CH575466A5 - 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

  

  
 



   The present invention relates to a method for producing a mixture of SF-837 substance, SF-837-A2 substance, SF-837-A3 substance and SF-837-A4 substance or the individual components SF- 837-A2 substance, SF-837-A3 substance and SF-837-A4 substance, and acid addition salts thereof.



   In the Swiss patent no. 563 454 describes a process for the preparation of SF-837 substance and its acid addition salts, which is characterized in that a strain of Streptomyces mycarofaciens or its mutants or variants in a culture medium which contains assimilable N and C sources is shown below cultivates aerobic conditions to produce and accumulate the SF-837 substance in the culture, and then recover it from the culture. The SF-837 substance has a strong growth-inhibiting effect against gram-positive pathogenic bacteria and against antibiotic-resistant pyogenic bacteria.



   It has now been found that still other new substances with antibiotic activity can be obtained from the culture of the said microorganism and that these new antibiotic substances are remarkably effective for inhibiting the growth of various kinds of pathogenic germs such as Staphylococcus aureaus.



   These new substances, namely the substances SF-837-A2, SF-837-A3 and SF-837-A4, are effective for the growth inhibition of gram-positive bacteria and pathogenic bacteria which are resistant to the known antibiotics. The substances SF-837-A2, SF-837-A3 and SF-837-A4 are practically non-toxic and have a therapeutic effect against infections by gram-positive bacteria in humans and animals.



   The inventive method for producing a
Mixture of SF-837 substance. SF-837-A2-substance, SF-837-A3-substance and SF-837-A4-substance or the individual components SF-837-A? -Substance, SF-837-Ar-substance and SF-837-A4-substance as well as acid addition salts thereof is characterized by having a trunk of
Streptomyces mycarofaciens or its mutants or variants in a culture medium which contains assimilable nitrogen and carbon sources, cultured under aerobic conditions to produce a mixture of SF-837 substance, SF-837-A2 substance, SF-837-A3 substance and SF-837-A4 substance in the culture and to accumulate, and this mixture is then obtained from the culture and, if necessary, the SF-837-A2 substance,

   SF-837-A3 substance and SF-837-A4 substance isolated therefrom.



   The substances obtained can be converted into the acid addition salts as well as into the corresponding acetyl derivatives, e.g. B. the diacetyl derivative of SF-837-A2 substance, the monoacetyl derivative of SF-837-A3.Substoff and the monoacetyl derivative of SF-837-A4 substance, which have the same activity.

   Each of the substances SF-837-A2, SF-837-A3 and SF-837-A4 is soluble in methanol, ethanol, acetone, chloroform, ethyl acetate, butyl acetate, acidified water, benzene, ethyl ether and carbon tetrachloride, but slightly soluble in Petroleum ether, n-hexane and neutral water, is a basic compound, forms salts with acids, results in a positive reaction in the erythromycin test with 50% sulfuric acid, but a negative reaction with ninhydrin and ferric chloride contains only the elements carbon, hydrogen and nitrogen and oxygen, is levorotatory in ethanol and has the characteristics of macrolid antibiotics.



   The new compounds prepared according to the invention are likely to have the following formulas:
EMI1.1
 Compound Rg R2 R3 R4 Substance SF-837-A2 COCH2CH3 H H COCH2CH2CH3 Diacetyl derivative of Substance SF-837-A2 COCH2CH3 COCH3 COCH3 COCH2CH2CH3
EMI2.1
 Compound R1 R3 R4 Substance SF-837-A3 COCH2CH3 H COCll2CH3 Monoacetyl derivative of substance SF-837-A3 COCll2CH3 COCH3 COCH2CH3 Substance SF-837-A4 COCH2CH3 H COCH2CH2CH3 Monoacetyl derivative of substance SF-837-A4 COCH2CH3 COCH2CH3 COCH2CH3
Based on these formulas, the new compounds can be designated as follows: SF-837-A2 substance:

   7- (Formylmethyl) -4,1 0-dihydroxy-5-methoxy-9,1 6 dimethyl-2-oxooxacyclohexadeca-1 1,1 3-dien-6-yl-3,6-dideoxy-4-0- ( 2,6-dideoxy-3-C-methyl-a-kribohexopyranosyl) -3- (demethylamino) -ss-D-glucopyranoside-4-monopropionate ester-4-monobutyric acid ester.



