SE455705B - PROCEDURE FOR THE PREPARATION OF ANTIBIOTIC SUBSTANCES AKLACINOMYCIN A AND B BY CULTIVATION OF STREPTOMYCES LAVENDOFOLIAE CBS 26183 AND STREPTOMYCES LAVENDOFOLIAE CBS 26183 - Google Patents

Description

455 705 Aclacinomycins A and B belong to the anthracycline antibiotic group and are active against various gram-positive bacteria and prevent the growth of tumors in animals. The aclacinomycins A and B and the processes for their preparation were described for the first time in JP patent specification 864851, application date 27.7.1974. Equivalents to said JP patent are, for example, GB patents 1,491,266 and U.S. patents 3,988, 315 and 4,07l, 411.

In the above-mentioned patent, Streptomyces galilaeus MA 144 M1 is used as the aclacinomycin-producing strain, which is deposited in the American Type Culture Collection under the A.T.C.C. No. 31133, and at the Japanese Institute Fermentation Research Institute under FERM No. 2455.

Said strain has the following characteristics: weakly developed aerial mycelium on solid agar plant substrate, to the color gray. Forms spiral-shaped spore carriers, the spores have a smooth surface, are oval-shaped, to the size 0.4 - 0.8 Fm.

The colonies are pleated and the substrate mycelium is yellow-brown. Soluble pigment is missing or is light brown. On some agar plant substrates, yellow-green soluble pigment is formed.

Streptomyces galilaeus is grown aerobically, the growth optimum is 24-30 ° C, at 50 ° C there is no growth. It hydrolyzes gelatin, peptonizes milk and hydrolyzes weak starch. Melanoid pigments are formed on organic substrates. It grows on Pridheim's and Gottlieb's medium which contains D-glucose, D-xylose, L-arabinose, L-ramnose, D-galactose or inositol, but does not grow on a medium which contains D-mannitol.

Then the strain is grown in a nutrient solution, which contains soybean meal, glucose, starch and various salts, under aero-. ._.._, - _ _..-, _ ._.- .. -. ... ... all 455 705 ba conditions, after 36 hours 46 mg / l aclacinomycin A and 23 mg / l aclacinomycin B are obtained.

The antibiotics are found in both the mycelium and the culture solution. They are extracted from the mycelium with acetone and from the culture solution with ethyl acetate. The extracts are combined and the aclacinomycins A and B are separated by column chromatography. The resulting aclacinomycins A and B contain as impurities antibiotics from the E-pyrromycin group, which are removed by treatment with copper ions.

A disadvantage of the above-mentioned known method is the weak production capacity of the microorganism. In addition to the aclacinomycins, certain antibiotics are formed from the E-pyrromycin group, which complicates the chemical purification technique.

The main object of the present invention has been to find new cultures, aclacinomycin A and B-producing cultures, which show a greater aclacinomycin A-producing activity compared to the previously known ones, and which synthesize these antibiotics as main components.

As a result of these efforts, a soil strain 12/3-A was obtained from a soil sample from Central Asia, which belongs to the species Streptomyces lavendofoliae, and which forms aclacinomycin A as a main component.

This strain of Streptomyces lavendofoliae is deposited in the strain collection of the Antibiotic Institute under VNIIA No. 1670 on 20.1.1982. This institute's (VNIIA) microorganism stock collection has been registered in the International 455 795 Federation of Tribal Collections in 1972, No. 337.

The strain 12/3-A of Streptomyces lavendofoliae was also deposited at the depository institute Centraalbureau voor Schimmelcultures (CBS) on 31.3 1983 under no.

CBS 261.83.

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

MOROLOGICAL CHARACTERISTICS The Streptomyces lavendofolíae strain 12/3-A forms an abundant purple-pink (X 3) aerial mycelium. The spore carriers are helical, the spores have a smooth surface, to the size 0.7-1.4 Fm. The substrate mycelium is beige-pink, t.o.m. to dirty purple (Kl + M2). The pigment secreted in the plant substrate is dark brown, t.o.m. to brown-pink-violet (Kl + f2 + H2). The colors are valued in accordance with Bondarzev's color scale, the USSR Academy of Sciences' publication of 1954.

