DE2065297A1 - Antibiotics from streptomyces mycarofac - iens - Google Patents

Abstract

Cmpds. designated SF.837, -837A2, -837A3 and -837A4, their salts, the diacetylates of SF837 and -837A2 and the monoacetylates of SF837A3 and -837A4 are characterised by their m.pts. pKa value. opt. rotation, U.V. and I.R. spectra and elemental analysis. The compds. are antibiotics active against Gram-positive and bacterial pyrogens resistant to other antibiotics.

Description

Antibiotics and their method of manufacture The priority becomes claimed due to: September 25, 1969, Japan 75845/69 - elimination from P 20 04 686.0 -The invention relates to new and valuable antibiotic substances that SF-837-A2, SF-837-A3 and SF-837-A4 substances can be mentioned; the invention relates to also processes for the production and isolation of these antibiotic substances.

It has been found that new antibiotic substances that have a strong growth-inhibiting or growth-preventing effect against gram-positive pathogens Bacteria and against pus-producing bacteria have that otherwise against antibiotics are resistant, can be produced in a culture of a milk organism belongs to the genus Streptomyces that these new antibiotic substances from the Culture this microorganism can be obtained and that this new antibiotic substances extremely effective in preventing or Inhibition of the growth of various types of pathogenic bacteria such as Stapylococcus aureus sing. As mentioned above, these new and valuable antibiotics are as SF-837-A2 substance, SF-837-A3 substance or

SF-837-A4 substance.

All of these SS-837-A2, SF-837-A3 and SF-837-A4 substances are effective to prevent or inhibit the growth of gram-positive bacteria as well to prevent or inhibit the growth of pathogenic bacteria that act against known Antibiotics are resistant. The SF-87) 7-A2, SF-837-A3 and SF-837-A4 substances are essentially non-toxic and.

have a therapeutic effect on infections caused by gram-positive Bacteria in human and animal life.

The invention relates to macrolide antibiotics of the general formulas: These substances are soluble in methanol, ethanol, acetone, chloroform, ethyl acetate, butyl acetate, acidified water, benzene, ethyl ether and carbon tetrachloride and only slightly soluble in petroleum ether, n-hexane and neutral water. They are basic and form salts with acids, which react positively in the erythromycin test with 50% sulfuric acid and, in contrast, react negatively with anhydrin and iron chloride reagents. They contain only the elements carbon, hydrogen, nitrogen and oxygen, are levorotatory in ethanol and have the characteristics of macrolid antibiotics.

The exact properties of the SF-837-A2 substance are as follows: - The SF-837-A2 substance free base forms a white powder with a. Melting point from 125-128 ° C; the rKa value in 50μ aqueous ethanol is 6.8; the Elemental analysis gives C 60.58, H 8.85, N 1.72% and 0 28.85 (equilibrium); the molecular weight determined by mass analysis is 827; - the molecular formula is C42H69015N; - at a concentration of 1% in ethanol, the optical is Rotation at (α) D22-68 °; - when dissolved in ethanol, there is a characteristic Absorption maximum at a wavelength of 232 m / l (E1cm1% = 320) in the ultraviolet Range of spectrum; 1cm - if granulated in the form of the free base in potassium bromide, the characteristic absorption bands lie in the infrared range of the spectrum with the following wave numbers in cm 3500, 2970, 2935, 1737, 1460, 1410, 1377, 1360, 1300, 1275, 1195, 1170, 1123, 1082, 1052, 1017, 990, 920, 910, 860, 840, 805, 780, 740 and 700; The diacetyl derivative of the SF-837-A2 substance forms white needles with a melting point of 130-1340 C and gives an elemental analysis of C 60.68%, H 8.23%, N 1.49 f0 and 0 29.60 Vo (equilibrium) and therefore has the empirical Formula C46H73O17N; - the exact properties of the SF-837-A3 substance are as follows: - the free base of the Sf-837-A3 substance forms a white powder with a melting point from 122-125 ° C; - in 50% aqueous ethanol the pEa value is 7.0; - the Elemental analysis gives C 60.53%, H 8.23%, N 1.87%, 0 29.32% (Balance); the molecular weight determined by mass analysis is 811; - the molecular formula is C41H65015N; - at a concentration of 1% in ethanol if the optical rotation is (α) D22-42 °; - when dissolved in ethanol, lies a characteristic absorption maximum at a wavelength of 280µ (E1cm1% = 295) in the ultraviolet region of the spectrum; - if in the form of the free base in Granulated potassium bromide, the characteristic absorption bands are in the infrared Range of the spectrum at the following wave numbers in cm-1: 3500, 2970, 2930, 1738, 1680, 1640, 1600, 1460, 1378, 1360, 1300, 1275, 1252, 1190, 1168 ', 1121, 1083, 1052, 1015, 980, 863, 840, 805 and 780; forms the monoacetyl derivative of the SF-837-A3 substance sand-like crystals with a melting point of 182-185 ° C and has an elemental analysis of C 60.56, H 7.92%, N 1.68% and 0 29.84% (balance), while the empirical formula Is C43H67O16N.

- The exact properties of the SF-837-A4 substance are as follows: - the free base of the SF-837-A4 substance forms a white powder with a melting point from 120-122 ° C; in 50% aqueous ethanol the pKa value is 7.0; - the elemental analysis yields C 60.82%, H 8.52%, N 1.73% and 0 28.93% (balance); the molecular weight determined by wet analysis is 825; the molecular formula reads 42 67 15 N; - at a concentration of 1 in ethanol, the optical Rotation (α) D22 -40 °; - when dissolved in ethanol, there is a characteristic Absorption maximum at a wavelength of 280 mµ (E1cm1% = 285) in the ultraviolet Range of spectrum; - if granulated in the form of the free base in potassium bromide, there are characteristic absorption bands in the infrared range of the spectrum the following wave numbers in cm 1 3500, 2970, 2930, 1738, 1680, 1640, 1600, 1460, 1378, 1360, 1300, 1276, 1252, 1190, 1170, 1120, 1082, 1052, 1017, 980, 910, 863, 840 and 780; the monoacetyl derivative of the SF-837-A4 substance forms sand-like crystals with a melting point of 166-168 ° C, an elemental analysis of C gives 60.85 %, H 8.06%, N 1.65 o / o and 0 29.44% (compensation) and has a molecular formula of C44H69O16N.

