NO130362B - - Google Patents

Description

Process for the preparation of antibiotic B-5050 or its components B-5050-A, -B, -C, -D, -E or -F or mixtures of two or more of these.

The present invention relates to a method for producing antibiotic B-5050 or its components B-5050-A,

-B-, -C, -D, -E or -F or mixtures of two or more of these, and with the formula: where

The new antibiotic substances are produced by a microorganism belonging to the genus Streptomyces, and these substances accumulate in the culture medium. It has been found that the respective components of the new antibiotics are very closely related to each other both with regard to properties and chemical structure, but that they can be extracted from the culture medium in the desired degree of purity, and that they can be separated into individual components or in mixtures of two or more.

According to the present invention, the microorganism Streptomyces hygroscopicus (IFO 12995) is thus grown in a culture medium containing assimilable carbon and nitrogen sources under aerobic conditions, and then the accumulated antibiotic B-5050 is recovered, and, if desired, this is separated into antibiotic B-5050- A, -B,

-C, -D, -E and -F or a mixture of two or more of these components. Hereinafter, each of the active ingredients B-5050-A, -B, -C, -D, -E and -F or a mixture of two or more of these is collectively referred to as "antibiotic B-5050" unless otherwise indicated.

The microorganism used was isolated from a soil sample from Chichibu, Saitama, Japan. The microbiological characteristics of this organism are shown below. In the following description, the color names are denoted "Rdg." based on Ridgway's "Color Standards and Nomenclature", and observations regarding cultivation are at 28°C

for 14 days if nothing else is stated. The abbreviations "G", "A", "R" and "SP" mean "growth", "aerial mycelium", "backside" and "soluble pigment", respectively.

Morphological characteristics.

Spore-bearing hyphae have a width of 0.7_1.^ micron and are bent or spirally twisted. Each spore is elliptical to cylindrical, and the size is approx. 0.7 to 1.7 microns by 0.9 to 1.4 microns, and the grooves have smooth surfaces. Cultivation was carried out on glucose-asparagine agar." ■

Cultural characteristic ika.

( 1) Czapek's agar:

G.: Good, colorless to pale yellowish brown.

A.: Good, velvety, white to pale olive gray (Rdg. LI, 23""-f) or light olive gray (Rdg. LI, 23""-d), with black spots.

R.: Smoky gray (Rdg. XLVI, 21""-d).

SP.: Nothing

( 2) Glucose Czapek's agar:

G.: Moderate, colorless.

A.: Moderate, white to pale gray (Rdg. XLVI, 17""-f). R.: Colorless to pale gray (Rdg. XLVI, 17""-f).

SP.: Pale burgundy to brown (Rdg. XL, 13"'-f).

( 3) Glycerin Czapek's agar:

G.: Moderate, colorless.

A.: Moderate, powdery, white to pale gray

(Rdg. XLVI, 17""-f).

R.: Cream yellow (Rdg. XVI, 19'-f).

SP.: Nothing

( 4) Glucose asparagine agar:

G.: Moderate, colorless to pale yellow.

A.: Moderate, white to grey-brown with black spots. R.: Creamy yellow (Rdg. XVI, 19'-f) to smoky gray (Rdg. XLVI, 21""-d).

SP.: None or pale yellow-brown.

(5) Nutrient agar:

G.: Moderate, spreading, colorless to pale yellow.

A.: Moderate, white.

R.: Cream yellow (Rdg. XVI, 19<*->f).

SP.: Faint yellow.

( 6) Nutrient medium:

G.: Poor to moderate, colorless, sank to the bottom. A.: Nothing.

SP.: Nothing.

( 7) Glycerin nutrient agar:

G.: Good, wrinkled, colorless to pale yellow.

A.: Good, white.

R.: Yellow-brown (Rdg. XXX, 19"-f).

SP.: Yellow to golden yellow.

( 8) Glucose nutrient agar:

G.: Good, wrinkled, colorless to slightly yellow. A.: Good, white to mouse gray (Rdg. LI, 15""'-f). R.: Sorghum-brown (Rdg. XXXIX, 19"'-i).

SP.: Light brown (Rdg. XL, 13"').

( 9) Starch agar:

G.: Bad, colorless.

A.: Nothing or bad, white.

R.: Pale yellow.

SP.: Nothing.

(10) Yeast extract agar:

G.: Good, wrinkled, colorless to pale yellow. A.: Good, white.

R.: Light yellowish brown (Rdg. XXX, 19"-d).

SP.: Yellow to golden.

( 11) Eggs: ( grown at 37°C).

G.: Moderate, spreading.

A.: Moderate, powdery, white.

SP.: Nothing.

( 12) Potato plug:

G.: Good, wrinkled, raised, pale brown. A.: Poor, white to mouse gray (Rdg. LI, 15'""-f).

SP.: Nothing.

( 13) Guierot plug:

G.: Good, wrinkled.

A.: Moderate, white.

SP.: Nothing.

( 14) LaKmus-mel k: ( grown at 37°C).

G.: Formed ring, later sinks to the bottom. A.: Nothing.

Peptonization without coagulation.

(15) Loffler's serum: (cultivated at 37°C).

G.: Moderate, glistening, colorless.

A.-. Nothing or bad, white.

SP.: Nothing.

No liquid formation.

(16) Gelatin: (cultured at 24°C for 25 days).

G.: Poor, limited, pale yellow.

A.: Bad, white.

No liquid formation.

(1 7) Cellulose:

No growth.

(18) Kalsi ummaltagar:

G.: Moderate, colorless.

A.: Moderate, powdery, white.

R.: Light brown to pink (Rdg. XXVIII, ll"-d).

SP.: Wine red to cinnamon brown (Rdg. XXIX, 13"-b).

(1 9) Tyrosinegar:

G.: Moderate, colorless.

A.: Poor, white to light gray (Rdg. XLVI, 17""-b).

R.: Light gray (.Rdg. XLVI, 17""-d) to brownish gray

(Rdg. XLVI, 17""-b).

SP.: Nothing.

(2 0) Pepto Agar:

G.: Bad, colorless.

A. :• Bad, white.

R.: Fargelds.

SP.: Nothing.

(21) Nitrate reduction:

Positive on Czapek's medium and on peptone medium.

(2 2) Starch hydrolysis:

Growth zone: 8-10 mm.