The culture is grown at a temperature of 28 ° C for a period of 28 days. 455 705 5 Table 1 Plant substrate Parameter Characteristics 1 2 3 Czapeck's agar _1uftmycel well-developed1, purple-pink GK 3) substrate- beige-pink- mycelium purple (Kl + K2) soluble brown-pink (Kl) pigment Gause I agar aerial mycelium abundant plant, light pink (X3 + D3) substrate mycelium brown-dirty violet (F2 + H2) soluble brown-pink (Kl) pigment Krasiljnikov's _aucial mycelium abundant plant, purple plant substrate 1 pink (X'3) substrate- beige-dirty violet mycelium (Kl + H2) soluble brown-pink-violet pigment (Kl + f2 + H2) Starch agar 'aerial mycelium moderate plant, pink (X 3) substrate beige-pink (Kl) mycelial soluble beige ~ pink (Kl) pigment 455 705 Glucose asparagine Waxman's agar Yeast- and soluble starch agar Glucose yeast agar Oatmeal air mycelium substrate mycelium soluble pigment air mycelium substrate mycelium soluble pigment aerial mycelium substrate myth soluble pigment lnftmycel substrate substrate mycelium soluble pigment aerial mycelium substrate mycelium soluble pigment abundant plant, light gray (X6) sand brown to brown H7-K7) sand brown (J] 7+ K7) abundant vä xt, reddish gray (A 3) from brown-chestnut brown to dirty violet (B2-H2) brown-dark brown (B2) abundant plant, reddish gray (A3) brown (B7) sandy brown to brown (B72K7) abundant plant, light (DI) dark brown (115) brown (K7) good plant, reddish gray (A3) dark brown-brown (B2) light brown (B7) Malt agar Gause 2 agar MPA Tyrosine agar Tresner's agar with iron citrate solution iuftmycel substrate- mycel soluble pigment aerial mycelium substrate- mycel soluble pigment aerial mycelium substrate- mycel soluble pigment aerial mycelium substrate- mycel soluble pigment aerial mycelium substrate- mycel soluble pigment 455 705 abundant plant, reddish gray (A3) brown-chestnut brown (K7 + K4) dark brown-brown (B2 + K7) moderate plant, light gray (A6) brown (X7) brown (K7) .weak plant beige-sand-colored (B 5) lacks abundant plant, purple-pink (X 3) plum black (01) umbra-colored (07) good plant, purple-pink (RIB) dark umbra (H 2) dark umbra (H2) 455 705 8, Potato slices aerial mycelium good growth, from white to pink (03-X3) substrate- dark brown unt mycel (Kl + Bl) soluble dark brown pigment (Kl + Bl) I FYsIoLoG1sKA xåuuzrßcxßu Aerobic, optimal growth temperature 24-30 ° C. At the temperature of 50 ° C no growth. Moderately dissolves gelatin, peptonizes milk, which darkens, moderately hydrolyzes starch. Forms melanoids (Tresner's plant substrate with iron citrate, tyrosine agar).

Streptomyces lavendøfoliae 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 poorly or not at all the following compounds: L-ramnose, sucrose, raffinose and D-mannitol. Streptomyces lavendofoliae strain 12/3-A can be stored on snowy days under a layer of Vaseline oil at a temperature of + 5 ° C for a period of six months.

ANTAGONISTIC CHARACTERISTICS Streptomyces lavendotoliae 12/3-A is grown in a liquid nutrient medium in a shaker. After three days, an amount of approximately 56-60 mg / l aclacinomycin A has been formed which exhibits the following antimicrobial activity, cytotoxic activity and tumor binding effect in test animals (Tables 2-4).

Table 2 455 705 Antimicrobial spectrum of aclacinomycin A produced by Streptomyces lavendofoliae 12/3-A and of the comparative substance X) Test organisms MIC épg / ml Aclacinomycin A Aclacinomycin A produced by Strain produced by lavendofolíae Zaidanhojin B butsu Kagaku Kenkyukaí, Tokyo, Japan 1. 2. 3.