Does not contain any of these SF-837-A2, SF-837-A3 and SF-837-A4 substances the elements sulfur, phosphorus and halogens.

When the erythromycin test is done with 50% sulfuric acid the SF-837-A2 substance gives a brownish and reddish purple color, while the Sf-837-A3 substance and the SF-837-A4 substance - a pale yellowish brown Show color.

As acid addition salts of these SF-837-A2-, SF-837-A3- and SF-837-A4 substances according to the invention there can be mentioned, for example, salts of these substances with non-toxic organic and inorganic acids such as hydrochloric acid, sulfuric acid, Hydrobromic acid? Phosphoric acid, nitric acid, acetic acid, citric acid, maleic acid, Malic acid, tartaric acid, cinnamic acid, ascorbic acid, glycolic acid and the like.

The invention is described below with reference to the accompanying Drawing described in more detail.

1 shows a graph of the ultraviolet absorption spectrum the SF-837-A2 substance in the form of the free base dissolved in ethanol, Fig. 2 a Curve of the infrared absorption spectrum of the SF-837-A2 substance in the form of the free, free base granulated in potassium bromide, FIG. 3 is a graph of the ultraviolet absorption spectrum the SF-837-A3 substance in the form of the free base dissolved in ethanol, Fig. 4 a Curve of the infrared absorption spectrum of the SF-837-A3 substance in the form of the free, in base granulated in potassium bromide, FIG. 5 is a graph of the ultraviolet absorption spectrum the SS-837-A4 substance in the form of the free base dissolved in ethanol and Fig, 6 is an infrared absorption spectrum graph of the SF-837-A4 substance in the form of FIG free base granulated in potassium bromide.

When the SF-837-A2, SF-837-A3 and SF-837-A4 substances of a silica gel or aluminum thin layer chromatography using various solvent systems are subjected to, they result in a single spot with different Rf values, so that the purity and homogeneity of these substances is confirmed.

The Rf values of these substances in different solvent systems are listed in the table below.

T abe 1 1 e 1 Rf value SF-837-A2- SF-837-A3- SF-837-A4- Substance substance substance Longitudinal center systems Silica gel thin layer Chromatography Benzene acetone 2: 1 0.51 0.50 0. -55 n-butanol acetic acid Water (3: 1: 1) 0.68 0.68 0.69 Methanol 0.83 0.83 0.84 Aluminum thin film Chromatography Ethyl acetate-benzene 2: 1 0.40 0.45 0.52 Ethyl acetate 0.84 0.87 0.91 The antibacterial or bacteria-preventing spectra of the SF-837-A2, SS-837-A3 and SF-837-A4 substances and their acetyl derivatives are shown in Table 2 below.

The minimum inhibitory concentrations of these new antibiotics were determined by, as indicated in Table 2, the broth dilution method with various Culture media was applied.

Table 2 minimum inhibitory concentration (mcg / ml) SF-837-A2 diacetyl- SF-837-A3- mono- use Substance SF-837-A2 Substance acetyl. Substance acetyl. dete Substance SF-837-A3 SF-837-A4 Culture test Substance substance media Microorganisms Staphylococcus aureus 209P 0.39 0.73 0.39 0.78 0.39 0.78 1 Staphylococcus aureus 209P resistant to Penicilin 0.39 0.39 0.39 0.39 0.39 0.39 1 Staphylococcus aureus 209P resistant to Streptomycin & A.249 substance 0.39 0.78 0.39 0.39 0.78 0.78 1 Staphylococcus aureus 209P resistant to Novobiocin 12.5 12.5 12.5 12.5 12.5> 25 1 Staphylococcus aureus 209P resistant to Actinomycin - - - - - - 1 Table 2 (continued) Staphylococcus aureus 209P resistant to Kanamycin 0.78 1.56 0.78 1.56 0.78 1.56 1 Staphylococcus aureus Smith 0.1 0.2 0.2 0.2 0.2 0.4 1 Staphylococcus aureus Terajima 0.39 0.78 0.78 1.56 0.78 1.56 1 Staphylococcus aureus resistant to Streptomycin, Penicilin & Tetracycline 0.78 1.56 1.56 3.125 1.56 3.125 1 Staphylococcus aureus 193 - - - - - 1 Bacillus subtilis ATCC 6633 0.2 0.4 0.39 0.78 0.39 0.39 1.56 1 Sarcina lutea 0.05 0.1 0.1 0.2 0.1 0.1 1 Escherichia coli>25>25>25>25>25> 25 1 Pseudomonas aeruginosa>25>25>25>25>25> 25 1 Proteus vulgaris>25>25>25>25>25> 25 1 Table 2 (continued) Klebsiella pneumoniae - - - - - - 1 Shigella dysenteriae - - - - - - 1 Mycobacterium smegmatis 607 12.5>25>25>25>25> 25 2 Mycobacterium phlei - - - - - - 2 Candida albicans>25>25>25>25>25> 25 3 Penicillium chrysogenum>25>25>25>25>25> 25 3 Aspergillus niger - - - - - - 3 Saccharomyces cerevisiae - - - - - - 3 Referring to Table 2, the following culture media were used: 1: Heart infusion medium 2: Glycerine broth medium 3: Sabouraud medium From the values in Table 2 it can be seen that the SF-837-A2-, SF-837-A3- and SF-837-A4 substances have useful antibacterial and bactericidal activity against gram-positive bacteria as well as pus-producing bacteria resistant to known antibiotics. It has not been found that the anti-bacterial activity of the SF-837-A2, SF-837-A3 and SF-837-A4 substances can be inactivated by serum.

Experiments on toxicity in the case of oral and parenteral administration found that the SF-837-A2, SF-837-A3 and SF-837-A4 substances all have one have low toxicity.

When the free bases of these substances were administered orally to mice, they had LD50 values of 3100 mg / kg, 2900 mg / kg and

2850 mg / kg.