  SF-837-A3 Substance: 7- (formylmethyl) -4-hydroxy-5-methoxy-9,1 6-dimethyl2,10-dioxooxacyclohexadeca-11,13-dien-6-yl-3,6-dideoxy-40 - (2,6-dideoxy-3α-methyl-α-L-ribohexopyranosyl) -3- (dimethylamino) - ss D-glucopyranoside4,4-dipropnonic acid ester.



  SF-837-A4 substance: 7- (formylmethyl) -4-hydroxy-5-methoxy-9,1 6-dimethyl-2,10-dioxooxacyclohexadeca-11,13-dien-6-yl-3,6-dideoxy-4 , 0- (2,6-dideoxy-3-C-methyl-α-kribohexopyranosyl) -3- (dimethylamino) -ss-D-glucopyranoside-4-monopropionic acid ester-4-monobutyric acid ester.



   The specific properties of the SF-837-A2 substance are that, as the free base, it is a white colored powder which has a melting point of 125 to 128 C, a pKa 'value of 6.8 in 50% aqueous ethanol , an elemental analysis of C 60,580 / 0, H 8,850 / 0, N 1,720 / 0 and 0 28,850 / o (remainder) gives, determined by mass analysis gives a molecular weight of 827 and consequently has the empirical formula C42Hó9OssN, an optical rotation of [rx ] 22 --68 has a characteristic absorption maximum in ultraviolet light of 232 mu (E% = 320) at a concentration of 1% in ethanol

   in ethanol solution and absorption bands in infrared light as free
Base in potassium bromide has the following wave numbers in cm- ': 3400, 2970, 2935, 1737, 1460, 1410, 1377, 1360, 1300, 1275, 1190, 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-colored needles with a melting point of 130 to 134 C, results in an elemental analysis of C 60.680 / 0, H 8.23%, N 1.490 / 0 and 0 29.600 / 0 ( Remainder) and consequently has the empirical formula C46H73O.7N.



   The specific properties of the SF-837-A3 substance are that, as a free base, it is a white colored powder with a melting point of 122 to 125 C, has a pKa 'value of 7.0 in 50% aqueous ethanol, has an elemental analysis of C 60.53 / o, H 8.23 / o, N 1.870 / 0 and 0 29.320 / 0 (remainder), and a molecular weight of 811, determined by mass analysis, and consequently the empirical formula C4, i { 65OsN has an optical rotation of [a] D -42 at a concentration of 1% ethanol, a characteristic absorption maximum in ultraviolet light at 280 m (E1 cmê = 295) in ethanolic solution and characteristic absorption bands in infrared light in the form of free base as beads in potassium bromide at the following wave numbers in cm-Ú:

   3500, 2970, 2930, 1738, 1680, 1640, 1600, 1460, 1378, 1360, 1300,
1275, 1252, 1190, 1,168,1121,1083,1052, 1015, 980, 910, 863, 840, 805 and 780. The monoacetyl derivative of SF-837-A3 substance forms sand-like crystals, melting point 182 to 185 ° C, gives a Elemental analysis of C 60.560 / 0, H 7.920 / 0, N 1.680 / 0 and 0 29.840 / 0 (remainder) and consequently has the empirical formula C43H67O.6N.