Staphylococcus aureus 209P 15.56 7.8 Staph. aureus 209P streptomycin-resistant 15.56 15.56 penicillin-resistantp 15.56 15.56 gentamycin-resistant 15.56 15.56 kanamycin-resistant 11.6 11.6 oleandomycin-resistant 15.56 11.6 lincomycin-resistant 15.56 23.3 violarine-resistant 23.3 19.0 erythromycin-resistant 31.2 23.2 dactinomycin-resistant 31.2 70.0 novobiocin-resistant> 250.0> 250.0 rifampicin-resistant 125.0 125.0 vancomycin-resistant 125.0 125.0 E. coli 675> 250.0> 250.0 Pseudomonas aerugínosa> 250.0> 250.0 Klebsiella pneumoniae 444> 250.0> 250.0 Providence aartií A-rcc 7240> 2sø, o> 250.0 Candida albicans> 250.0> 250.0 455 7ÛÉ 10 x) MIC, the minimum growth inhibitory drug concentration is determined by culturing in two parallel samples a microbial standard transfer 105 pcs / ml in a test tube series containing peptone -broth- nutrient medium and growing drug concentration, 20 hours at a temperature of 37 ° C and pH 7. 'Table 3 Tumor-preventing effect of aclacinomycin A in animal experiments Strain - Administra- Optimal dose Medium- Minsk ning animal tenion time (mg / kg) ning of of tumor- (day) lifelong- growth- the-%) the density (§) li carl 1 ___; 9 2,5 92 LLc _ carl 1 ___ ¿9 p 67 S-37 _ mice of l --g 5 2,5 80 mixed breed Table 4 Cytostatic effect of aclacinomycin A and its DNA complex on tumor cells P-388 in suspension culture ' / ml X) Preparation ED5u_J P9 Aclacinomycin A 0.01 Aclacinomycin A - DNA complex 0101 x) The dose that reduces the production of nucleic acids (DNA + RNA) by 50%. 455 705 11 The results of the comparison of the characteristics of strain 12/3 according to the invention with the characteristics of the known aclacinomycin A and B producer, Streptomyces galilaeus MA 144M1, confirm the differences between the morphological and physiological characteristics of the respective cultures.

On the other hand, the strain according to the invention corresponds in its mentioned characteristics to the properties of Streptomyces lavendofoliae according to Bergey's definition (Table 5).

Table 5 Comparison of the morphological and physiological properties of a culture of strain 12/3-A according to the invention with the properties of the prototype Str. Galílaeus MA 144Ml and Str. Lavendofoliae.

Parameter Stem \ Str. Galílaeus Str.lavendofo- 12/3-A MA l44Ml liae (prototype) 1 2 '3' 4 Spore carrier spiral shape. spiral shape. spiral shape.

Spores smooth, oval- smooth, oval- smooth, oval- 'shaped shaped Synthetic plant substrates: Aerial mycelium good to rich- moderate plant, good plant, lig growth, b1å- from light gray purple-pink touched (X 3) to gray 455 705 12 Substrate- from beige-yellow-brown, in from yellow-brown mycelium pink certain substrates to dark brown to mute-yellow-green-sight purple. (Jl 7-142) Soluble from dark brown missing or yellow-brown or pigment to dark brown-brown 'dark brown-red-violet (Kl + f2 + H2) Organic substrates: Aerial mycelium good plant, red- moderate plant, good plant, from like gray-light-gray-purple pink to (A 3) violet Substrate- from sand- yellow-brown dark brown or mycelial brown to 'very dark brown-brown (fl7 + B7-K7) Loosely dark brown-brown dark brown or pigment çn7 ~ B2 + K7) very dark brown Potato slices: Aerial mycelium good plant, from good plant, light-good plant, dark-white to gray-brown-pink reddish m3- X2) Substrate- dark brown-brown-yellow dark-brown mycelium (Kl + Bl) Soluble pigment dark brown (Kl + Bl) Melanoids are formed Growth good on cellulose Milk _peptonized to brown Starch - moderate hydrolysis Gelatin- moderate hydrolysis Cane sugar - D-glucose D-xylose +++ L-arabinose L-rhamnose D-fructose D-galactose ++ Refined - D-mannitol - Inositol - 13 chestnut-brown formed moderate peptonized moderately weak l ++++++++ + 455 705 dark brown is formed good I is peptonized to brown moderate moderate + For the taxonomic determination of culture 12/3-A the following literature was used: Krasíljníkov H.A. "The ray fungi", publ. "Nauka", l970, pages 172, 328; Ber- gey's Manual of Determinative Bacteriology, Editors: Buchanan R.E. , Gibbons N.E. et al., pp. 808-810, 455 705 14 As a nutrient substrate, any substrate used by an aclacinomycin-producing strain belonging to the species Str. lavendofoliae can be used.