It was further found that the inventive SF-837-A2, SF-837-A3 and SF-837-A4 substances (free bases) and their acetyl derivatives are suitable Antibiotics are.

When treating ICR mice that are infected with a lot of the Streptococcus haemolyticus Ti-125 Gr-A Type I breed had been infected that were 100 times larger was than the LD50 value, the oral application of the free bases of-S? -837-A2-, SF-837-A3 and SF-837-A4 substances CD50 values of 135 mg / kg, 170 mg / kg and 155 mg / kg.

The therapeutic effect of the di-acetyl derivative-the SF-837-A2 substance, the mono-acetyl derivative of the SF-837-A3 substance and the mono-acetyl derivative of the SF-837-A4 substance was examined in the following manner: An aqueous suspension of bacteria of the Staphylococcus aureus Smith breed was administered intraperitoneally to ICR mice in a Injected dose that was 100 times greater than the LD50 value. Thirty minutes after The infected mice were then injected by oral application of an aqueous Treatment suspension containing the active test substance and a small amount of gum arabic In each group 10 mice were treated in this way and after Treatment fed for 7 days. The results of a series of attempts became found, or estimated, that these acetyl derivatives have useful CD50 values of 180 mg / kg, 200 mg / kg and 200 mg / kg, respectively.

For therapeutic purposes, the free bases, the acid salts and the acetyl derivatives of SF-837-A2, SF-837-A3 and SF-837-A4 substances of Invention for oral use suitably in tablets or capsules be shaped while an aqueous solution or suspension suitable for injection where, if necessary, pharmaceutically acceptable additives such as Tragery excipients, Suspending agents and the like can be added. For making a aqueous solution suitable for injection, preferably due to its high Water solubility, acid addition salts such as tartrates used. The acid addition salts and the acetyl derivatives of the SF-837-A2, SF-837-A3 and SS-837-A4 fabrics of the present invention are usable not only in their isolated form, but for therapeutic purposes also in the form of a mixture of two or more of these substances.

The invention also relates to a method for production the SS-837-A2, SF-837-A3 and SF-837-A4 substances. This procedure consists of that a breed of Streptomyces mycarofaciens in a culture or nutrient medium, containing assimilable nitrogen and carbon sources, under aerobic conditions cultivated or grown to the SF-837-A2 substance, the SF-837-A3 substance and to produce and propagate the SF-837-A4 substance in the culture, and that then these antibiotics, which are present in a mixture, are removed from the culture and, if necessary, separated from each other.

The microorganism that, during breeding, simultaneously produces the SF-837 substance, produces the SF-837-A2 substance, the SF-837-A3 substance and the SF-837-A4 substance, was first isolated by the inventors from a soil sample and named Streptomyces mycarofaciens nov. sp. referred to, which has been deposited with the American Type Kultur Collection, Washington D.C., under ATCC. No. 21454.

The Streptomyces mycarofaciens has the following biological properties; (I) Morphological observations 1.) Air mycelium: Buft mycelium, produced on glycerine Czypek's agar, Glycerin calcium maleate agar and synthetic starch agar etc. abundant open Spirals.

2.) Spores: The spores are spherical, oval or elliptical Shape, the surface structure is thorny or prickly, (relatively thinner and longer sting 3 and the size of the spore is 0.5-0.7 Micron - at 0.8 to 1.0 micron.

(II) The properties on different cultures result in nutrient media from the following table 3.

Table 3 Culture medium growth aerial mycelium soluble pigment Secrose-Czapek- low growth sparse, frizzy none Agar colorless, (frizzy) to off-white whitish yellow Glycerin-Czapek- dark brown white to cream-colored, none or very much Agar partially with pale pink formation of greyish-colored and frizzy aerial mycelium Krainsky glucose light brown to white, with rosé none or weak Asparagine agar reddish-brown color, brownish yellow Ushinsky glucose brown with reddish pink to lavender Asparagine agar chem paint colors light brown Calcium Maleate Low Growth None None Cream colored agar rose, Glycerin-calcium-light brown gradually transitioning none maleate agar too greyish (curled) Table 3 (continued synthetic dark brown with rosé with strong red none Starch-agar with a purple, all- stroked like a transition in grayish (curled) Meat broth - yellowish-brown, very sparse, none white Agar Glucose meat - thick, good and white to yellowish - none broth-agar brown to dark-cream colored pale brown Glucose peptone brown sparse, white none Agar Tyrosine agar reddish brown white none Potato-thick, good and plentiful, blackish- snippets brown to grayish-brown brown blackish Brown Potato brown white to cream brown on the snippets of the periphery of the Growth Skimmed milk ring-shaped, sparse, none white Surfaces- Growth, light brown Table 3 (continued) Egg pale brown none none Glucose Czapek surfaces - sparse, none white Solution and soil growth pale brown Loeffler's coagulated pale brown none none serum Gelatin up to cream color (20 ° C) pale brown none none Bennett's brown to rose with reddish blackish-tinted light brown Brown Cellulosic Medium no growth --- --- Note: Unless otherwise stated, the incubation temperature is generally 28 ° C.

(III) Physiological properties: Formation of hydrogen sulfide: negative formation of tyrosinase: negative formation of nitrite: positive coagulation of Skimmed milk: positive peptonization of skimmed milk: negative hydrolysis of starch : positive liquefaction of gelatine: positive (weak) decomposition of Loeffler's coagulated serum: negative chromogenic effect; negative (IV) Use of carbon sources: 1.) Suitable: glucose, galactose, fructose, maltose, lactose, dextrin, starch, Glycerin, inositol, mannose, salicin, sodium acetate, sodium citrate, sodium succinate.

2.) Doubtful: arabinose and L-rhamnose.

3.) Not to be used: xylose, sucrose, raffinose, dulcitol, sorbitol, Mannitol and cellulose.

- (V) Growth temperature: 15 to 380 C.