   The specific properties of the SF-837-A4 substance are that, as the free base, it is a white-colored powder with a melting point of 120 to 122 ° C., has a pKa 'value of 7.0 in 500 / above aqueous ethanol, an elemental analysis of C 60.82 O / o, H 8.52%, N 1.73% and 0 2893K (balance) and a molecular weight of 825.

   determined by mass analysis, and thus has the empirical formula C42H67O.5N, an optical rotation of [t] [2.2 -40 "at a concentration of 1% in ethanol, a characteristic absorption maximum in ultraviolet light at 280 m, u (i $ / Cm = 285) in ethanolic solution and has characteristic absorption bands in infrared light in the form of the free base as spheres in potassium bromide at the following wave numbers in cm- ': 3500, 2970, 2930, 1738, 1680, 1640, 1600, 1460, 1378, 1360, 1300, 1276, 1252, 1190, 1170, 1120, 1082, 1052, 1017, 980, 920, 910, 863, 840 and 780.



  The monoacetyl derivative of the SF-837-A4 substance forms sand-like crystals with a melting point of 166 to 168 C, gives an elemental analysis of C 60.850 / 0, H 8.060 / 0, N 1.650 / 0 and 0 29.44% (remainder) and consequently has the empirical formula C44H690.6N
It was found that none of the substances SF-837-A2, SF-837-A3 and SF-837-A4 contain the elements sulfur, phosphorus or halogen.



   When carrying out the erythromycin test with 500/0 of your sulfuric acid, the SF-837-A2 substance gives a brownish or reddish-purple color, while the substances SF-837-A3 and SF-837-A4 give a pale brown color.



   The acid addition salts of the substances SF-837-A, SF-837-A3 and SF-837-A4 are z. B. the 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, room acid, ascorbic acid, glycolic acid and the like.



   In the accompanying drawings:
Fig. 1 is a graph of the ultraviolet absorption spectrum of the SF-837-A2 substance dissolved as a free base in ethanol,
Fig. 2 is a graph showing the infrared absorption spectrum of the SF-837-A2 substance as a free base in beads in potassium bromide;
Fig. 3 is a graph showing the ultraviolet absorption spectrum of the SF-837-A3 substance dissolved as a free base in ethanol.
Fig. 4 is a graph showing the ultraviolet absorption spectrum of the SF-837-A4 substance dissolved as a free base in ethanol.
Fig. 5 is a graph showing the infrared absorption spectrum of the SF-837-A3 substance as a free base in beads in potassium bromide;
Fig. 6 is a graph showing the infrared absorption spectrum of the SF-837-A4 substance as a free base in beads in potassium bromide.



   When substances SF-837-A2, SF-837-A, and SF-837-A4 are subjected to silica gel or aluminum oxide thin layer chromatography using different solvent systems, they give a single spot with different Rf values, so that the purity and l-lomogc nitrite of these substances can be confirmed.



   The Rf values of these substances for various solvent systems are compiled in Table 1 below.



   Table I Rf value
Solvent- SF-837-A2- SF-837-A - SF- 37-A4- systems substance substance substance
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 alumina.



   Thin layer chromatography ethyl acetate benzene (2 1) 0.40 0.45 0.52 ethyl acetate ¯¯ 0.84 0.87 0.91
The antibacterial spectrum of the substances SF-837-A2,
SF-837-A3 and SF-837-A3, as well as their acetyl derivatives, are shown in Table 2 below. The minimum growth inhibitory concentrations of these new antibiotics were determined using the broth dilution method with various culture media as shown in Table 2.