As a carbon source in the nutrient medium, fructose, glucose, arabinose, xylose, galactose, starch and others are advantageously used.

As a nitrogen source, you can use meat extracts, peptone, corn extract, yeast extract, casein hydrolyzate, corn flour, soy flour, cotton flour and more. , as well as nitrogen compounds containing organic and inorganic compounds, such as ammonium salts (nitrates, sulphates, phosphates, etc.), urea and others.

The plant substrate can be added mineral salts, such as calcium carbonate, sodium and potassium phosphates, sodium and potassium chloride, magnesium salts, copper and others.

For the production of larger amounts of aclacinomycin A * and B, the culture is carried out as an aerated deep culture in two stages.

The inoculation of the fermentor takes place with vegetative mycelium.

The substrate used for growing vegetative mycelium may be the same as that used in the biosynthesis of aclacinomycin A and B, but may also be different.

A check of different substrate alternatives showed that the plant substrates B-2 and B-3 were the most advantageous: Component Å plant substrate B-2 (%) B-3 (%) 1. Ammonium sulphate 0.1 1.5 2. Soybean meal 1.1 1 , 5 3. Potato starch 2.6 _ 3.0 455 705 15 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 plant substrates are sterilized at a temperature of 124-126 ° C for a period of 30- 40 minutes, after which the pH of the substrate is neutral.

Size lavendofoliae, strain 12/3-A, when grown under aerated conditions, gives 1 liter of air / liter of plant substrate / min at a temperature of 20-30 ° C and a pH of about 6-8.5, preferably 6.9-. 7.9, after 48-96 hours aclacinomycin A in an amount of up to 60 mg / l and aclacinomycin B in an amount of 4-10 mg / l.

Table 6 Comparison of the physicochemical properties of acacinomycin A from Str. Galílaeus (antibiotic from the company Zaidanhojin Biseibutsu Kagaku Kenkyukai, Tokyo, Japan) and from Str. Lavendofoliae. Q Antibiotic-producing strain Str.galilaeus MA l44Ml Str.lavendofo1iae l 2 3 X) UV spectrum l 8 / K nm / E / max l cm 90% MeOH 43l, 5 / l46, l / 290 / ll2,1 / 43l, 5 / 148.6 /, 289 / 137.4 / 257 / 285.3 /, 228 / 485.7 /; 257 / 302.9 /, 228 / SO2 /; __ ”__ n: _ _....-._, _..- _. 455 705 16 01N HCl1 290 / 112.1 /, 228 / 485.7 /: 431.5 / 146.1 /, 257 / 285.3 /, 431.5 / 151.4 /, 289 / 137.4 / , 257 / 302.9 / 228 / 499.2 / 7 O, 1N NaOH 521.3 / 118.9 /, 317.3 / 64.5 /, 521.3 / 130 /, 318.3 / 78.5 /, 285 / 101.9 /, 236 / 424.6 xx) IR spectrum γ cm -1 3450, 2980, 2945, 2020, max 2760, 1735, 1675, 1620, 1600, 1010. xxx) PMR spectrum Å ppm 12.55, 1H, s .; 12.0, 1H, s .; 7.15-7.85, 43, m .; 5.58, 1H, s., 5.31, 1H, s .; 4.95-5.20.2H, m .; 4,35-4, ss, ën, m .; 4.11, 1H, s .; 3.68, 3H, S .; 2.25-2.65.5H, m .; 2.20, SH, s .; 1.45-2.15.8H, m .; 1.00-1, 36.12H, m .; 284 / 126.2 /, 236 / 440.3 /. 3480, 2980, 2950, 2825, 2775, 1735, 1680, 1625, 1600, 1010. 12.7, 1H, p .; 12.0, 1H, s ,; 7.83.1H, J1 = 1.8 Hz, J2 = 7.8 Hz; 7.68, 1H, t.

