NO157825B - PROCEDURE FOR THE PREPARATION OF ANTIBIOTIC AKLACINOMYCIN A AND B WITH ANTITUMUM EFFECTS. - Google Patents

Description

The present invention relates to a method for producing

ling of the antibiotic aclacinomycin A and B with antitumor effect,

and it further relates to the preparation of mixtures thereof.

Aclacinomycin has the structural formula:

in aclacinomycin A, R is:

in aclacinomycin B, R is:

The above-mentioned method is characterized by cultivating the Streptomyces lavendofolia strain 12/3-A, deposited at the "Central Bureau voor Schimmelcultures (CBS) on 31 March 1983 under accession no. CBS 261.83, under aerobic conditions,

in a nutrient medium containing carbon- and nitrogen-containing compounds as well as mineral salts at a temperature of approx. 20-30°C and at a pH value of approx. 6-8.5, preferably 6.9-7.9, for 4-8 hours, after which the formed aclacinomycins A and B are stored, the aclacinomycins A and B are separated and purified by methods known per se.

The aclacinomycins A and B belong to the anthracycline antibiotic group and are active against various gram-positive bacteria and prevent tumor growth in animals. The aclacinomycins A and B and compounds for their preparation were first described in JP application 864851. Corresponding applications led to GB-PS 1,491,266 and US-PS 3,988,315 and 4,071,411.

In the above-mentioned patent, Streptomyces galilaeus MA 144 M1, which is deposited in the American collection "American Type Culture Collection" under the designation ATCC No. 31133, and in the Japanese institute "Fermentation Research Institute" was used as aklacinomycin-producing strain " under the designation FERM no. 2455.

The strain has the following characteristics: weakly developed aerial mycelium on a solid agar growth substrate, to colored grey. Forms spiral-shaped spore carriers, the spores have a smooth surface, are oval shaped and size 0.4 - 0.8 ym. The colonies are branched and the substrate mycelium yellowish brown. Soluble pigment is missing or is light brown. On certain agar growth substrates, a yellow-green soluble pigment is formed.

Streptomyces galilaeus is grown aerobically, the growth optimum is at 24-30°C, at 50°C no growth occurs. It hydrolyzes gelatin, peptonises milk and hydrolyses weak starch. Melanoid pigment is formed on organic substrates. It grows

on Pridheim and Gottlieb's medium containing D-glucose, D-xylose, L-arabinose, L-rhamnose, D-galactose or inositol, but does not grow on a medium containing D-mannitol.

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

These antibiotics are found both in the mycelium and in 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 obtained aclacinomyeins A and B contain as contaminants antibiotics from the E-pyrromycin group, and these can be removed by treatment with copper ions.

A shortcoming of the above-mentioned known method is the microorganism's weak production capacity. Next to the acla-cinomycin, certain antibiotics from the E-pyrromycin group are formed, which complicates the chemical purification.

The main purpose of the present invention has therefore been

to find new, aklacinomycin A- and B-producing cultures that

show a greater aclacinomycin A-producing activity compared to the previously known ones, and as main components these synthesize antibiotics.

As a result of these tests, it is from a soil sample from

Central Asia obtained a new strain 12/3-A, which belongs to the species Streptomyces lavendofoliae, and forms aklacinomycin A as its main component.

This Streptomyces lavendofoliae strain is deposited in the antibiotic institute's strain collection under the designation

VNIIA No. 1670 on 20.1.1982. This institute's (VNIIA) microorganism strain collection is registered in the International Federation of Strain Collections in 1972, No. 337.

Strain 12/3-A of Streptomyces lavendofoliae has also been deposited in the deposit institute Centraalbureau voor Schimmelcultures (CBS) on 31.3.193 under no. CBS 261.83.

The Streptomyces lavendofoliae strain 12/3-A exhibits the following morphological, physiological and antaphonistic characteristics:

MORPHOLISTIC CHARACTERISTICS:

Strepromyces lavendofoliae strain 12/3-A forms an abundant purple-pink ()K3) aerial mycelium. The spore carriers are spiral-shaped,

the tracks have a smooth surface of the order of 0.7-1.4 ym. The substar mycelium is beige-pink, even to dirty purple (K1+M2). The pigment secreted in the growth substrate is dark-brown to brown-pink-violet (K1+T2+H2). The colors are assessed according to Bondarzev's color scale, of the USSR Academy of Sciences

publication from the year 1954.

