str. Galilaeue (Zaidanhojln Biseibutsu Kagaku Kenkyukai, Tokyo, Japan) and Str. Lavendofollae.

li 72534 - 15 - _Antibiotic strain_ _Str. galllaeus MA144M1 Str. lavendofollae_ 1_2_3_ UV spectrum x) N 1% X nm (E / v 1 cm max 90% MeOH 431.5 / 146.1 / 290 / 112.1 / 431.5 / 148.6 /, 289 (137.4), 257 (285.3), 228 (485.7); 257 (302.9), 228 (502): 0.1 N HCl 431.5 (146.1), 257 (285.3), 431.5 (151.4), 289 (137.4), 290 / 112.1 / 228 / 485.7 /; 257 / 302.9 / 228 / 499.2 /; 0.1 N NaOH 521.3 (118.9), 317.3 (64.5), 521.3 (130), 318.3 (78.5), 285 (101.9), 236 (424), 6 /. 284 / 126.2 / 236 / 440.3 /.

xx) IR spectrum ν cm -1 3450, 2980, 2945, 2820, 3480, 2980, 2950, 2825, max 2760, 1735, 1675, 1620, 2775, 1735, 1680, 1625, 1600, 1010, 1600, 1010.

xxx) PMR spectrum δ ppm 12.55, 1H, s .; 12.7: 1H: s; 12.0, 1H, 12.0, 1H, s .; s .; 7.83, 1H, J-1.8 Hz, 7.15-7.85, 4H, m; J2 '7.8 Hz; 7 * 68 »1H» 5.58, 1H, s., J ** 7.8 Hz; 7.68, 1H, s; 7.30, 5.31, 1H, s; 1H, J1-1.8 Hz, J2 '7.8 Hz;

4.95-5.20, 2 H, m; 5.52, 1 H, m; 5.28, 1 H

4.35-4.65: 2H: m .; m .; 4.95-5.20, 2 H, m; 4.11, 1H, B ·; 4.40-4.70: 2H: m .; 4.11, 1H, s; 3.68, 3H, p .; 3.70: 3H: s .; 2.3-2.7: 5H: m .; 2.25-2.65: 5H: m .; 2.23: 6H: 8; 1.40-2.15, 2.20, 6H, s; 8H, m .; 1.32, 3H, d., J 6 6.6 Hz; 72534 - 16 - 1_2_3_ 1.45-2.15, 8H, m; 1.29, 3H, d., J-6.4 Hz; 1.00-1.36, 12H, m. 1.17, 3H, d., J-6.2 Hz; 1.08, 3H, t, J-7 Hz.

xxxx)

Mass spectrum o / z 376/100% /, 114 / 2.6 Z /, 377/50% /, 376/100 Z /, 794/15 Z /, 377 (42.8 Z), 812/30 Z (394 / 5.1 Z), 416 (1.5 Z), 794 (10.2 Z), 812 (28.8 Z).

x) - UV spectra were recorded on a Pye Unleam SP-8-100, calibrated on a standard filter HO-720540 and Di-720538 using a Pye Unicam.

xx) - IR spectra were recorded on a UR-20 KBr tablet. xxx) - PMR spectra were recorded on a 90 M Hz "Bruker-WH-90" for CDCl 3, TMS reference, xxxx) - Field desorption mass spectra were recorded on a Varian Mat 311-A, calibration with PFK. Current in the emitter 5-20 mA.

Table 7

Comparison of the physicochemical properties of aklaslnomy-eiln B obtained from Str. Galilaeus and Str. Lavendofollaer.

_Antibiotic strain_ _Str. galilaeus MA144M1 Str. lavendofollae_ 1_2_3_

UV spectrum 1 Z

Λ nm (E / max 1 cm 90 Z MeOH 432/159), 289.5 (137), 431.5 (149.2), 290 (119), 259 (313), 229.5 (498); 257/296 /, 228/508 /; 72534 - 17 - 1_2_3_

Of1N HCl 431/162 /, 289.5 / 156 /, 431.5 / 149 /, 290/119 /, 259/355 /, 229/586 /; 257/296/228/503; 0.1 N NaOH 522 (145), 315 (101), 521.3 (127), 316.3 (77.3), 284 (160), 239 (510). 285/119/236/453 /.