J = 7.8 Hz; 7.68, 1H, s .; 7.30, 1H, J1 = 1.1B Hz, J2 = 7.8Hz S, 52.1H, m .; 5.28.1H m .; 4.95-5.20.2H, m .; 4.40-4.70.2H, m .; 4.11, 1H, s .; 3.70, 3H, p .; 2.3-2.7 SH, m .; 2.23.6H, s .; 1.40-2.15, BH, m .; 1.32, BH, d., J = 6.6 Hz; 1.29 H, d, J = 6.4 Hz; 1,17,3H, d., J = 6.2 Hz; l, 08.3H, t., J = 7 Hz. 455 705 17 xxxx) Mass spectrum w / 2 x) xx) xxx) xxxx) Table 7 376/100% /, 377 / S0% /, 794/15% /, 812/30% /, 114 / 2.6% / , 376/100% /, 377 / 42.8% /, 394 / 5.1 8 /, 416 / 1.5% /, 794 / 10.2% /, 812 / 28.8% /.

The UV spectrum was recorded with a Pye Unícam SP-8-100 device calibrated with the standard filters HO-720540 and Di-720538 Pye Unicam.

The IR spectrum was recorded with a UR-20 device from a KB: tablet.

The PMR spectrum was recorded with a 90 MHz "User-WH-90" device in CDCI3, TMS reference.

The mass spectra of the field desorption were recorded with a Varian Mat 311-A apparatus, calibrated with PPK. The current in the emitter is 5-20 mA.

Comparison of the physicochemical properties of aclacinomycin B from Str. Galílaeus and from Str. Lavendofoliae Antibiotic-producing strain strigalílaeus MA 144M1 Str. Lavendofoliae 2 3 455 705 18 1. 2. 3.

UV spectrum 1% Å nm / s / max 1 cm 90% MeOH 432/159 /, 289.5 / 137 /, 431.5 / 149.2 /, 290/119 /, I 259/313 /, 229, 5/498 /; 257/296 /, 228/508 /; 0.1N HCl 431/162 /, 289.5 / 156 /, 431.5 / 149 /, 290/119 /, 259/355 /, 229/586 /; 257/296 /, 228/503; 0.1N NaOH 522/145 /, 315/101 /, 521.3 / 127 /, 316.3 / 77.3 /, 284/160 /, 239/510 /. 285/119 /, 236/453 /.

IR spectrum 3450, 2980, 2945, 2820 3490, 2980, 2950, 2830, 17 cm -1 2760, 1730, 1675, 1620, 2780, 1735, 1680, 1625, max 1600, 1010, 1605, 1015.

PMR spectrum 12.2, 1H, s .; J ppm 11.1, 5.1H, s .; 7.2-7.9, 4B, 5.5, 1H, s .: 5.1-5.3, 3H, 4.6-4.9, 2H, 4.5, 1H, s .; 4.3-4.4.1 fl, 4.15, 1H, s, 3.8-4.0, 3.7, 3H, 2.2-2.7, 2.2, 6H, 1.4 -2.0, 0.9-1.4, 23, p .; 4H, s .: BH, III-F 12 H, m .; 12.68, 1H, s .: 12.02, 1H, s .; 7.24-7.89, 4H, 5.48, 1H, 5 .; 5.09-5.27, 3H, 4.62-4.90, 2H, 4.53, 18, p .; 4.36, 1H, q: 4.11, 1H, p .; 3.90-4.03, 2H, 3.75, 1H, s ,; 3.69, 3H, p .; 2.58, 2H, d .; 2.46, 1H, d .; 2.35, 1H, s .; 2.15, 6H, p .; 1.45 - 2.01, 8H, m .; 0.89-1.45, 12H, m .. 10.7 455 705 The UV, IR and PMR spectra of aclacinomycin A and B are shown in Figures 1-6 (FIGS. 1-6).