Enzymatic zone: 27 to 30 mm.

Thus, when this strain is grown on a synthetic medium, the vegetative mycelium will be colorless or pale yellow to light brown, and soluble pigment will not form, or when formed, will be pale burgundy to light brown. (Rdg. XL, 13"'-f) or pale purple to pale yellow-brown. The aerial mycelium is first white and later pale olive gray (Rdg. LI, 23""'-d) to light brownish gray (Rdg. XLVI, I7""- f), or in certain cases it eventually becomes black. When grown on a proteinaceous medium, this strain is colorless or yellow or faintly yellowish-brown (Rdg. XL, 13"'), but no melanoid pigments are formed, indicating that this strain is a non-chromogenic type.

Application of carbon sources:

The observations took place according to Pridham and Gottlieb, (Journal of Bacteriology, Vol. 59, p. 107 (19^8)) on cultures that had been incubated at 28 C for 10 days, and the results are shown in table 1.

Notes for Table 1:

+++: Strong growth.

+ +: Good growth-.

+: Moderate growth.

+: Poor growth.

No growth.

When comparing said microbiological characteristics of strain No. B-5050 with corresponding characteristics of known organisms (reference is made here to S.A. Waksman's "The Actinomycetes", Vol. 2, published by The Williams & Wilkins Co. in 1961), then it is noted that the said strain is very close to Streptomyces hygroscopicus-

with regard to most properties, except for the production of soluble pigment on synthetic media, cellulose decomposition, nitrate reduction, etc. It is considered from these observations that B-5050 is a strain of Streptomyces hygroscopicus. A sample of this strain

has been deposited in The Institute for Fermentation, Osaka, Japan, under accession number IFO-12995- In the present description this strain is thus designated as "Streptomyces hygroscopicus no. B-5050" or simply "strain no. B-5050".

The antimicrobial spectrum for antibiotics produced by strain No. B-5050 using the cross-hatching method is shown in Table 2. The results were obtained by streaking said strain on a nutrient agar plate (pH 7.0) or on a glycerin nutrient agar plate ( pH 7.0), grow the streaked organism at 28°C for 4 days, cross-streak a test organism of the type indicated in Table 2

and grow it all at 37°C for 18 hours (in the case of normal bacteria) or for 40 hours (in the case of acid-fast bacteria), and measure the length of the inhibition zone. It appears from the results that strain No. B-5050 very strongly inhibits the growth of Gram-positive bacteria, in addition to inhibiting Mycobacterium avium.

Notes for table 2:

(<*>1): Strain resistant to oleandomycin and erythromycin.

- : Inside the sample.

An examination of this strain by means of the cross-strip agar plate method (Motoo Shioata: Annual Reports of the Takeda Research Laboratories r Vol. 20, pages 222 et.seq. (1956'), using Bacillus subtilis as the test organism and nutrient agar plates as test media, suggests that strain No. B^-5050 produces antibiotics.

The results are -stated in table 3-

The nutrient medium used in the present method can be liquid or solid, but liquid media is more appropriate. Said medium is supplemented with one or more assimilable carbon sources such as glucose, sucrose, maltose, dextrin, glycerin, starch or the like, and one or more degradable nitrogen sources, such as peptone, soybean meal, corn extract, meat extract, rice extract, wheat extract, urea, 'various ammonium salts, (e.g. NH^.Cl, NH^NO.,, CNH^^SO^) and the like,, as well as small

amounts of other inorganic salts as appropriate are used for the cultivation of microorganisms, e.g. sodium chloride, phosphates or special metal salts of calcium, magnesium, zinc, manganese, iron and the like. In addition to this, you can add growth-promoting substances, in addition to anti-foaming agents, such as oils, fats or mineral oils as needed.

The culture medium preferably contains approx. 3 to 5% of the carbon source and from approx. 1 to approx. 2% of an organic nitrogen source.

Cultivation can be carried out with the help of a stationary culture, but one will usually use submerged cultivation under aeration and stirring. In the latter case, it is preferred that the culture medium's pH lies around the neutral point, i.e. at approx. 6 to approx. 8, most preferably from approx.' 6.8 to 7.4, and the cultivation temperature is between 20 and 43°C, preferably in the range from 23 to 32°C.

The fermentation liquid obtained, as indicated above, contains the components of antibiotic B-5050. In order to isolate this group of antibiotics, different methods can be used which are known for extracting metabolites from microorganisms in nutrient media. One can thus use the property that this type of antibiotic is weakly basic and fat-soluble substances, and that they can be separated by a technique that utilizes these properties.

As antibiotic B-5050 preferentially accumulates in the liquid phase in the nutrient medium, this can first be filtered to eliminate the mycelium, and the active ingredients can be extracted at a slightly basic pH with a water-immiscible organic solvent from the nutrient medium. The filtered medium can e.g. adjusted to a pH of from approx. 7 to approx. 10, and then extracted with an organic solvent which is immiscible, or not completely miscible with water (e.g. a lower fatty acid ester, e.g. ethyl acetate, n-butyl acetate), halogenated aliphatic hydrocarbons (e.g. chloroform , ethylene chloride), ketones (e.g. methyl ethyl ketone, methyl isobutyl ketone), aromatic hydrocarbons (e.g. benzene, toluene), alcohols (e.g. n-butanol) or the like, or a mixture of two or more of these.

The antibiotics that may be present in the mycelium can, after filtration, be extracted from this with a water-soluble organic solvent, such as acetone or methanol, or a dilute aqueous acid solution. From the organic solvent extracts, the active ingredients can be recovered by means of filtration, concentration in vacuo and extraction with a water-immiscible organic solvent, as indicated above. From the aqueous acid solution they can be recovered by filtration, adjustment to a neutral or slightly basic pH and extraction with said water-immiscible organic solvent.......

The active ingredients in the nutrient medium can be absorbed on an adsorbent, such as activated charcoal, colloidal silicon dioxide or the like. The adsorbed active ingredients can then be eluted with a suitable eluent, such as e.g. can be an aqueous alcohol (e.g. aqueous methanol, aqueous ethanol), aqueous acetone, a mixture of such aqueous solutions as well as an acid, an organic solvent (e.g. ethyl acetate, chloroform) or the like.