The above mentioned micro-biological properties of the SF-837-A2, SF-837-A3 and SF-837-A4 substance-producing breed (hereinafter referred to as SF-837 breed designated) can be summarized as follows: The spore carrier is spiral-shaped, the spore is prickly. A cream-colored can be made from synthetic culture or nutrient media to brown to reddish brown colored growth can be observed, whereby a rose colored aerial mycelium and a frizzy gray colored aerial mycelium forms are. No substantial formation of soluble pigment can be observed while the formation of soluble pigment with a light brown color at a limited group of synthetic culture media can be observed. With organic In culture media, on the other hand, brown colored growth is generally observed with the formation of white or cream-colored aerial mycelium, but without formation a soluble pigment. However, aerial mycelium is abundant in potato schnitzel formed of greyish brown color and the formation of blackish colored, soluble Pigment observed.

In view of the description of Waksman (Waksmants "? He Actinomycetes" Volume 2, 1961) it can be seen that the above-mentioned cultural characteristics of the SF-857 breed partly similar to the properties of the Fradiae species, the Ruber species and the Flavus species of the genus Streptomyces.

Since the SF-837 breed is negative in melanin formation and formation of scented mycelium showing pink in color or tinge, the SF-837 breed becomes first compared with the races of the Fradiae species, namely Streptomyces fradiae, Streptomyces luridus, Streptornyces roseus and Streptomyces fuscus.

The SF-837 breed is different from the breeds of the Fradiae species in that in the SF-837 breed the scented mycelium with pink color or paint occurs temporarily in the initial stage of incubation and in many cases during the middle and later stages of incubation turn gray and frizzy air niycelium changes, while the pink color or tint, the one at the Races of the Fradiae species are observed, is very stable.

The aerial mycelium of Streptomyces fradiae is straight and differentiated get off the spiral of the SF-837 breed. Streptomyces fradiae is also different distinct from the SF-837 breed in that Streptomyces fradiae is an abundant Formation of aerial mycelium on sucrose-Czapek agar and colorless growth Starch agar shows.

The SF-837 breed is different from Streptomyces fuscus and Streptomyces luridus in that Streptomyces fuscus does not have the spiral formation, while Streptomyces luridus only shows spirals in a limited group of culture media, while the SF-837-Ras5e shows abundant spiral formation on many culture media. Streptomyces roseus is comparable to the SF-837 breed insofar as the spiral formation can be observed, but these two are distinctly different from each other distinguish Streptomyces roseus a colorless growth on synthetic However, starch agar does not show the formation of aerial mycelium on potato chips having.

Because the SF-837 breed is negative in melanin formation and growth of red paint or paint, the SF-837 breed is compared second with Streptomyces albos-poreus, Streptomyces erythraeus, Streptomyces niveoruber and Streptomyces purpurascens, etc. belonging to the Ruber species.

S-treptomyces niveoruber clearly distinguishes silch from the SF-837 breed in that the surface structure of the spore of the first mentioned type is uniform while that of the last-mentioned At is prickly. Streptomyces albosporeus further differs from the SF-837 breed in that Streptomyces albosporeus Forms aerial mycelium, which is mainly straight, with a pronounced red; colored Growth on sucrose Czapek agar and on glycerine calcium maleate agar is present while no aerial mycelium is formed on starch agar. Streptomyces erythraeus does not match the SF-8T7 breed in that it is Streptomyces erythraeus on sucrose Czapek agar and on potato pulp agar a red colored one Shows growth while showing an off-white growth on glucose-asparagine agar and synthetic starch agar is present on which the SF-837 breed is growing shows with reddish paint. Streptomyces purpurascens is consistent with the SF ~ 837 breed regarding the prickly or

fibrous structure of the surface of the spores, however Streptomyces purpurascens differs from the SF-837 breed in that the first The named species has a clearly soluble pigment of red color or paint synthetic culture medium, while the SF-837 breed does not form such soluble Having pigment.

The SF-837 breed continues with Streptomyces microflavus and Streptomyces roseflavus, which belong to and are accepted by the Flavus species becomes that they are closely related to the SF-837 breed. Like the cultural characteristics show it, Streptomyces microflavus and Streptomyces roseoflavus are related Certain points close to the SF-837 breed, but with the first two mentioned Breeds form spores, the surface structure of which is even or smooth, (H.D. Tresner and others "Journal of Bacteriology" Volume 91, pages 1993-2005, 1966) was a clear difference to the prickly or fibrous spore structure of the SF-837 breed means.

It follows that there is no race among the known Species of the genus Streptomyces can be found that coincide with the SF-837 breed.

On the other hand, the new antibiotic SF-837-A2-, SF-837-A3- and SF-837-A4 fabrics produced by the SF-837 breed typical of basic macrolide part -biotics. Of the new antibiotic substances according to the invention the SF-837-A2 substance shows an absorption maximum at a wavelength of 232 m in the ultraviolet spectrum, from which it can be concluded that this substance an antibiotic of the same kind as leucomycin, josamycin, spiramycin, miamycin and tertiomycin. The SF-837-A3 and SF-837-A4 substances also show high Absorption maximum near the wavelength of 280 m in the ultraviolet spectrum, from which it can be concluded that these two substances are antibiotics of the same Kinds are like Niddamycin, Oarbomycin B, Tylosin, Relomycin and Macrocin. For this The microbiological properties of the SF-837 breed are now based on the Properties of microorganisms compared to the above known antibiotics form.

It is easy to understand that the SF-837 breed is different from the leucomycin producing microorganism Streptomyces kitasatoensis; the josamycin producing microorganism Streptomyces narbonensis var. Josamyceticus; dem, spiramycin producing microorganism Streptomyces Anbofaciens; the miamycin producing microorganism Streptomyces Anbofaciens; the tertiomycin producing microorganism Streptomyces eurocidicus; Streptomyces albireticuli; the carbomycin B producing microorganism Streptomyces halstedii; the tylosin and macrocin producing microorganism Streptomyces fradiae and the relomycin-producing microorganism Streptomyces hygroscopicus, there are significant differences between the morphological characteristics of the SF-837 breed and the properties the known microorganisms are present, as shown in Table 4 below.