Fig. 1 shows the UV spectrum of aclacinomycin A hydrochloride in 90% methanol, Fig. 2 shows the UV spectrum of aclacinomycin B hydrochloride in 90% methanol, Fig. 3 shows the IR spectrum of aclacinomycin A hydrochloride from a KBr tablet, Fig. 4 shows IR spectrum of aclacinomycin B hydrochloride from a Ksrft tablet, FIG. 5 shows the PMR spectrum of aclacinomycin A hyarokioria (cnci3-rms; -r = 30 ° C) and mc; s mn spectrum of aclacinomycin B hydrochloride (CDCl 3 -TMS; T = 30 ° C).

Aclacinomycins A and B are found in the mycelium and in the plant substrate. After fermentation, the plant substrate is filtered at pH 4.5-6.0. The antibiotics are extracted from the plant substrate with ethyl acetate (per 10 parts by volume of plant substrate, 1 part by volume of ethyl acetate is used).

The antibiotic-containing organic layer is separated from the mycelium. After removal of the solvents, the aqueous residues are combined and neutralized to pH 6.9. The antibiotics are then extracted with toluene and with twice the volume of 0,01N HCl solution. The antibiotics are extracted from the aqueous solution with chloroform, which is dried over anhydrous Na 2 SO 4. The chloroform extract is concentrated in a rotary evaporator to a 0.1-fold volume and the residue is fed with 10-12 parts by volume of dry hexane. The precipitated antibiotics are separated and dried. Purification and separation of the antibiotics takes place chromatographically with aqueous silicic acid in a solvent system consisting of carbon tetrachloride and isopropanol.

Tables 6 and 7 show the comparative results for the aclacinomycins A and B, which were obtained by UV, IR, PMR-455 705 zo and mass spectroscopy, and which confirm the similarity between the antibiotics obtained with the strain according to the invention and those obtained with the already known strains.

The antibiotics aclacinomycin A and B, which are produced by the strain 12/3-A of Str. Lavendofoliae, are thus identical with the antibiotics obtained by culturing Str. Galilaeus MA l44Ml. The ability to produce aclacinomycin A and B was not previously known in Str. Lavendofoliae. The process according to the invention can be illustrated by the following examples.

Example 1 Streptomyces lavendofoliae strain 12/3-A is grown on a solid oat-agar plant substrate at a temperature of 28 ° C for 12 days until an abundant aerial mycelium has developed. The culture is transferred to a 750 ml Erlenmeyer flask in a B-2 plant substrate, which does not contain copper sulphate or defoamers. The flask is agitated by shaking at 250 rpm at a temperature of 28 ° C for a period of 2 days. The culture grown in the Erlenmeyer flask is transferred in an amount of 5% in a 10 liter inoculum fermenter in a B-2 plant substrate. which does not contain copper sulphate. The fermenter is stirred at a speed of 200 rpm and aerated at 10 liters / min at a temperature of 28 ° C for 24 hours. Three liters of culture grown in this fermentor are transferred to a 100 liter fermenter in 60 liters of B-2 plant substrate. The fermenter is stirred at a speed of 200 rpm and aerated at 60 l / min at a temperature of 27 ° C for three days. The levels of aclacinomycin A and B in the plant substrate are determined by the following method. Determination of the levels of aclacinomycin A and B in mycelium I After fermentation, the mycelium is separated from 5 ml of plant substrate by centrifugation at 2000 rpm for a period of 10 minutes. From the mycelium thus obtained, the antibiotics are extracted for 2 hours with 4 ml of acetone, the mixture being stirred at 15-20 minute intervals.