Alternatively, the active ingredients can be adsorbed on a cationic exchange resin, such as "Amberlite IRC-50", "Amberlite IR-120" or "Dowex 50". The active ingredients adsorbed on such a resin can then be eluted with a suitable eluent, e.g.

a dilute aqueous acid solution, a dilute aqueous basic solution, an aqueous acidic methanolic solution, an aqueous basic methanolic solution, an aqueous acidic acetone solution, an aqueous basic acetone solution or the like.

The organic extract obtained can then be transferred to an aqueous acid solution, preferably after washing with water. The acid solution for this purpose can be a dilute aqueous acid solution or an acidic buffer solution. The thus obtained aqueous extract can then be adjusted to a neutral or slightly basic pH, and can again be extracted with a water-immiscible organic solvent, as indicated above. . When the content of the active ingredients in the organic solvent extract obtained is relatively high, antibiotic B-5050 can only be crystallized by concentrating the extract in vacuum. When this is not the case, a non-polar organic solvent such as petroleum ether or n-hexane can be added to the concentrate, whereby an impure, powdery antibiotic B-5050 is secreted. This powder product can then be recrystallized from an organic solvent, such as benzene, diethyl ether, ethyl acetate, or a mixture of ethyl acetate-n-hexane, acetone:n-hexane, acetone:water or ethanol water.

When the impure powder contains certain colored impurities, these can be removed using activated charcoal.

In these special cases where the antibiotic B-5050 cannot be crystallized even with the aforementioned treatment, a further purification can be used using an absorbent such as silicon dioxide gel or aluminum oxide. The adsorbed components can then be eluted with a fatty acid ester (e.g. methyl acetate, ethyl acetate, n-butyl acetate), a 'mixed ■ solvent (e.g. benzene:acetone, benzene:ethyl acetate, benzene:methanol, chloroform: .methanol) or similar.

The antibiotic B-5P50 mixture, as well as its respective components, are basic substances and can consequently form salts with water-soluble acids such as hydrochloric acid, sulfuric acid, tartaric acid, nicotinic acid or the like.

The following is indicated to show the general characteristics

by the antibiotic B-5050 mixture, but it should be noted that the specific values were obtained by performing the measurements on a sample of the antibiotic B-5050 mixture, which was prepared as is

described in example 7, and that certain deviations from these specific values can be found on other samples of antibiotic B-5050.

(1) Melting point 137-141°C.

(2) Elementary analysis:

Recrystallized from benzene:

C: 58.14 + 0.5; H: 7.88 + 0.3; N: 1.73 + 0.3 Recrystallized from ethyl acetate: C: 57.83 + 0.5; H: 8.14 + 0.3; N: 1.33 <+> 0.3-

(3) Molecular weight measured by vapor pressure cosmos:

880 + 90 (in ethyl acetate).

(4) Specific rotation:

(a)p<4>:-76.4° + 8° (c=1, ethanol).

(5) Color reactions:

The Dragendorff reaction: positive.

Erythromycin test: positive with a reddish purple color, while the chloroform layer turned slightly blue.

Carbomycin test: negative.

(6) Solubility properties:

Easily soluble in methanol, ethanol, acetone, ethyl acetate, methyl ethyl ketone, chloroform or an aqueous acid solution;

Soluble in benzene or diethyl ether;

Hardly soluble in n-hexane or neutral water;

(7) Ultraviolet Absorption:

No significant maximum in methanol.

(8) Infrared absorption:

As shown in fig. 1 (KBr disk) the main absorption bands (cm 1 ) are the following: 3448(s), 2924(m), 1736(s), l453(m), 1376(m), 1295(m), 1239(m), ll67 (s), 108l(s), 1050(s), 968(m), 912(m), 86l(w), 840(w).

Notes: The abbreviations in brackets "vs", "s", "m",

"sh" and "w" mean "very strong", "strong", "medium", "shoulder" and "weak" respectively, and the same applies to the description below with regard to infrared spectra.

As mentioned above, the present antibiotic consists of several components, and so far at least six components have been discovered which are very closely related both in terms of chemical and biological properties. This fact can be easily confirmed by subjecting the antibiotic mixture to paper chromatography or thin layer chromatography. The following are examples of such an identification of the respective components.

(1) Paper Chromatography:

Paper: Toyoroshi No. 50, impregnated with 1.2% liquid

paraffin solution in naphtha.

Developer: M/15 phosphate buffer solution with a pH 8.0 saturated

with n-butyl acetate.

Detection: Bioautography using Bacillus subtilis

as a test organism.

Rf values:

B-505-0-A 0.32 + 0.05-

B-5050-B 0.38 + 0.05-

B-5050-C 0.66 + 0.05-

B-5050-D = 0.72 + 0.05.

B-505.0-E = 0.74 + 0.05.

B-5050-F = 0.82 + 0.05.

(2 ) Thin layer chromatography:

Thin layer: (a) Silicon dioxide gel ("Spotfilm f"), preheated to 105°C for 1 hour. (b) Silica gel ("Kieselgel G"), (c) same as (b).

Developer: (a) Benzene:acetone (3:2 by volume).'

(b) Benzene-acetone (3:2 by volume).

(c) Benzene:methanol (10:1 by volume),

but development was repeated 3 times.

Detection: (a): Spraying with concentrated H^SO^.

(b) and (c): Treated as follows:

1) 5% phosphomolybdate solution in ethanol was sprayed on and dried for 5 minutes at 110°C, 2) A 5% cerium sulfate solution in N-H^SO^ was sprayed on and then dried at 110°C for 5 minutes, 3) A 10% aqueous H^SO^ solution was sprayed on and dried at 110 C for 5 minutes, and 4) Concentrated H^SO^ or a 5% ^ solution in chloroform was sprayed on.

Rf values:

In order to separate or isolate these components on an industrial scale, it is recommended to use a technique such as adsorption chromatography, partition chromatography or counter-current distribution.

Suitable adsorbents for adsorption chromatography can e.g. be silica gel or aluminum oxide. When using silicon dioxide gel, you can e.g. use for the elution a mixture of a non-polar organic solvent and a polar organic solvent, whereby the respective ingredients are fractionated with regard to their affinity towards the non-polar solvent, i.e. antibiotic B-5050-A, -B , -C, -D, -E and -F. Suitable mixed solvents that can be used as eluent can be listed as benzene-ethyl acetate, benzene-acetone, n-hexane-methanol and chloroform-methanol.