ICable 4 Surfaces- Educated aerial structure of the Antibiotic microorganism celium spore SF-837-A2- SF-837-A3- SF-837-breed spiral prickly SF-837-A4- substance leukomycin Streptomyces whorl qatt kitasatoensis Josamycin Streptomyces straight narboensis var. linear smooth josamyceticus Spiramycin Streptomyces spiral smooth ambofaciens Miamycin Streptomyces spiral smooth ambofaciens Tertiomycin Streptomyces whorl smooth eurocidicus Tertiomycin Streptomyces whorl smooth albireticuli Carbomycin B Streptomyces spiral smooth halstedii Tyrosine Streptomyces straight-smooth fradiae linig Macronin Streptomyces straight fradiae lined smooth Relomycin Streptomyces spiral smooth hygroscopicus The SF-837 breed also differs markedly from the niddamycin-producing microorganism Streptomyces djakartensis (which is described in German Patent 1,077,381), of which the spore surface structure is not described, the difference being that Streptomyces djakartensis produces melanin is positive, but there is no peptonization or coagulation of skimmed milk.

Have the above microbiological comparisons and considerations tremble that the SF-8) 7 breed is not just a new microorganism that the basic macrolid antibiotics, but also the SF-837 races are different from all of them known races, even from the point of view of natural science System knowledge of all Actinomycetes.

Therefore, this SF-837 breed was named Streptomyces mycarofaciens nov. sp. designated.

The SF-837 breed has traits that are prone to change, like it can commonly be seen in other Streptomyces. So can the SF-837 breed for example, variants and mutants form when they are known with different Mutant generators is treated, e.g. B. ultraviolet rays, X-rays, high frequency magnetic waves, radioactive rays, chemicals, etc. All natural and artificial variants and mutants of the SF-837 breed can be used in conjunction can be used with the method of the invention as long as they are suitable to form one of the SF-837-A2, SF-837-A3 or SF-837-A4 substances according to the invention.

According to the method of the invention, the SF-837 breed or their variants or mutants in a known manner in a culture or nutrient medium be bred or cultivated, the contains the nutrients that used for common microorganisms. All of them can be used as sources of nutrients use known nutrients that are commonly used in breeding or cultivation have been used by Streptomyces. As a carbon source, b-e.e. Glucose, starch, glycerine, dextrin, sucrose or sucrose, saccharified, are used Starch, molasses and the like. As a nitrogen source, for example, can be used become soybean meal, wheat seedlings, meat extract, peptone, corn water, soluble vegetable protein, dry yeast, ammonium sulfate, sodium nitrate and the like. If necessary, the culture or Nutrient medium inorganic salts such as calcium carbonate, Sodium chloride, potassium chloride, phosphates and the like can be added. Additionally Organic and inorganic substances can be added to support the growth of the Promote SF-837 breed and the formation of at least one of SF-837-A2- ,, SF-837-A3-, or accelerate SF-837-A4 substances.

As a method of breeding or cultivating the SF-837 breed is the liquid culture and especially the liquid culture under submerged aerobic conditions most advantageous, in a manner similar to conventional procedures for the production of the known antibiotics. The breeding or culture can take aerobic Conditions take place at a suitable fermentation temperature of about 20-30 ° C. For the commercial or laboratory production of these SF-837-A2-, SF-837-A3- and SF-837-A4 substances, however, it is often preferable to use the culture method to be carried out at a temperature in the vicinity of 280 C. Under these circumstances reach the concentrations of the SF-837-A2, SF-837-A3, SF-837-A4 substances in of the culture broth a maximum at the end of a fermentation time of 2 - 5 days, under either in shake cultures or tank cultures.

The SF-837 substance is the main metabolic product of Streptomyces mycarofaciens and the SF-837-A2, SF-837-A3 and SF-837-A4 substances are usually formed in smaller proportions overall, on the order of magnitude from about 5 to 20 percent by weight of the SF-837 substance produced.

The SF-837-A2, SF-837-A3 and SF-837-A4 substances can be used in the Breeding of the SF-837 breed can be produced simultaneously and, as mentioned above, have comparable physicochemical properties; these substances can come from the culture can be removed, purified and separated by generally known Procedures are used that are commonly used for recovery, purification and isolation of the known basic antibiotics are available.

The recovery of the SF-837-A2, SX-837-A3 and SF-837-A4 substances from the culture can be done in different ways.

The SF-837-A2, SF-837-A3 and SF-837-A4 substances produced by breeding of the SF-837 breed are both in the solid phase, the iliac cakes as well as in the liquid phase of the culture. The culture can be in be filtered in a known manner in order to remove the filter cake (i.e. the solid phase which Containing mycelial cake) and the filtrate (i.e.

the liquid phase of the fermentation broth) separately. the Active substances present in the filtrate .können with a water immiscible organic solvents, e.g. ethyl acetate, butyl acetate, chloroform, Benzene, ethyl ether, methyl isobutyl ketone, butanol, etc., under neutral or weakly alkaline conditions.

The active substances present in the filter cake can also be used acidified water or extracted with a suitable water-miscible organic solvents such as methanol, ethanol and acetone, etc. Optionally, the Culture directly with a water-immiscible organic solvent previous filtration of the mycelial cake extracted so that the active ingredients pass directly from the culture into the organic solvent used.

The active ingredients that are in solution in the organic solvent can then be back extracted with acidified water. : 3e accruing Extract in the acidified water can then be neutral or weakly alkaline be made by using basic substances like sodium hydroxide, potassium hydroxide, sodium carbonate or sodium bicarbonate can be added, followed by a shaking treatment with a suitable organic solvent, so that the active ingredients pass back into the organic solvent phase. By doing this operation of the Reverse or reverse extraction can be repeated to a certain extent Impurities are separated from the active substances. The extract in the organic Solvent can be concentrated to dryness under reduced pressure, see above that a raw powder is obtained which contains the SF-837-A2, SF-837-A3 and SF-837-A4 substances contains. The solution of the active ingredients in the acidified water can optionally either be freeze-dried to preserve the acidic salts of the active ingredients, or the aqueous solution can be made neutral or weakly alkaline by a suitable basic material is added to the active ingredients in the form of the precipitate free bases.

The SF-837-A2, SF-837-A3 and SF-837-A4 substances are as above mentioned, besisch by nature, so that the culture or, its filtrate or solution of the Active substances can also be treated with ion exchangers, e.g. 3. an ion exchange resin, for example Amberlite IRC-50 etc., or an ion exchange cellulose, so that the active substances are adsorbed by the ion exchanger, which is then used with can be eluted with a suitable solvent.