IR spectra 3450, 2980, 2945, 2820, 3490, 2980, 2950, 2830,? Cn "1 2760, 1730, 1675, 1620, 2780, 1735, 1680, 1625, max 1600, 1010. 1605, 1015.

PMR spectrum 12.2, 1H, s: 12.68, 1H, α, δ ppm 11.5, 1H, s .; 12.02, 1H, 8; 7.2-7.9: 4H: m .; 7.24-7.89: 4H: m .; 5.5: 1H: 8; 5.48, 1H, s; 5.1-5.3: 3H: m .; 5.09-5.27: 3H, m; 4.6-4.9: 2H: m .; 4.62-4.90: 2H: m .; 4.5, 1H, 8; 4.53, 1H, p .; 4.3-4.4: 1H: m .; 4.36, 1H, q .; 4.15, 1H, 8; 4.11, 1H, 8; 3.8-4.0: 2H: m .; 3.90-4.03: 2H: m; 3.7: 3H: e .; 3.75, 1H, 8; 2.2-2.7: 4H: m .; 3.69, 3H, 8; 2.2: 6H: s .; 2.58, 2R, d .; 1.4-2.0: 8H: m .; 2.46, 1H, d; 2.35, 1H, p .; 0.9-1.4, 12H, m. 2.15, 6H, e .; 1.45-2.01: 8H: m .; 0.89-1.45, 12H, p.

The UV, IR and PMR spectra of aclasinomycins A and B are shown in Figures 1-6 of HHS (FIGS. 1-6).

FIG. 1 shows the UV spectrum of the hydrochloride of aclasinorayslin A in 90 X methanol, FIG. 2 shows the UV spectrum of the hydrochloride of aclasinorcilin B in 90% methanol, FIG. 3 shows the UV spectrum of aclasinomycline A

/ - 18 - 72534 IR spectrum of hydrochloride from KBr tablet, FIG. 4 shows the IR spectrum of aclaeminosil B hydrochloride KBr tablet, FIG. 5 shows the PMR spectrum of aclaiminomycin A hydrochloride (CDCl 3; TMS; T »303 ° C) and FIG. 6 achloromylellin B hydrochloride PMR spectrum (CDClyTMS; δ T-303 ° C).

Aklaslnomysilnit A and B are in the mycelium and culture medium. At the end of the fermentation, the medium is filtered at a pH of 4.5-6.0. Antibiotics are extracted from the medium with ethyl acetate (1 volume of medium per volume of ethyl acetate is taken per 10 volumes of medium).

The organic layer containing antibiotics is separated from the mycelium. After removal of the solvents, the aqueous residues are combined and neutralized to ρΉ 6.9. The antibiotics are then extracted with toluene and twice the volume of 0.01 N HCl-15 solution. From the aqueous solution, the antibiotics are extracted with chloroform, which is dried over anhydrous Na 2 SO 4. The chloroform extract is concentrated on a rotary evaporator to 0.1 volume and 10-12 volumes of dry hexane are added to the residue. The antibiotics in the precipitate are separated and dried. Purification and separation of antibiotics is performed by chromatography with aqueous pilic acid in a solvent system consisting of carbon tetrachloride and isopropanol.

Tables 6 and 7 show the comparative results of aclasinomyins A and B obtained by UV, IR, PMR and mass spectrometry, proving the similarity of these antibiotics obtained with the strains according to the invention and already known.

Thus, the antibiotics A and B generated by Str. Lavendofollae strain 12/3-A are identical to the antibiotics obtained from the culture of Str. Galllaeus MA144M1. The previously known ability of Str. Lavendofollae to produce aclazinomycins A and B. The method of preparation according to the invention can be illustrated by the following examples.