One can also use the difference with regard to distribution between two liquid phases, and in this respect one can use a combination of a lipophilic organic solvent (eg ethyl acetate) and an aqueous buffer solution. By using a buffer with pH 5.5, antibiotic B-5050-D, -E and -F can be transferred to the buffer, while B-5050-A, -B and -C remain in the organic phase.

From the latter solvent phase, B-5050-C can be extracted with a buffer with a pH of 4.9. With a buffer with a pH of 4.4, B-5050-B and -C can be extracted from the organic layer, while B-5050-A remains in the organic solvent phase. In this way, the respective components can be isolated.

Distribution chromatography is another good technique. Materials such as cellulose powder or diatomaceous earth (e.g. "Celite" or "Hyflo Super Gel") can be used as a carrier for the stationary phase.

Characteristics and properties of the respective active ingredients in antibiotic B-5050 are stated below:

(1) Melting point (with decomposition):

Measured on samples recrystallized from a mixture of • acetone and n-hexane (1:3 per 1 volume).

B-5050-A: 129 - 132°C.

B-5050-B: 134 - 136°C

B-5050-C: 135 - 138°C.

B-5050-D: 143 - 146°C.

B-5050-E: 144 - 149°C

B-5050-P: 149 - 154°C.

(2) ■ Elementary analysis:

B-5050-A: C 59.40+0.5; H 8.15+0.3; N 1.40+0.3 B-5050-B: C 58.67+1.0; H 8.02+0.5;' N 1.86+0.5 B-5050-C: C 57.93 + 1.0; H 8.18 + 0.5; N 1.69±0.5 B-5050-D: C 57.48+1.0; H 8.17+0.5; N 1.64+0.5 B-5050-E: C 57.34+1.0; H 8.25+0.5; N 1.34+0.5 B-5050-P: C 58.59+0.5; H 7.82+0.3; M 1.62+0.3-

(3) Molecular weight:

All components have a molecular weight of approx. 800 to 900, as this can be measured by vapor pressure chromatography in ethyl acetate.

(4) Specific rotation:

B-5050-A: ct^3:-72.3° + 7° (c = 1, ethanol)

B: ajp:-71.9°j;7<0> (c = 1, ethanol)

C: ctjp :-76.0° + 8° (c=1, ethanol)

D: ctQ3:-76.20 + 8° (c = 1, ethanol)

E: a^3:-73.6°+7° (c=1, ethanol)

F: ajp :-77.7° + 8° (c=1, ethanol)

(5) Color reactions:

All components are positive for the Dragendorff reaction and the erythromycin test, but negative for the carbomycin test.

(6) Solubility ratio:

All components are soluble in methanol, ethanol, acetone, methyl ethyl ketone, ethyl acetate, benzene, diethyl ether, chloroform or an aqueous acid solution, but only very weakly

soluble in n-hexane or neutral water.

(7) Ultraviolet Absorption:

None of the components have. some significant maximum

absorption.

(8) Infrared absorption (KBr disk):

B-505O-A: As shown in fig. 2, where the main absorption bands are the following given in wavenumber (cm 1): 3448(m), 291»l(m), 1739(s), l458(w), 137Mm), 1295(m), ll88(s), ll67(s), 1120(m), 1086(s), 1053(s), 1033(s), 97Km), 917(w), 862(m). B-5050-B: As shown in fig. 35 where the main absorption bands is the following indicated in wavenumber (cm ): 31»00(s), 2990(s), 2960(s), 1740(vs), 1454(m), 1370(m), 1296(m), 1233(s) , ll88(sh), ll66(vs), 1120(s>, 1080(s), 1052(vs), 1025(vs), 968(m), 9l6(w), 858(w), 842(w) B-5050-C: As shown in Fig. 4, where the main absorption bands are the following, given in wavenumber (cm ): 3460(s), 2980(s), 2940(s), 1735(vs), l455(m), 1375(s), 1357(s), 1295(s), 1270(s), 1240(m), ll83(sh), ll62(vs), 1080(vs), 1050(vs), 1028(sh), 10l8(sh), 972(m), 910(m), 862(w), 840(w). B-5050-D: As indicated in fig. 5, where the main absorption the bands are the following given in wavenumber (cm "'"): 3440(s), 2946(m), 1735(vs), 1450(m), 1373(m), 1357(sh), 1296(m), I274( m), 1237(s), ll63(s), 1127(m), 1083(s), 1047(vs), 1031(sh), 1012(sh), 972(m), 913(m), 862( w), 843(w). B-5050-E: As shown in fig. 6, where the main absorption bands are the following, given in wavenumber (cm 1): 3430(s), 2920(m), 1732(vs), 1450(m), 1373(m), 1354(m), 1296(m), 1239(s) , ll63(s), 1127(m), 108Ms), 1048(vs), 1032(sh), lOll(sh), 970(m), 9l4(m), 862(w), 840(w). B-5050-F: As shown in fig. 1, where the main absorption bands are the following, given in wavenumber (cm -1-): 3425(s), 299Mm), 17l5(s), 1730(s), 1460(w), 1379(m), 1362(m), 1300(m) , 1242(s), ll66(s),

1133(m), i089(m), 1053(s), I031(m), 973(w),

919(w), 864(w).

Antimicrobial spectra for the recovered antibiotic B-5050 mixture' and its respective constituents (ie, B-5050-A, -B, -C, -D, -E and -F) are shown in Table h. measurements, common bacteria were grown on a nutrient agar medium, while the Mycobacterium species were grown on a glycine nutrient agar medium. Yeast and other fungi were grown on a glucose nutrient agar medium.

Notes for Table 4:

(<x>l): The strain was resistant to oleandomycin and erythromyc in.

(<*>2): The strain was resistant to streptomycin, chloramphenicol, tetracycline, oleandomycin and erythromycin.

( 3): The strain was resistant to streptomycin.

(<x>4): The strain was resistant to dextromicin.

- : Not tried.

It is clear from the above results that antibiotic B-5050 belongs to the so-called "macrolide antibiotics" viewed from the background of its properties, such as color reactions (e.g. the erythromycin sample), solubility conditions (e.g. lipophilic and basic) , infrared spectra (e.g., the values of V \_, — _ O or V ^ ), antimicrobial spectra (e.g., the difference in minimum inhibitory concentration against an erythromycin- and oleandomycin-resistant strain of Staphylococcus aureus), etc.