The resulting raw powder, which is obtained in this way, with each other mixed active substances can be further extracted by extraction with one suitable organic solution are cleaned medium, z. 3. with benzene, which the The existing SF-83-7-A2, SF-837-A3 and SF-837-A4 substances can be dissolved in order to to remove any remaining impurities, or by washing with a suitable one Solvents, e.g. 3. Petroleum ether, ligroin or n-hexane, the SF-837-A2, SF-837-A3 and SF-837-A4 substances hardly or not at all, but can dissolve impurities well, and / or by adding petroleum ether, ligroin or n-He = an to a solution of the Active ingredients in an organic solvent such as 3. Benzene.

In this way, a single mixture of SF-837-A2, SF-837-A3 - and SF-837-A4 substances are obtained in which the total proportion of SF-837-A2, SF-837-A3 and SF-837-A4 substances usually on the order of 5-20 Weight percent of the proportion of the SF-837 substance varies depending on on the fermentation conditions, the method of recovery, the purification methods and other various factors. This purified mixture is ready as such for approach and application for therapeutic Purposes without that each substance component must be separated from it.

If desired, the purified mixture of SF-837-A2-, SF-837-A3- and SF-837-A4 substances are directly converted into a mixture of their Srea storage salts be done by the genic of free bases in an organic solvent like Ethyl ether is dissolved, the solution with a solution of an organic or inorganic acid such as hydrochloric acid, sulfuric acid, tartaric acid or citric acid is dissolved in a suitable solvent such as ethyl ether, whereby the Acid addition salts are precipitated in the form of a mixture that is filtered and Can be dried and then used for approach and application for therapeutic Purposes is ready. The purified mixture of the free bases can also be used as a mixture of the abovementioned acetyl derivatives are converted by means of a suitable Acetylation process, e. 3. by dissolving in pyridine, treating the solution with Acetic anhydride at room temperature or at a slightly higher temperature, this treatment being carried out for a sufficient time to produce the diacetyl derivative the SF-837-A2 substance and the monoacetyl derivatives of the SF-837-A3 and SF-837-A4 substances to obtain, and then by adding the acetylation products by adding ice water to be precipitated into the reaction mixture. The mixture of acetylated derivatives can then be filtered and washed with water to remove impurities to become free, and this purified mixture is ready for use as an eolch and for use for therapeutic purposes.

To the SF-837-A2, SF-837-A3 and SF-837-A4 substances from each other to be separated are chromatographic methods suitable, being a suitable adsorbent, e.g. activated aluminum oxide or silica gel or the like and an appropriate developing solvent may be used, or others different types of countercurrent distribution methods either alone or in combination To be used for the isolation of the components, it may be appropriate that the Purified mixture of free bases first using a countercurrent partition process is subjected to a fraction rich in the SF-837 substance from a Separate fraction rich in a mixture of SF-837-A2, SF-837-A3 and SF-837-A4 substances is, and that these two fractions are then separated by chromatography with silica gel or alumina to be subjected to complete isolation of each Reaching substances from each other.

In general, the separation of the SF-837-A2, SF-837-A3 and SF-837-A4 substances be carried out in a suitable manner such that, for. B a solution to the above Raw powder containing the active, recovered substances mixed together contains, in a suitable organic solvent of a chromatography in a column of activated alumina or silica gel is subjected by a mixture of ethyl acetate-benzene (1: 1) or benzene-acetone (4: 1) as a developing solvent is used, if necessary, these chromatographic separation methods several times be repeated. Usually the active ingredients can be extracted from the alumina column one after the other eluted in the order SF-837-A4 substance, SF-837-A3 substance, SF-837-A2 substance when ethyl acetate benzene (1: 1) is used as the developing medium. A A small portion of each of these fractions of the eluate is continuously layered by means of an aluminum oxide thin layer Chromatography examined. Fractions found to be associated with one of the active ingredients this aluminum thin-layer chromatography result in a single-spot characteristic, so that it can be assumed that they contain only one active ingredient, are combined with each other and then under reduced pressure to dryness one, these operations being repeated with each of the SF-837-A2 substance fraction containing alone with each of those containing the SF-837-A3 substance alone Fraction and with each of the fraction containing the SF-837-A4 substance alone. In this way, the pure free bases of the SF-837-A2, SF-837-A3 and SF-837-A4 substances separated from each other in the form of each white powder.

For cleaning and separating the SF-837-A2, SF-837-A3 and SF-837-A4 substances it should be added that every known method that has hitherto been used for purification and separation of macrolid antibiotics was used, also used in the previous case can, since the substances according to the invention also belong to the genus of macrolides Include antibiotics. The generation, recovery or isolating purification and separating the SF-837-A2, SF-837-A3 and SF-837-A4 substances from the culture the SF-837-Xasse and the subsequent steps to separate the individual substances each other, however, are preferably performed in such a way that the SP-837 breed at a temperature of 20 - 300 C in a well-ventilated liquid medium is grown or cultivated, the known assimilable nitrogen and carbon sources until the culture broth has significant antibacterial or anti-bacterial properties.