D

5

0

5

PHYSIOLOGICAL CHARACTERISTICS :

Aerobic, optimum growth temperature 24-30°C. At a temperature of 50°C no growth. Moderately dissolves gelatin, peptonizes darkening milk, moderately hydrolyzes starch. Forms melanoids (Tresner's growth substrate with ferric citrate, tyrosine agar).

The Streptomyces lavendofoliae strains 12/3-A make good use of the following carbon sources: D-glucose, D-xylose, L-arabinose, D-fructose, D-galactose, inositol. It utilizes poorly or not at all the following compounds: L-rhamnose, sucrose, raffinose and D-mannitol. The Streptomyces lavendofoliae strain 12/3-A can be stored under a layer of petroleum jelly at a temperature of +5°C for 6 months.

ANTAGONIST CHARACTERISTICS:

Streptomyces lavendofoliae 12/3-A is grown in a liquid nutrient substrate in a shaking device. After three days, a quantity of approx. 56-60 mg/l aklacinomycin A which exhibits the following antimicrobial activity, cytostatic activity and anti-tumor activity in test animals

(tables 2-4).

The results of the comparison of the different characteristics of the strain 12/3 according to the invention and the corresponding characteristics of the known aclacinomycin A and B producer, Streptomyces galilaeus MA 144M1, confirm the difference between the morphological and physiological characteristics

in the respective cultures.

On the other hand, the different characteristics of the strain according to the invention correspond to the corresponding characteristics of Streptomyces lavendofoliae according to Bergey's definition (table 5).

For the taxonomic determination of the culture 12/3-A was

the following literature was used: H.A. Krasiljnikov "Radiation Mushrooms", "Nauka", 1970, pages 172, 328; Bergey's Manual of Determinative Bacteriology, Publishers: R.E. Buchanan, N.E. Gibbons et al., pages 808-810.

As nutrient substrate, any substrate that is utilized by a species Str. lavendofoliae associated, aklacinomycin-producing strain.

Fructose, glucose, arabinose, xylose, galactose, starch etc. were advantageously used as a carbon source in the nutrient substrate.

As a hydrogen source, you can use meat extract, peptone, corn extract, yeast extract, casein hydrolyzate, corn flour, soya flour, cotton flour etc., and also nitrogen compounds containing organic and inorganic compounds, such as ammonium salts (nitrates, sulphates, phosphates etc.),

urea and others.

Mineral salts can be added to the growth substrate, such as calcium carbonate, sodium and potassium phosphates, sodium and

potassium chloride, magnesium salts, copper etc.

For the production of larger quantities of aklacinomycin A and B, the cultivation is carried out as an aerated reptile bed in two stages. The fermentation tower is grafted with vegetative mycelium. The substrate used for the cultivation of 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 shows that the growth substrate B-2 and B-3 were the most beneficial:

The growth substrate was sterilized at a temperature of 124-126°C during 30-40 minutes, after which the substrate's pH value was neutral.

Str. lavendofoliae, strain 12/3-A, when cultivated under aerated conditions with 1 liter of air/liter of growth substrate/min. at a temperature of 20-30°C and a pH value of approx. 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.

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 KÉr tablet, Fig 4

shows the IR spectrum of aclacinomycin B hydrochloride from a KBr tablet, Fig. 5 shows the PMR spectrum of aclacinomycin A hydrochloride (CDC13"TMS; T=303°C) and Fig. 6 shows the PMR spectrum of aclacinomycin B hydrochloride (CDCl ^TMS;

T = 303°C).

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

The antibiotic-containing organic layer is separated from the mycelium. After removing the solvents, the remaining water is combined and neutralized to pH 6.9. After this, the different antibiotics are extracted with toluene and. with the double volume of 0.01N HCl solution. The different antibiotics are extracted from the water solution with chloroform,

which is then dried with anhydrous Na2S0^. The chloroform extract is concentrated in a rotary evaporator to 0.1 of the

flowable volume and 10-12 parts by volume of dry hexane are added. The precipitated antibiotics are separated and dried. Purification and separation of these takes place chromatographically with aqueous <S>i02 in a solvent system consisting of carbon tetrachloride and isopropanol.

Tables 6 and 7 show comparison results for the aclamycins A and B, obtained with UV, IR, PMR and mass spectroscopy, and which confirm the similarity between the different antibiotics obtained using the strain according to the present invention and those obtained with the already known tribes.

The indicated antibiotics aklacinomycin A and B, which are produced by the strain 12/3-A of Str. lavendofoliae, are thus identical to the antibiotics obtained by cultivating Str. Galilean MA 144M1. The ability to produce aklacinomycin A and B was previously unknown in Str. lavendofoliae. The method according to the invention shall be illustrated by the following non-limiting examples.