The acetone is evaporated from 1 ml of 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 chromatographic separation of the antibiotic complex. For this purpose, a disc is used, which is coated with an adsorbent layer, and whose left side and lower edge have been left free for a distance of 2 cm. On the disc, apply with a capillary the whole chloroform extract as a band with a width of 1-1.5 cm. A standard solution of acacinomycin A and B is applied to the same disk. The disk is placed in a chromatography chamber which contains a chloroform-ethyl acetate-methanol mixture in the ratio 7: 2: 1. When the liquid front has reached the edge of the disc, remove the disc and air dry for 20 minutes.

The yellow zones, which correspond to the mobility of aclacinomycins A and B, are eluted separately with 4 ml of methanol (pH = 6) and measured spectrophotometrically at a wavelength of 430 nm. The amounts of aclacinomycin (mg / l culture fluid) are calculated on the basis of the extinction values of the aclacinomycins (fi lglcm for aclacinomycins A and B are 161 and 159, a. Antizšiotics, 1979 §_2_, no s, p. Val-son) according to the following formula 455 705 22 aclacinomycin AC mg / ml = 199 D aclacinomycin BC mg / ml = 201 D 2. Determination of the content of aclacinomycin A and B in the plant substrate I From 5 ml of plant substrate the mycelium is separated by centrifugation. Extract with ethyl acetate at pH 6 using the ratio of 5 ml of plant substrate and 5 ml of ethyl acetate. 2 ml of ethyl acetate extract are evaporated in vacuo and the residue is extracted with 2 ml of chloroform. The reprocessing takes place as in point 1.

The amounts of aclacinomycin A and B are calculated on the basis of the extinction values of the antibiotics according to the following formula: aclacinomycin AC mg / ml = 124.2 D aclacinomycin BC mg / ml = 125.8 D After three days of fermentation in the plant substrate B-2, the culture produces 12 / 3- A aclacinomycin A in an amount of about 56 mg / l and aclacinomycin B in an amount of about 4-8 mg / l. .

To separate aclacinomycin A and B from the plant substrate, 60 l are filtered on a suction filter at pH 4.5-5.0.

The antibiotics in the plant substrate are extracted with ethyl acetate in a ratio of 10: 1. The organic layer is separated and evaporated in vacuo in a rotary evaporator at a temperature of not more than 40 ° C until the residue is an aqueous solution. The moist mycelium (5 kg) is extracted twice with 10 l of 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 a 10 -8 NaOH solution and the antibiotics are extracted with 3 x 10 l toluene. The toluene extracts are combined and the aqueous layer is discarded. The toluene extract (30 l) is evaporated in vacuo at a temperature not exceeding 40 ° C until the residue is 3 liters. The antibiotics are extracted from this concentrate with a 0.5-fold volume of 0.01N HCl. The emulsion formed in the extraction stage is separated by centrifugation.

The antibiotics in the aqueous solution are extracted with a 0.5-fold volume of chloroform. The extraction time is 20-30 minutes while stirring the mixture continuously.

The resulting emulsion is decomposed by centrifugation. The chloroform layer (1.5 L) is recovered and dried for 4 hours over anhydrous Na 2 SO 4 (10 g Na 2 SO 4/100 ml extract). Na 2 SO 4 is filtered off and the chloroform is distilled in vacuo in a rotary evaporator to a 0.1-fold volume. The residue is fed with 10-12 parts by volume of dry hexane, forming a precipitate which is allowed to settle (30 minutes). The precipitated antibiotics are filtered on glass filter no. 4 and dried. The filtrate is evaporated to dryness in a rotary evaporator at a temperature not exceeding 40 ° C. The residue is slurried in hexane and the hexane is removed by filtration. Both precipitates are combined and purified by chromatography. The yield is 2.5 g of crude product.