bactericidal activity that then only the fractions combined and concentrated in vacuo to dryness be that at aluminum oxide thin-layer chromatography has a single-spot characteristic for the SF-837 substance give rise to the free base of the SF-837 substance in the form a raw powder is obtained that the fractions are combined and in vacuo to Concentrated dry state, the outside of the silica gel column according to the above Fractions containing the single characteristic stain of the SF-837-A4 substance, however in front of the above-mentioned fractions, which represent each characteristic spot of the SF-837 substance are eluted, and the two SF-337-A2- and SF-837-A3 substances along with a smaller proportion of the SF-837 substance contained, creating a powder mixture of the SF-837-A2 and SF-837-A3 substances with a smaller proportion of the SF-837 substance it is obtained that this7 powder is dissolved in benzene and the resulting solution of this powder in benzene a Is subjected to chromatography in an activated alumina column, being a mixture of ethyl acetate-benzene and then a mixture of ethyl acetate-acetone is used as a developing solvent that the eluate thereof in fralc-tionen is collected, each of these Frakti.onen by aluminum oxide thin layer chromatography it is investigated that these small fractions are combined and in vacuo to dryness are concentrated in the last-mentioned aluminum thin-layer chromatography result in the single spot characteristic of the SF-837-A3 substance, whereby the Free base of the SF-837-A3 substance is obtained in the form of a raw powder that then the remaining fractions of the eluate from the activated alumina column mentioned above combined and concentrated in vacuo to dryness to give a powder, which mainly contains the fact that this powder is again dissolved in benzene and these Benzene solution, in turn, undergoes chromatography in a column of activated alumina is subjected, using a mixture of ethyl acetate-benzene as the developing solvent it is used that the eluate thereof is collected into fractions and each of these fractions by means of aluminum oxide thin-layer chromatography is examined, and then that only the fractions, cie in the last mentioned aluminum oxide thin-layer chromatography result in the single spot characteristic of the SF-837-A2 substance, combined and concentrated in vacuo to dryness, releasing the free base of the SF-837-A2 substance is obtained in the form of a raw powder, The SF-837-A2, SF-837-A3 AND SF-837-A4 substances, which, as described above, have been separated from one another into the pure state, could, if desired, each subsequently be converted into the corresponding acid addition salt or converted into the above-mentioned acetyl derivative. The conversion to the Acid addition salt can for example be done by adding the free base is dissolved in a suitable organic solvent such as ethyl ether and then a saturated solution of a suitable organic or inorganic acid such as Tartaric acid, citric acid, acetic acid, hydrochloric acid, sulfuric acid and the like, namely dissolved in ethyl ether, is added.

The conversion into the diacetyl derivative of the SF-837-A2 substance or the monoacetyl derivatives of the SF-837-A3 and SF-837-A4 substances can, for example be carried out by dissolving the free base in pyridine and then dissolving the solution with a large excess of acetic anhydride in the manner described above is treated.

The invention is illustrated by the following examples.

Example 1 The SF-837 breed, namely Streptomyces' mycarofaciens, deposited under ATCC No. 2145.4, was inoculated into 300 l of a liquid culture medium, the 2.0% glucose, 1.5% peptone, 0.5% meat extract, 0.4% corn water, 0.2% NaCl, Contained 0.3% CaCo3 and 0.2% pork fat at pH 7.0; afterward was stirred at 28 ° C. for 70 hours with ventilation.

The fermented broth or nutrient solution was filtered directly and the filter cake containing the mycelium cake is washed with dilute hydrochloric acid. The culture filtrate combined with the washing liquid was in a total volume of 280 l obtained (strength 320 mcg / ml). The filtrate was extracted with 70 l of ethyl acetate and 71% of the resulting ethyl acetate phase were reduced to about 20 1 concentrated.

The concentrate was diluted with 10 l of water by adding 5N Hydrochloric acid adjusted to pH 2 and then shaken vigorously. The watery Phase was separated from the organic phase by adding 3N sodium hydroxide adjusted to a pH value of 9 and then extracted with 4 l itthyl acetate. Of the Resulting ethyl acetate extract was then in the same way together with 2 liters of aqueous Hydrochloric acid shaken to transfer the active ingredients into the latter, which then was again extracted with 1 l of ethyl acetate at a pH of 8. This ethyl acetate extract was then dried over anhydrous sodium sulfate and under reduced pressure concentrated to give 92 g of a crude yellow powder.

90 g of this raw powder were dissolved in 1 l of ethyl acetate and the solution sent through a column b.zwX column of 1.5 1 carbon powder, which with Ethyl acetate was impregnated. the Development was using carried out by ethyl acetate as solvent. and the active fractions of the eluate collected in a total amount of 5.5 1 and then under reduced pressure to Evaporated dry to give 55 g of a white powder (strength 700 mcg / mg). This powder was countercurrently distributed with benzene and 0.3 M phosphate buffer (pH 4.40). (2.5 liters of each solvent were used and the lower one movable layer removed 10 times.) The SF-837 substance was in the first through third Pipes and mainly distributed in the second pipe, and larger proportions (about 80%) of the SF-837-A2, SB-837-A3 and SF-837-A4 substances were in the first Pipe withheld.

The portion (2.4 1) in the first tube was concentrated under reduced pressure, to give 7.7X of a white powder (strength 720 mcg / mg). This powder was dissolved in 15 ml of benzene, any insoluble substances present were filtered off and the filtered solution then sent through a column or column of 800 ml of silica gel, which with Benzene was impregnated. The absorbed active substances were developed chromatographically, by using a solvent mixture of benzene-acetone (4: 1) and the eluate in Fractions of 50 ml each were collected. A smaller proportion of each of these obtained fractions was examined by performing an alumina thin layer chromatography was subjected (with 2: 1 ethyl acetate-benzene as a developing solvent). the Fractions 28 to 30, which in the aluminum oxide thin-layer chromatography a Individual spots were combined and concentrated in vacuo to give 140 to give mg of a white powder with a melting point of 120-1220 C; this Powder was identified as the pure free base of the S3P-837-A4 substance by analysis.

Fractions 38 to 57 obtained from aluminum oxide thin-layer chromatography gave three spots, were combined and concentrated in vacuo to give 1.1 g of a white To give powder that, according to analysis, the SF-837-A2 and SF-837-A3, substances together with a smaller amount of the SP-837 substance.

Example 2 In 4 ml of benzene, 1 g of the white powder was dissolved which the SS-837-A2 and SF-837-A3 substances along with a smaller amount of the SF-837 substance and obtained by silica gel chromatography according to Example 1 was.

The solution was subjected to a chromatographic separation process, by passing them through a column of 100 ml of aluminum oxide which was impregnated with benzene and by using a mixed solvent from ethyl acetate-benzene (1: 1) was developed. The eluate was in fractions of 20 ml each are taken and each fraction is examined by using an alumina thin-layer chromatography (using ethyl acetate-benzene (2: 1) as a developing solvent). Fractions 30 to 48 were combined and concentrated in vacuo to give 230 mg of one to give white powder with a melting point of 122 to 125.0 C; the analysis found that this powder consisted of the pure SF-837-A3 substance free base.