Il% - 19 -

Example 1 7 2 5 3 4

Streptomycee lavendofollae strain 12/3-A is grown on solid oat agar medium at 28 ° C for 12 days until the development of a rich mycelium. The culture is transferred to a 5 750 ml conical flask of B-2 medium containing copper sulphate or antifoam. The flask is mixed by shaking at 250 rpm at 28 ° C for 2 days. The culture grown in an Erlenmeyer flask is transferred in an amount of 5% to a 10 L medium of B-2 medium containing 10 copper sulfate. The fermentor is stirred at 200 rpm and aerated at 10 l / million at 28 ° C for 1 day.

3 liters of culture grown in a transfer fermentor are transferred from 100 l of fermentor to 60 l of B-2 medium. The fermentor is stirred at 200 rpm and aerated at 60 1 / million at 27 ° C for 3 days.

The concentration of aclasnomycin A and B in the medium is determined according to the following method: 1. Determination of the concentration of aclasnomycin A and B in mycelium 20 At the end of the fermentation, mycelium is separated from 5 ml of medium by centrifugation at 2000 rpm for 10 min. From the resulting mycelium, the antibiotics are extracted for 2 hours with A ml of acetone, the mixture is stirred at 15-20 min intervals. The acetone is evaporated from 1.0 ml of the extract in vacuo at a temperature below 40 ° C and the residue is extracted with 1.0 ml of chloroform. The concentrated chloroform extract is used for the chromatographic separation of the antibiotic complex.

For this purpose, take a plate to which an adsorbent tier has been applied, leaving the left side and the lower edge free for a distance of 2 cm. Apply the entire chloroform extract 30 to a 1-1.5 cm wide line on the plate with a capillary. A standard solution of Alaslomyelin A and B is applied to the same plate. The plate is placed in a chromatography chamber with a 7: 2: 1 mixture of chloroform and ethyl acetate-methanol. When the liquid front has progressed to the edge of the plate, the plate is taken out and air-dried for 20 min.

- 20 - 725 34

The yellow bands corresponding to the mobile eclasinomycins and A and B are separately eluted with 4 ml of methanol (pH-6) and measured spectrophotometrically at 430 nm. The amounts of akiae1 nominyl (mg / l of culture) are calculated from their extinction values (E 1 cm of aclaelomyclin A and B are 161 and 159, J. Antibiotics, 1979 32 ^, No. 8, slv. 781-800) according to the following formula :

aclasinomycin A Cmg / 1 199 D

aclasinomycin B Cmg / 1 * 201 D

10 2. Determination of aclasinomycin A and B content 5 ml of medium is separated from the culture medium by centrifugation. Extract with ethyl acetate at pH 6 in a ratio of 5 ml of medium to 5 ml of ethyl acetate. 2 ml of ethyl acetate extract are evaporated off under vacuum and the residue is extracted with 2 ml of chloroform. The follow-up is as in 1.

The amounts of aclasinomysUs A and B are calculated from the extinction values of the antibiotics according to the following formula:

aclasinomycin A C mg / 1 * 124.2 D 20 aclasinomycin B C mg / 1 · 125.8 D

After 3 days of fermentation in medium B-2, the culture 12/3-A produces about 56 mg / l of aclasinomycin A and about 4-8 mg / l of aclasinomycin B.

To separate aclacinomyinins A and B from the medium, 60 l of 25 is filtered through an air filter at a pH of 4.5-5.0. The antibiotics in the medium are extracted with ethyl acetate in a ratio of 10: 1. The organic layer is separated and evaporated in vacuo on a rotary evaporator at a temperature not exceeding 40 ° C until the residue is an aqueous solution. The moist mycelium (5 kg) is extracted twice with 30 10 1 acetone. The mycelium is separated by filtration and discarded. The acetone extracts are combined and evaporated. The aqueous residues from the ethyl acetate and acetone evaporations are combined and the concentrate (5 L) thus obtained is neutralized to pH 6.8 with 10 Z NaOH solution and the antibiotics are extracted 3 x 10 1 with toluene.

li - 21 - 72534

The toluene extracts are combined and the aqueous layer is discarded. The toluene extract (30 L) is evaporated in vacuo at a temperature of not more than 0 ° C until the residue is 3 L. Antibiotics are extracted from this concentrate 0.5 times with 0.01 N HCl. The emulsion formed from the new step 5 is decomposed in a centrifuge.