Example 1

Streptomyces lavendofoliae strain 12/3-A is grown on a

solid oat-agar growth culture at a temperature of 28°C for 12 days, up to a rich aerial mycelium had developed.

The culture was transferred to a 750 ml Erlenmeyer flask in a B-2 growth medium, which did not contain copper sulfate or antifoam. The flask was agitated by shaking at a rate of 250 rpm. at a temperature of 28°C for a period of 2 days. The culture grown in the Erlenmeyer flask was transferred in an amount of 5% in a 10 liter inoculum fermenter in a B-2 growth substrate that did not contain copper sulfate. The fermenter was stirred at a rotational speed of 200 rpm and was aerated at 10 liters/min. at a temperature of 28°C during the day.

Three liters of the culture grown in this fermenter was transferred to a 100 liter fermenter in 60 liters of B-2 growth medium. The fermenter was stirred at a speed of 200 rpm and aerated at 60 l/min. at a temperature of 270C for three days.

The content of aklacinomycin A and B in the growth substrate is determined by the following method. 1. Determination of the content of aklacinomycin A and B in the mycelium.

After fermentation, the mycelium is separated from 5 ml of growth substrate by centrifugation at 2000 rpm for 10 minutes. From the thus obtained mycelium, the relevant antibiotics are extracted over the course of 2 hours with 4 ml of acetone, whereby the mixture is stirred at 15-20 minute intervals. The acetone is evaporated from a 1 ml extract in vacuum 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 disk is used which is coated with an adsorbent layer, and whose left side and lower edge are left free for a stretch of 2 cm. The entire chloroform extract is applied to the disc with a capillary tube as a band with a width of 1-1.5 cm. A standard solution of aklacinomycin A and B is applied to the same disc. The disc is placed in a chromatography chamber containing a chloroform-ethyl acetate-methanol mixture in a ratio of 7:2:1. When the liquid front had reached the edge of the slice, the slice was taken out and dried in air for 20 minutes.

The yellow zones corresponding to the mobility of the 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 in mg/l culture fluid were calculated on the basis of the extinction values of the aclacinomycins (E„ 11c%ra;.. for aclacinomycin A and B are 161 and 159, "J. Antibiotics',' 1979 32, no. 8 , pp. 781-800) according to the following formula: aklacinomycin A C mg/ml = 199 D aklacinomycin B C mg/ml = 201 D 2. Determination of the content of aklacinomycin A and B in the growth substrate.

The mycelium is separated from 5 ml of growth substrate by centrifugation. One extracts with ethyl acetate at pH 6 by using the ratio 5 ml of growth 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. Processing takes place as in point 1.

The amounts of aklacinomycin A and B are calculated on the basis of the extinction values of the different antibiotics according to the following formula: aklacinomycin A C mg/ml = 124.2 D aklacinomycin B C mg/ml = 125.8 D

After three days of fermentation in the growth substrate B-2, the culture 12/3-A gives aklacinomycin A in an amount of approx. 56 mg/l and aclacinomycin B in an amount of approx. 4-8 mg/l.

To separate aklacinomycin A and B from the growth substrate, 60 liters are filtered on a suction filter with a pH value of 4.5-5.0. The various antibiotics in the growth substrate are extracted with ethyl acetate in a ratio of 10:1. The organic layer is separated and evaporated under vacuum in a rotary evaporator at a temperature of no more than 40°C, until the remainder consists of a water solution. The moist mycelium in an amount of 5 kg is extracted twice with 10 1 of acetone. The mycelium is separated by filtration and processed. The acetone extracts are combined and evaporated. The water residues from the ethyl acetate and acetone evaporation are combined and the thus obtained concentrate in a quantity of 5 liters is neutralized to a pH value of 6.8 with a 10% NaOH solution and the various antibiotics are extracted with 3 x 10 liters of toluene . The toluene extracts are combined and the aqueous layer is discarded. The toluene extracts in a quantity of 30 liters are evaporated in a vacuum at a temperature of no more than 40°C until the remainder is 3 litres. The various antibiotics are extracted from this concentrate with a 0.5-fold volume of 0.01N HCl. The emulsion formed in the extraction step is finely divided by centrifugation. The antibiotic substances that are in water solution are extracted with a 0.5-fold volume of chloroform. The extraction time is 20-30 minutes with continuous stirring of the mixture.