Among the macrolide antibiotics, antibiotic B-5050 differs from those that have maximum absorption at wavelengths of 225, 230, 240 or 280-290 millimicrons. Erythromycin and oleandomycin have relatively weak maximum absorption in the range from 280 to 290 millimicrons, but differ from antibiotic B-5050 due to their infrared absorption spectra, their molecular weights, melting points, etc.

Until now, carbomycin, relomycin and tylosin have been known as macrolide antibiotics produced by Streptomyces hygroscopicus.

Carbomycin A and carbomycin B are thus known by the general formulas

There is no literature that states the formula for relomycin, but the formula is the same as for tylosin with the exception of

The -CH2COH group in the 6-position is -CH2CH2OH.

The above known antibiotics have a maximum absorption

at a wavelength of 238, 282 and 289 millimicrons, resp., and thus clearly differs from antibiotic B-5050. It should therefore be clear that antibiotic B-5050 comprises a new group of antibiotics, of which each of the components is also new.

Antibiotic B-5050 regardless of whether it is a mixture of the above-mentioned active ingredients or is seen as an isolated active ingredient, has very strong antimicrobial activity, especially against Gram-positive bacteria, and is also characterized by an exceptionally low toxicity towards warm-blooded animals, this includes humans. It can, for example, be mentioned that a mean effective dose (ED^q) of antibiotic B-5050 mixture in mice infected with Staphylococcus aureus was 640 mg/kg when administered orally. Nevertheless, the lethal dose (LD^-q) in the same mouse could not be determined due to the low toxicity of the antibiotic, and can only be expressed as "higher than 10,000 mg/kg" by an oral administration. Furthermore, rats were daily given 1 g/kg body weight of the antibiotic B-5050 orally for 1 month. No differences from a control group could be observed in these rats, nor could a subsequent autopsy find any significant changes in organs in the tested rats.

Staphylococci are pyogenic or pus-forming bacteria.

They tend to produce well-defined lesions, e.g.

in the form of boils or the like, which often appear on the skin. Staphylococci are the cause of pimples and carbuncles and other common wound infections. The new products produced according to the present invention can be used in topical preparations for the treatment of this type of infection in mammals (dogs, cats, humans, etc.).

Due to their bactericidal and bacteriostatic properties, the new products produced according to the present invention can be used to disinfect hospital equipment, etc., which are usually exposed to pathogenic Gram-positive bacteria of the type that are sensitive to such products, e.g. of the type mentioned above.

With regard to the composition of the aforementioned cultivation media, the percentages are stated in weight per volume basis, i.e. in grams per 100 milliliters. All other percentages are per weight basis unless otherwise stated.

Example 1.

500 ml of a pre-culture medium (pH 7.0) consisting of 2% glucose, 3.0? soluble starch, 1.0% soybean meal, 1.0% corn extract, 0.5% "Polypeptone" (a casein hydrolyzate), sodium chloride (0.3%),

0.3% calcium carbonate and water were added to 2000 ml Sakaguchi flasks and sterilized. The sterilized medium was inoculated with a Streptomyces hygroscopicus (IFO-12995) from an agar culture that

was cultured for at least 4 days, and the flasks were incubated at 28°C in a shaker with a stroke length of 10 cm and a motor rotation speed of 115 rpm for 48 hours. 1000 ml of a thus

prepared medium hie used as pre-culture.

The pre-culture was inoculated in 100 1 of a culture medium

(pK 7.0) which was added and sterilized in a 200 L stainless steel tank, and where the medium consisted of 3% glucose, 0.5% corn extract, 1.0% defatted soybean meal, 0.5% sodium chloride , 0.05% magnesium sulfate, 0.3% calcium carbonate' and water. The incubation was carried out at 28°C with an aeration of 100 1 per minute and with stirring at 200 rpm for a total of 24 hours.

100 L of the resulting pre-culture was transferred to 1000 L of a sterilized culture medium of the same composition as stated above, and the whole was added to a 2000 L stainless steel tank. The incubation was continued at 28°C with an aeration of 1000 1 per minute and a stirring of 120 rpm. for 66 hours. During the incubation, "Actocol" (a polyoxypropylene sample mixture with an OH number of 56) was added as an antifoam agent in a concentration of approx. 0.05% in relation to the weight of the medium. The culture medium obtained has an antimicrobial effect of 350 units expressed as dilution units against Bacillus subtilis as the test micro-organism.

The medium was filtered. The filtered medium (950 L) was adjusted to pH ■ 8.5 with a 2-N. aqueous NaOH solution, and the whole extracted with 300 l of ethyl acetate. The ethyl acetate layer was separated, washed with water and extracted twice with 70 L of 0.005N-HCl. The aqueous extracts were combined, adjusted to pH 8.5 with an aqueous dilute ammonia and again extracted twice with 45 L of ethyl acetate. The extracts were washed with water, dehydrated and concentrated in vacuo to 50 ml. The concentrate was then added to 750 ml of n-hexane, whereby 95 g of impure powdery antibiotic B-5050 mixture was obtained.

The powdery product was dissolved in 450 ml of benzene under heating. After addition of 2 g of activated carbon, the mixture was heated, filtered, after which the filtrate was cooled on standing, and 19.0 g of the antibiotic B-5050 mixture was separated as colorless needles.

10 g of the crystalline antibiotic B-5050 mixture was dissolved in 50 ml of ethyl acetate and subjected to column chromatography on silica gel (450 g, grain size from 0.05 to 0.2 mm). The added substances were eluted with a mixture of ethyl acetate and benzene (2:1 per volume). The first 600 ml of the eluate showing antimicrobial activity was concentrated to dryness, after which the residue was dissolved in hot benzene. When the solution stood still, 1 g of colorless crystals were separated which consisted mainly of antibiotic B-5050-A.

After the column was eluted with 4 L of ethyl acetate benzene, it was then eluted with 3 L of ethyl acetate, after which the eluates were concentrated in vacuo. The residue was dissolved in hot benzene. On standing, 550 mg of colorless crystals were secreted which essentially consisted of antibiotic B-5050-F.