After fraction 80 was eluted from the column, was changed the developing solvent to a mixture of ethyl acetate-benzene (2: 1) and fractions 90-125 collected and concentrated in vacuo to give 280 mg of a to give white powder which, according to analysis, contains about 70% by weight of the SF-837-A2 substance contained. 250 mg of this powder were then in ml of benzene dissolved and subjected the solution to chromatography by passing it through a column or Column of 50 ml of aluminum oxide, which was impregnated with benzene, sent and with .500 ml of ethyl acetate-benzene (1: 3) and then with a mixture of ethyl acetate-benzene (2: 1) was developed. The eluate was taken in fractions of 5 ml each and each fraction examined by alumina thin layer chromatography. The fractions 36 to 47, which in this aluminum oxide thin-layer chromatography spotted were combined and concentrated in vacuo to yield of 80 mg of a white powder having a melting point of 125-128 ° C; this powder was determined by analysis to be the pure SF-837-A2 substance free base identified.

Example 3 The SF-837 breed was grown in 30 liters of a liquid culture medium inoculated, the 3.0% glucose, 5 5S soluble vegetable protein, 0.2 S KOl and 0.3 cXs CaCO3 at pH 7.0 and with aeration for 25 hours in a stand or

Bottle fermenter stirred at 300 a. The fermented broth or Nutrient solution was filtered directly and the filter cake containing the mycelium cake washed with dilute hydrochloric acid.

The combined with the washing liquid culture filtrate was in a Get a total of 28 1 (strength 300 mcg / ml).

The filtrate (pH 8.4) was extracted with 7 1 butyl acetate and the obtained: butyl acetate extract then in a similar manner to example 1 treated to carry out the re-extraction of the active ingredients, producing a yellow Raw powder was obtained in a yield of 15.2 g. 15 g of this raw powder were dissolved in 200 ml of butyl acetate and the solution a chromatography subjected by passing through a column of 400 ml of butyl acetate impregnated carbon passed through and then developed with butyl acetate became. The active fractions of the eluate were up to a total of 1.8 1 and then concentrated in vacuo to about 220 ml. The concentrate was with 50 ml of distilled water diluted, with the addition of 6N hydrochloric acid to a p, -H value set from 2 d and shaken vigorously, the aqueous phase was removed from the organic Phase separated and the aqueous solution by adding 1N sodium hydroxide to one A pR value of 9.0 was used to precipitate a precipitate. This precipitate was filtered and dried in a drier to give 6.3 g of a white Powder (strength 720 mcg / mg) was obtained, This powder was in 12 ml of benzene dissolved and the insoluble substances filtered off. The filtered solution became one Subjected to chromatography by passing through a column of 600 ml with benzene impregnated silica gel was passed through; subsequently the adsorbed Active ingredients with a solvent mixture of benzene-acetone (6: 1) ntf wraps. The eluate was taken in fractions of 20 ml each. The SF-837-A2-, SF-837-A3- and SF-837-A4 substances were eluted from the SF-837 substance in the column these fractions were examined by means of silica gel thin layer chromatography (using benzene-acetone (2: 1) as developing solvent). The factions 64 to 82 were combined and concentrated in vacuo to give 800 mg of a white powder to erge'ben, of which the analysis showed that it is the SF-837-A2, SF-837-A3 and SF-837-A4 substances together with a smaller amount of the SF-837 substance. This white powder was then subjected to a chromatographic separation subjected in a silica gel column as in 3 example 1, and then subsequently a chromatographic separation with the aluminum oxide column as in example 2, whereby 15 mg of the pure free base of the SF-837-A2 substance w 85 mg of the pure SB-837-A3 free base and 35 mg of pure SF-837-A4 free base each obtained in the form of a white powder.

Example 4 If in each case 200 mg of the free bases according to the examples 1 and 2 isolated SF-837-A2, SF-837-A3 and SS-837-A4 substances with a saturated Solution of citric acid was treated, the corresponding citrates were the SF-837-A2, SF-837-A3 and SF-837-A4 substances in yields of 210 mg each, Obtained 220 mg and 190 mg.

Example 5 In 20 ml of distilled water, 300 mg of SS-837-A3 substance was added placed in suspension and the resulting suspension an aqueous solution of tartaric acid added. The mixture was stirred well and adjusted to pH 4 and filtered to remove some insolubles. The filtrate was freeze-dried, around 340 mg tartrate of the SF-837-A3 substance in the form of a white powder with a To give melting point of 115 ° C.

Example 6 300 mg of the free base were added to each 1.5 ml of pyridine of the SF-837-A2, SF-837-A3 and SF-837-A4 substances are dissolved and the resulting solution 1.0 ml acetic anhydride was added.

The mixture was stirred, and then at room temperature for 20 hours ditched. The reaction mixture was then poured into ice water and stirred to collect a precipitate to create. This precipitate was filtered and dried in a dryer, to give 330 mg of a white powder. The recrystallization of this powder from carbon tetrachloride, gave the di-acetyl derivatives of SF-837-A2 substance and the mono-acetyl derivatives of SF-837-A3 and SF-837-A4 substances in the form of white Crystals in yields of 3-10 mg (melting point 130-134 ° C), 295 mg (melting point 182-185 ° C) or 300 mg (melting point 166-168 ° C).

Claims (2)

P a t e n t a n 5 p r ü c h e 1. Macrolide antibiotics of the general formulas: in which R1 is the radical -COCH2CH3, R2 and R3 are hydrogen or a radical COCH3 and R4 is a radical -COCH2CH2CH3 and their acid addition salts. 2. Process for the preparation of the substances SP-837-A2 and their Diacetyl derivative, SF-837-A3 and SF-837-A4 and their monoacetyl compounds by conventional aerobic cultivation of a breed of Streptomyces mycarofaciens in one Nutrient medium containing assimilable nitrogen and carbon sources thereby characterized in that a strain of the breed Streptomyces mycarofaciens is deposited under the AD No. 21454, breeds and extracts the individual components from the culture. L e r s e i t e