The antibiotics in aqueous solution are extracted with 0.5 times the volume of chloroform. The extraction time is 20-30 min with the mixture stirring constantly.

The resulting emulsion is disintegrated in a centrifuge. The chloroform layer 10 (1.5 L) is collected and dried over anhydrous for 4 hours

Na 2 SO 4 (10 g N 2 SO 4/100 ml extract). Na 2 O 4 is filtered and the chloroform is distilled off in vacuo on a rotary evaporator to 0.1 volume. 10-12 volumes of dry hexane are added to the residue to form a precipitate which is allowed to settle (30-15 min). The antibiotics in the precipitate are filtered through a No. 4 glass filter and dried. The filtrate is evaporated to dryness on a rotary evaporator at a temperature not exceeding 40 ° C. The residue is slurried in hexane and the hexane is filtered off. The two precipitates are combined and purified by chromatography. The yield of crude product is 2.5 g.

Chromatographic separation of aclasinomycins

The column (8 x 95 cm) is packed with 5.5 l of a suspension consisting of 1.8 kg of aqueous pilic acid (250-400 μm) and 5.5 l of chloroform in which 0.5 Z of triethylamine has been dissolved and vinegar-25 acid. After packing, the column is rinsed with 3 l of chloroform. Add the crude product described above (10 g) dissolved in 100 ml of chloroform. After absorption of the antibiotics, the column is eluted. To separate the antibiotics, a stepwise gradient is performed. To achieve this, the following is passed through the column: - 22 - 72534 1) 4 l of carbon tetrachloride * Jia 2) 8 l of a solvent system consisting of carbon tetrachloride and 25% of leopropanol (25: 1) 3) 8.5 l of solvent cetethane (20: 1) 4) 20.5 l of a solvent system consisting of CCl 4 and an isopropano strip (10: 1)

Aclasmnomycin B is taken from the column with CCl 4: isopropanol (20: 1) and aclasmomycin A with CCl 4: isopropanol 10 (10: 1). Eluates (5-7 1) containing aclasinomycin A

and B, are extracted separately with 0.2 volumes of 0.01 N HCl solution.

Antibiotics are extracted from the aqueous phase with 0.2 times the volume of chloroform. The chloroform extracts A and B of Aklaslnomysilnlen A and B are dried separately over anhydrous Na 2 SO 4 (2 g / 100 ml solution), the sodium sulphate is filtered off and the filtrate is evaporated in vacuo at 40 ° C to 0.1 volume. Add 10 to 10 times the volume of dry hexane and allow to stand for about 30 minutes. The precipitate is filtered through a No. 4 glass filter and the resulting material is air-dried. Thus, from 0.7 g of crude product, 0.7 g of aclasinomycin A and 0.1 g of aclasinomycin B are obtained.

Example 2

Streptomyces lavendofoliae strain 12/3-A is grown for 12 days on oat agar. The culture is transferred to a vessel in which the growth medium is B-2 without copper sulphate. The vessel is shaken at 250 rpm at 28eC for 2 days. The culture is transferred to an Erlenmeyer flask. The inoculation is typically 5-10 Z of the volume of medium. The growth medium is B-3, which consists of the following substances: soy flour 30 -1.5 Z, potato starch - 3 Z, calcium carbonate - 1.4 Z, sodium chloride - 0.3 Z, copper sulphate - 0.007 Z. The growth conditions are the same as in Example 1 and the rearing time is 72 hours. In this case, the activity of the medium is 60 mg / l aklasl-t nomycin A and 10 mg / l aclasinomycin B.

11 - 23 - 72534

The chemical separation of aclasinomycline A and B is performed in the same manner as in Example 1.

As can be seen from the examples, the process for the preparation of aclasinomycins A and B according to the invention has the following advantages over the known methods: 1. Higher yield of aclasinomycin A. The prototype strain produces 46 μg / ml and the strain according to the invention up to 60 μg / ml.

2. Sinerubin A and B are not formed and thus further purification is avoided. In the known process, the above-mentioned by-products must be removed as metal ion complexes.

3. Less acacinomycin B is formed. The prototype strain produces 23 ug / ml and the strain according to the invention 4-8 ug / ml.

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