500 mg of the crystals, which essentially consisted of antibiotic B-5050-A, were dissolved in 1.5 ml of ethyl acetate, and again chromatographed on a column of silica gel (50 g, properties as indicated above). The added material was eluted with a mixture of ethyl acetate and benzene (1:1 per volume), whereby the material was fractionated. Each fraction was tested for content of active ingredients by thin layer chromatography on silica gel ("HF2^^"), using a mixture of benzene and acetone (3:2 by volume). The fractions that exclusively contained antibiotic B-5050-A as active ingredient were collected and concentrated in vacuo, whereby a residue was obtained. This was recrystallized from benzene, and 88.5 mg of antibiotic B-5050-A were obtained as colorless prisms.

Furthermore, 350 mg of the crystals which essentially consisted of antibiotic B-5050-F were subjected to column chromatography on silica gel (50 g) in the same manner as stated above. The column was eluted with 700 ml of ethyl acetate-benzene (2:1 by volume), and was further eluted with a mixture of ethyl acetate and benzene (3:1 by volume), after which each fraction was tested for active ingredients using thin layer chromatography. The fractions that exclusively contained antibiotic B-505O-F as active ingredient were collected and concentrated in vacuo to a residue. This residue was recrystallized, whereby 75.6 mg of B-5050-F were obtained as colorless prisms.

Example 2.

A series of 2 liter Sakaguchi flasks, each containing 500 ml of sterilized culture medium of the same composition as indicated in Example 1, were inoculated with Streptomyces hygroscopicus (IFO-12995), and the inoculated flasks were incubated in the same manner as indicated in Example 1, thereby creating a pre-culture.

A 500' liter stainless steel tank was charged with 200 1 of

a culture medium of the same composition as stated in example 1 for the pre-culture. After a sterilization at 121°C for 20 minutes, the culture was inoculated with 2 l of the above pre-culture. The incubation was carried out at 28°C under aeration of 200 1 per minute and a stirring of 200 rpm. for 24 hours, whereby a pre-culture was obtained for the main fermentation.

200 L of the pre-culture thus prepared was transferred to 4000 L of a culture medium of the same composition as stated above, and the whole was transferred to a 6000 L stainless steel tank, and the cultivation was carried out at 28°C under an aeration of 2400 L per minute and a stirring with a revolution speed of

l80 rpm. for 48 hours, During the incubation "Actocol" was used as ant ice agent.

The culture medium was filtered, and the filtered medium

(4000 L) was adjusted to a pH of 9 with 2N NaOH, then extracted with 1330 L of ethyl acetate, followed by washing with water to give 1052 L of ethyl acetate extract. The ethyl acetate solution was concentrated in vacuo to 100 L. The concentrated solution was extracted twice with 50 L each of a 1.5 M aqueous KH 2 PO 3 solution previously adjusted to pH 4.0 with phosphoric acid. The extracts were combined, adjusted to pH 8.5 with a dilute aqueous ammonia solution and then extracted with 50 L of ethyl acetate. The ethyl acetate solution was washed with water, dehydrated and concentrated in vacuo to give a syrup. Said syrup was added to 3 1 n-hexane, and 330 g of an impure powder was precipitated. 300 g of the powder was dissolved in 1.1 1 benzene under heating, and a cooling of the solution resulted in the precipitation of 117 g of crystals, which essentially consisted of antibiotic B-5050-C, -D and -E (crystal IN).

Furthermore, the ethyl acetate layer extracted with the phosphate solution was further extracted twice with 50 L of a N/200 HpSO 4 , and the aqueous layers were combined, adjusted to pH 8 with a dilute aqueous ammonia solution, and then extracted three times with 30 1 ethyl acetate. The ethyl acetate extracts were combined, washed with water, dehydrated and concentrated in vacuo to give a syrup. Said syrup was added to 1.5 l of n-hexane, and 160 g of an impure powder was precipitated. 150 g of the powder was recrystallized from hot benzene, yielding 72.5 g of crystals, consisting essentially of antibiotic B-5050-A and -B together with a minor amount of antibiotic B-5050-C (crystal II). By thin-layer chromatography as indicated under (a) or c) above, gave. crystal II two points, i.e. Rf values of 0.48 and 0.54 under conditions (a) or Rf values of 0.66 and 0.71 under conditions indicated under (c)..

2.0 g of crystal II was dissolved in 20 ml of ethyl acetate, after which 14 ml of benzene was added. The solution was subjected to column chromatography on silica gel (grain size from 0.05 to 0.2 mm), which had previously been filled with a corresponding volume of a mixture of benzene and ethyl acetate. The chromatogram was first developed with ethyl acetate-benzene (1:1 per volume) until 200 ml of eluate was collected, then with ethyl acetate-benzene (3"-2 per volume), thereby obtaining fractions showing antimicrobial activity against Bacillus subtilis. The respective fractions were combined, concentrated and recrystallized from benzene, whereby 435 mg of crystals of antibiotic B-5050-A, and 185 mg of crystals of antibiotic B-5050-B were obtained.

10 gave crystal II was dissolved in 50 ml of ethyl acetate, followed

of an addition of 25 ml of benzene. The solution was chromatographed on silica gel (400 g) in the same manner as above. The column was eluted first with 3 1 ethyl acetate-benzene (1:1 by volume), then with ethyl acetate-benzene (2:1 by volume). The eluate was fractionated into 500 ml fractions. The fractions obtained using the 1:1 mixture contained antibiotics B-5050-A and -B. The first fraction obtained by using the 2:1 mixture contained antibiotics B-5050-B and -C, the second fraction antibiotics B-5050-C and -D, the third fraction antibiotics B-5050-D and - E and the last fraction antibiotics B-5050-E and -F, respectively.

2 g of the crystals obtained from the fraction containing antibiotics B-5050-B and -C were again dissolved in 15 ml of ethyl acetate, after which 10 ml of benzene was added. The solution was subjected to column chromatography with 250 mg of silica gel, and the column was eluted with ethyl acetate-benzene (3:2 by volume). The eluate was fractionated into 20 ml portions. Fractions containing the same component were pooled, concentrated and recrystallized from benzene or a mixture of ethyl acetate and n-hexane to give 83 mg of crystals of antibiotic B-5050-B, 940 mg of crystals of antibiotic B-5050-C and 75 mg crystals of antithiotic B-5050-D.