Chromatographic separation of the aclacinomycins A column (8 x 95 cm) is filled with 5.5 l of suspension consisting of 1.8 kg of aqueous silicic acid (250-400 μm) and 5.5 l of chloroform in which 0.5% of triethylamine and acetic acid are dissolved . After filling, the column is rinsed with 3 455 705 24 l of chloroform. The above crude product (10 g) is added, dissolved in 100 ml of chloroform. After absorption of the antibiotics, the column is eluted. For separation of the antibiotics, a gradual elution is performed. For this purpose, column 1) 4 l of carbon tetrachloride 2) 8 l of solvent system, consisting of carbon tetrachloride and isopropanol (25: 1) _ 3) 8.5 l of solvent system, consisting of carbon tetrachloride and isopropanol (20: 1 ) 4) 20.5 l solvent system, consisting of carbon tetrachloride and isopropanol (10: 1).

Aclacinomycin B is obtained from the column using the CCl4: isopropanol (20: 1) system and aclacinomycin A with the CCl4: isopropanol (10: 1) system. The eluates (5-7 L), which contain aclacinomycin Å and B, are extracted separately with 0.2 volumes of 0.01 N HCl solution.

The antibiotics are extracted from the aqueous phase with 0.2 volumes of chloroform. The chloroform extracts of the aclacinomycins A and B are dried separately with anhydrous Na 2 SO 4 (2 g / 100 ml solution), the sodium sulphate is filtered and the filtrate is evaporated in vacuo at 40 ° C to a 0.1-fold volume. A 10-fold volume of dry hexane is added and the mixture is allowed to stand for about 30 minutes.

The precipitate is filtered with glass filter no. 4 and the resulting substance is air-dried. Thus, from 2.5 g of crude product, 0.7 g of aclacinomycin A and 0.1 g of aclacinomycin B are obtained.

Example gel 2 Streptomyces lavendofolíae strain 12/3-A is grown for 12 days on oatmeal. The culture is transferred to a vessel which contains B-2 without copper sulphate as plant substrate. The vessel is shaken at a speed of 250 rpm at a temperature of 28 ° C for a period of two days. The culture is transferred to an Erlenmeyer bottle. The transferred part typically corresponds to 5-10% of the volume of the plant substrate. B-3 is used as the plant substrate, which consists of the following substances: soy flour - 1.5 8, potato starch - 3%, calcium carbonate - 1.4 8, sodium chloride - 0.3%, copper sulphate 0.007 S. The culture conditions are the same as in Example 1 and the culture time is 72 hours. The activity of the plant substrate is 60 mg / l aclacinomycin A and 10 mg / l aclacinomycin B.

The chemical separation of the aclacinomycins A and B takes place in the same manner as in Example 1.

As can be seen from the examples, the process for the preparation of aclacinomycin A and B has the following advantages over the known processes: 1. Higher yield of aclacinomycin A. The prototype strain produces 46 pg / ml and the strain according to the invention up to 60 pg / ml. - 2. Sínerubín A and B are not formed, so further cleaning becomes superfluous. In the known processes, said by-products must be removed as metal ion complexes. 3. A small amount of aclacinomycin B is formed. The prototype strain produces 23 pg / ml and the strain according to the invention 4-8 pg / ml.

Claims (2)

455 755 .ik PATENTKRAV A process for the preparation of the antibiotic compounds aclacinomycin A and B having anticancer activity or mixtures thereof, wherein said aclacinomycin has the formula: wherein R for aclacinomycin A has the formula: CH3 m3 N (cH3) ¿0 H that Streptomyces lavendofoliae strain 12/3-A with the accession number CBS 26183 is grown under aerobic conditions in a nutrient medium containing carbon and nitrogen-containing compounds and mineral salts, at a temperature of about 20-30 ° C and at a pH of about 6 - 8.5, preferably 6.9 - 7.9, for 48-96 hours, after which the formed aclacinomycins A and B are recovered, and the aclacinomycins A and B are separated and purified by methods known per se, 2. Streptomyces layendofoíiae strain 12/3-A with the deposit number CBS 26183.