The resulting emulsion is separated by centrifugation. The chloroform layer in a quantity of 1.5 liters is preserved and dried 4 times over anhydrous Na^O^ (10 g Na2S0^/100 ml extract). Na2S0^ is filtered off and the chloroform is distilled in vacuum in a rotary evaporator to a volume of 0.1. The residue is added to 10-12 parts by volume of dry hexane, whereby a precipitate is formed, which is allowed to precipitate within 30 minutes. The precipitated antibiotics are filtered on a glass filter No. 4 and dried. The filtrate is evaporated to dryness in a rotary evaporator at a temperature of no more than 40°C. The residue is slurried in hexane and the hexane is removed by filtration.

The two precipitates are combined and purified chromatographically. The yield is 2.5 g of crude product.

Chromatographic separation of the aclacinomycins.

A column with dimensions 8 cm x 95 cm was filled with 5.5 l of suspension, which consisted of 1.8 kg of hydrous silicic acid (250-400 ym) and 5.5 liters of chloroform in which was dissolved

5.8 liters of triethylamine and acetic acid. After filling, the column was rinsed with 3 liters of chloroform. The above-mentioned crude product in an amount of 10 g was added, dissolved in 100 ml of chloroform. After absorption of the various antibiotics

the column was eluted. To separate the different antibiotics, the column was eluted. A stepwise gradient elution was performed to separate the different antibiotics. For this purpose, the following were added to the column: 1) 4 1 of carbon tetrachloride; 2) 8 1 solvent system which consisted of carbon tetrachloride : isopropanol in the ratio 25:1; 3) 8.5 1 solvent system, which consisted of carbon tetrachloride:; isopropanol in a ratio of 20:1; 4) 20.5 1 solvent system which consisted of carbon tetrachloride: isopropanol in the ratio 10:1.

Aclacinomycin B is obtained from the column with the system CCl^:isopropanol in the ratio 20:1 and aclacinomycin A with the system CC14:isopropanol in the ratio 10:1. Eluates 5-7 1 containing aclacinomycin A and B were extracted separately with 0.2 volumes of 0.01 N HCl solution.

The indicated antibiotics are extracted from the anhydrous phase with

0.2 volumes of chloroform. The chloroform extract of the aclacinomycins A and B is dried separately with anhydrous Na2S0^

(2,0100 ml of solution), sodium sulfate is filtered and the filtrate is evaporated in vacuo at 40°C to a 0.1! random volume quantity. A 10 times greater volume of dry hexane is added and the mixture is allowed to stand for approx. 30 minutes.

The precipitate is filtered with glass filter No. 4 and the obtained sample is dried in air. Thus, 0.7 g of aclacinomycin A and 0.1 g of aclacinomycin B are obtained from 2.5 g of raw product.

Example 2

Streptomyces lavendofoliae strain 12/3-A is cultivated for 12 days

on oat agar. The culture is transferred to a container, which as growth substrate contains B-2 without copper sulphate.

The vessel is shaken at a speed of 250 rpm at a temperature

of 28°C within two days. The culture is transferred to an Erlenmeyer flask. This transferred part corresponds characteristically

5-10% of the growth substrate volume. B-3 is used as a growth substrate, which consists of the following substances: soy flour - 1.5%, potato starch - 3%, calcium carbonate - 1.4%, sodium chloride - 0.3%, copper sulphate 0.007%. The cultivation conditions are the same as in example 1 and the cultivation time 72 hours. The activity of the growth 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 way as in Example 1.

As an example shows the method of manufacture

of aklacinomycin A and B have the following advantages compared to the known compounds: 1. A higher yield of aklacinomycin A. The prototype strain gives f and the strain according to the invention up to

60 y<g>/rol.

2. Sinerubin A and B are not formed, which is why further purification becomes redundant. In the known methods, the known by-products must be removed as metal ion complexes. 3. A smaller amount of aklacinomycin B is formed. The prototype strain gives 23 ug/ml and the strain according to the invention 4-8 ug/ml.

Claims (1)

1. Process for the production of antibiotic aclacinomycin A and B with antitumor activity, or mixtures thereof, this aclacinomycin having the formula: where R in aclacinomycin A is: and where R in aclacinomycin B is: characterized by cultivating the Streptomyces lavendofolia strain 12/3-A, deposited at the "Central Bureau voor Schimmelcultures (CBS) on 31 March 1983 under accession no. CBS 261.83, under aerobic conditions, in a nutrient medium containing carbon and nitrogen-containing compounds as well as mineral salts at a temperature of 20-30°C and at a pH value of 6-8.5, preferably 6.9-7.9 for 48 hours, after which the formed aclacinomycins A and B are taken care of, the aclacinomycins A and B are separated and purified by methods known per se.