In the same way, 1.2 g of the fraction containing antibiotics B-5050-D and -E was subjected to column chromatography on silica gel, whereby 600 mg of crystals of antibiotic B-5050-E were obtained.

The fraction obtained after the fraction containing antibiotics B-505O-B and -C contained antibiotic B-5050-C as the main component. 770 mg of the resulting crude crystals were again dissolved in 5 ml of acetone, and the solution was subjected to column chromatography with 100 g of silica gel in the same manner as above to obtain 220 mg of antibiotic B-5050-C as crystals. Example 3-

4000 1 filtered medium prepared in the same way as indicated

in Example 2, was adjusted to pH 8.0, and was extracted with re-

trained 1/3 volume of ethyl acetate. The ethyl acetate extract was concentrated to approx. 100 1. The concentrate was washed with 50 1 of water and extracted 3 times with 50 1 of a 3-molar aqueous KH^PO^ solution which had previously been adjusted to pH 3.0 with phosphoric acid. The aqueous extracts were combined, adjusted to a pH of 9 to 10 with an 8N aqueous ammonia solution and extracted with 75 L of ethyl acetate. The ethyl acetate extract was washed three times with 25 1 of water each time, and then concentrated to approx. 1.5 1. An addition of approx. 30 1 n-hexane to the concentrate gave 522 g impure powder. A recrystallization of the powder from benzene gave 272 g of crystals of the antibiotic B-5050 mixture.

If, in the above-mentioned methods, a 0.2-normal aqueous acetic acid solution is used instead of the KH2P0^ solution, essentially the same result is obtained.

19 g of the antibiotic B-5050 mixture thus obtained

was dissolved in 75 ml of acetone. The solution was subjected to column chromatography with 800 g of silica gel. The column was eluted with 3000 ml of ethyl acetate-benzene (1:1 per volume), whereby 7.25 g of crystals were obtained essentially consisting of antibiotics B-5050-A and -B. The column was then eluted with ethyl acetate-benzene (2:1

per volume). The first fraction of 1100 ml was treated as indicated above, whereby 2.22 g of crystals consisting essentially of antibiotic B-5050-B and -C were obtained. The subsequent fraction containing 200 ml gives 0.90 g of crystals which essentially consist of

of antibiotic B-5050-C. From the subsequent 300 ml, 1.4 g of a mixture consisting essentially of antibiotics B-5050-C and -D were obtained. From the last 600 ml, 1.99 g of crystals consisting essentially of antibiotics B-5050-D and -E were obtained. The column was then eluted with 600 ml of ethyl acetate-benzene (3:1 per volume), whereby 0.645 g of crystals were obtained consisting essentially of antibiotics B-5050-D and -E. From the last fraction, which was eluted with 1600 ml of ethyl acetate, 2.09 g of crystals consisting essentially of antibiotic B-5050-F were obtained.

The obtained antibiotics B-5050-A and -B crystals (7.25 g) were dissolved in 40 ml of acetone and subjected to column chromatography on 400 g of silica gel, followed by an elution with ethyl acetate-benzene (1:1 by volume), whereby 1.5 g of crystals of antibiotic B-5050-A and 0.65 g of crystals of antibiotic B-5050-B were obtained.

The above-mentioned antibiotics B-5050-B and -C crystals (2.22 g) were dissolved in 20 ml of acetone and column chromatographed on 300 g of silica gel, followed by an elution with benzene-acetone (3:1 by volume), whereby 0.398 g of crystals of B-5050-B were obtained. A further elution with benzene-acetone (2:1 by volume) gives 0.269 g of crystals of antibiotic B-5050-C.

The crystals, which essentially consisted of antibiotic B-5050-C (0.90 g), were dissolved in 5 ml of acetone and column chromatographed on 100 g of silica gel and eluted with ethyl acetate-benzene (2:1 by volume), which gave 0.258 g crystals of antibiotic B-505O-C.

The antibiotics B-5050-C and -D crystals (1.40 g) were dissolved in 10 ml of acetone and chromatographed on a column packed with 150 g of silica gel, eluting with benzene-acetone (2:1 by volume), whereby 0.596 g of antibiotic B-5050 and 0.187 g of crystals of antibiotic B-5050-D were obtained.

The antibiotics B-5050-D and -E crystals (1.99 g) were dissolved in 30 ml of acetone, and column chromatographed on 300 g of silica gel and eluted with benzene-acetone (2:1 per volume), whereby one . obtained 0.340 g crystals of antibiotic B-5050-D and 0.187 g crystals of antibiotic B-5050-E.

The crystals which essentially consisted of antibiotic B-5050-F (2.09 g) were dissolved in 15 ml of acetone and column chromatographed on 250 g of silica gel, after which they were eluted with ethyl acetate-benzene (3:1 per . volume), whereby 0.585 g of crystals of antibiotic B-5050-P were obtained.

Example 4.

A mixture of 120 ml of n-hexane, 80 ml of ethylene dichloride, 30 ml of methanol and 6 ml of water was shaken and set aside, whereby two layers were obtained. The lower layer was separated from the upper layer. To 6 ml of the lower layer was added 1 mg of chlorophenol red which dissolved. To the solution was added 5 g of "Celite" (a processed diatomaceous earth, it being preferred to use "Celite 535"), and 100 ml of the above-mentioned separated upper layer. The mixture was vigorously shaken, after which hydrogen chloride was introduced into the mixture until the violet color of the indicator had turned yellow. The suspension was then introduced into a glass column little by little, whereby a uniform and homogeneous "Celite" column was obtained. 25 mg of crystals of the antibiotic B-5050 mixture as obtained in Example 35 were dissolved in 0.4 ml of the above separated upper layer, and the solution was subjected to chromatography on the above prepared "Celite" column. The column was eluted with the upper layer, whereby the active components were separated and observed as respective violet bands. Further elution gives the respective fractions of antibiotics B-505O-A, -B, -C, -D and -E and -P in this order. 4 mg of B-5050-A, 3 mg of B-5050-B, 5 mg of B-5050-C, 4 mg of a mixture of B-5050-D and -E and 2 mg of B-5050-P.

Example 5»

In the same way as stated in example 1, a pre-culture of Streptomyces hygroscopicus (IFO-12995) was prepared. 2 ml of the pre-culture was inoculated into 50 ml portions of a main culture medium which had been sterilized in 200 ml Erlenmeyer flasks, and where said medium consisted of 3»0% glucose, 0.5% corn extract, 1.0% defatted soybean meal, 0, 5% sodium chloride, 0.05% magnesium sulfate, 0.1% dipotassium hydrogen phosphate, 0.05% potassium chloride, 0.3% calcium carbonate and water, all adjusted to pH 7.0. The fermentation was carried out in a rotary shaker at 28°C for 96 hours. The filtered medium has an antimicrobial effect of 350 dilution units against Bacillus subtilis.

1.6 1 of the filtered medium was adjusted to pH 7.8 and then extracted first with 800 ml of ethyl acetate and then with 600 ml of ethyl acetate.

The combined extracts were washed with water and extracted twice with 600 ml of a N/200 aqueous HCl solution. The aqueous layers were combined, neutralized to pH 7 to 8 with N/10 aqueous NaOH solution, and then extracted twice with 500 mL of ethyl acetate. The ethyl acetate extracts were washed with water, dehydrated with anhydrous sodium sulfate and concentrated in vacuo. 30 ml of n-hexane was added to the concentrate, whereby 59 mg of a crude powder was obtained. This powder was dissolved in hot benzene and the solution was set aside to give 15 mg of the antibiotic B-5050 mixture as colorless needles.

Example 6.

2 ml of a pre-culture prepared as indicated in Example 1,

was inoculated in 50 ml portions of a culture medium in 200 ml Erlenmeyer flasks, and where the culture medium was of the same composition as the main culture medium in example 1. The culture was carried out at 28°C in a rotary shaker for 96 hours. The filtered medium had an antimicrobial potency of 350 dilution units against Bacillus subtilis.

1.8 L of the filtered medium was adjusted to pH 8.0 and extracted twice with 400 ml of ethyl acetate. After washing with water and dehydration, the ethyl acetate solution was concentrated in vacuo, and 30 ml of n-hexane was added to the concentrate, whereby 210 mg of impure powder was obtained. This powder was dissolved in 15 ml of benzene under heating, and after standing, 59 mg of the antibiotic B-5050 mixture was precipitated as crystals.

Example 7.

900 l of filtered culture medium obtained in the same manner as stated in Example 1 was treated in the same manner as stated in this example, whereby 70 g of an impure antibiotic B-5050 mixture was obtained.

The impure sample was dissolved in a mixture of ethyl acetate and benzene (2:1 by volume), and chromatographed on a column packed with 2.1 kg of silica gel. The column was eluted with ethyl acetate-benzene (2:1 by volume), and colorless fractions were collected and concentrated to dryness. The residue was dissolved in 350 ml of hot benzene, and on standing gave 7.0 g of colorless needles of the antibiotic B-5050 mixture.

Example 8.

800 ml of filtered culture medium obtained in the same manner as in Example 6 was adjusted to pH 7.6 and passed through a column packed with 40 ml of "Amberlite XAD-2" (molecular sieve). The column was washed with 200 ml of distilled water, and then eluted with 200 ml of 30% aqueous methanol solution, whereby a brown eluate was obtained which showed essentially no antimicrobial activity. The elution was continued with 500 ml of 80% aqueous methanolic solution, whereby fractions containing most of the active ingredients were obtained. The fractions were concentrated in vacuo until the methanol was eliminated. The concentrate was adjusted to pH 7.5 to 8.5 and extracted twice with 100 ml of ethyl acetate. The ethyl acetate extracts were combined, washed with water, dehydrated and concentrated in vacuo. n-hexane was added to the concentrate, whereby 53 mg of an impure powder of the antibiotic B-5050 mixture was obtained. The powder was dissolved in hot benzene, and on standing, 17 mg of crystals were precipitated.

Example 9-

1.0 g of crystal I prepared as in Example 2 was dissolved

in 100 ml of ethyl acetate. The solution was extracted three times with 100 ml portions of Sørensen's buffer solution with pH 5.5, 4.9 and 4.4 (containing M/10 disodium hydrogen citrate) in the specified order. Each fraction was adjusted to pH 9.5 with dilute aqueous ammonia and then extracted with half a volume of ethyl acetate. The ethyl acetate layer was washed with water, dehydrated and concentrated in vacuo. From the fraction which had been extracted with a buffer solution of pH 4.9, 365 mg of antibiotic B-5050-C were thus obtained.

The ethyl acetate layer which remained after the extraction with these buffer solutions gave 110 mg of crystals consisting essentially of antibiotic B-5050-A and -B. The fraction extracted with the buffer solution of pH 4.4 gave 165 mg of crystals consisting essentially of antibiotic B-5050-B and -C, while the fraction extracted with the buffer solution of pH 5.5 gave 320 mg of crystals consisting mainly of antibiotic B-5050-C, -D,

-E and -F.

Example 10.

10 g of crystal II obtained as indicated in example 2 were dissolved in 1 liter of ethyl acetate. The solution was extracted twice with 800 ml portions, and then four times with 1 liter portions of Sørensen's buffer solutions with a pH of 4.9 and a pH of 4.4 in this order. Each fraction was adjusted to pH 955 with a dilute aqueous ammonia solution and extracted with half a volume of ethyl acetate. The ethyl acetate layer was washed with water, dehydrated and concentrated in vacuo. The respective fractions were treated as indicated, in example 9- The ethyl acetate layer that remained. again after the extra action-with the buffer solutions, yielded 4.2 g

crystals consisting essentially of antibiotic B-5050-A. The fractions which were extracted with the buffer solution of pH 4.4 gave 3.8 g of crystals consisting essentially of antibiotic B-5050-B. The fractions that were extracted with the buffer solution on

pH 4.9 gave 1.3 g of crystals consisting essentially of antibiotic B-5050-B and -C.

Claims (1)

Process for the manufacture of antibiotic B-5050 or its components B-5050-A, -B, -C, -D, -E or -F or mixtures of two or more of these, and with the formula: characterized by growing the microorganism Streptomyces hygroscopicus (IFO 12995) in a culture medium inside holding assimilable carbon and rut.roger.kiides under aerobic conditions, and then recover d'-i_ accumulated antibiotic B-5050, and, if desired, separate this x antibiotic B-5050-A, -B, -C, -D , -E and -F or a mixture of two or more of these components.