   Table 2
Minimum growth inhibiting concentration (mcglml) Test- SF-837- Diace- SF-837- Mono- SF-837- Mono microorganism A2-Sub- tyl-SF- A3-Sub- ace- A4-Sub- ace- culture 837- A2- stanz tyl-SF- stanz tyl-SF- medium
Sub- 837-A3- 837-A4 punch substance substance Staphylococcus aureus 209p 0.39 0.78 0.39 0.78 0.39 0.78 1 Staphylococcus aureus 209p resistant to penicillin 0.39 0.39 0.39 0.37 0.39 0.39 1 Staphylococcus aureus 209p resistant to streptomycin and A-249 substance 0.39 0.78 0.39 0.39 0.78 0.78 1
Minimum growth inhibiting concentration (mcg / ml) Test- Sl - 837- Diace- SF-837- Mono- SF-837- Mono microorganism A2-Sub- tyl-SF- A3-Sub- ace- A4-Sub-ace- culture - punch 837-A2- punch tyl-SF- punch tyl-SF- medium
Sub- 837-A3- 837-A4 punch substance substance 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 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, pencillin and tetracycline 0.78 1, 56 1.56 3.125 1.56 3.125 1 Staphylococcus aureus 193 1 Bacillus subtillis ATCC 6633 0.2 0.4 0.39 0.78 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 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
The culture media used according to Table 2 were as follows: Medium 1: Heart infusion medium.



   Medium 2: glycerin broth medium.



   Medium: Sabouraud Medium.



   The results in Table 2 clearly show that the substances SF-837-A2, SF-837-A3 and SF-837-A4 all have valuable antibacterial activity against Gram-positive bacteria, as well as against pyogenic bacteria, which compared to known ones Antibiotics are resistant. It was also found that the antibacterial effectiveness of these three substances cannot be rendered ineffective by serum.



   From the results of the toxicity studies by oral and parenteral administration, it was found that the substances SF-837-A2, SF-837-A3 and SF-837-A4 all have low toxicity. After oral administration to mice, the free bases of these substances gave LD50 values of 3100 mg / kg, or 2900 mg / kg or



  2850 mg / kg.



   It has also been observed that the substances SF-837-A2, SF-837-A3 and SF-837-A4, both as free bases and in the form of their acetyl derivatives, can all be used like the currently known antibiotics.



   In the treatment of ICR mice which had been infected with a dose of 100 times the LD50 of Streptococcus haemolyticus Ti-125 Gr-A type I, the oral administration of the free bases of the substances SF-837-A2, SF-837- A3 and SF-837-A4 have the following CD50 values of 120 mg / kg, 135 mg / kg, 170 mg / kg and 155 mg / kg, respectively.



   The therapeutic efficacy of the diacetyl derivative of the SF-837-A2 substance, the monoacetyl derivative of the SF-87-A3 substance and the monoacetyl derivative of the SF-837-A4 substance were also examined in the following manner:
An aqueous suspension of a strain of Staphylococcus aureus Smith was intraperitoneally injected into 1CR mice at a dosage of 100 times more than the LD 50 value. 30 minutes after this infection, the infected mice were by oral administration of an aqueous suspension containing the test active substance and a small
Amount of gum arabic contained. treated. Ten mice in each group were treated in this way and fed for 7 days after the treatment.

   From the results of a series of these studies, it was then estimated that these acetyl derivatives had useful CD0 values of
180 mg / kg, or 200 mg / kg, or 200 mg / kg.



   For therapeutic purposes, the free bases available according to the invention, the acid addition salts and the acetyl derivatives of the substances SF-837-A2, SF-837-A3 and
SF-837-A4 can be processed in the usual way into tablets or capsules for oral administration and into aqueous solutions or suspensions for injections, with or without pharmaceutically acceptable additives, such as carriers, diluents, suspending agents and the like. For the preparation of aqueous injection solutions, the acid addition salts, eg. B. the tartarate, used because it is more soluble in water.

   Furthermore, the free bases, the acid addition salts and the acetyl derivatives of the substances SF-837-A2, SF-837-A3 and SF-837-A4 can of course not only be used alone but also in the form of a mixture of two or more of these substances for therapeutic purposes be used.



   The microorganism which produces the substances SF-837-A2, SF-837-A3 and SF-837-A4 was isolated for the first time by the inventors of the present invention from a soil sample and named Streptomyces mycarofaciens nov.



  sp. designated and deposited in full in the American Type Culture Collection, Washington, D.C., under ATCC number 21454.



   Streptomyces mycarofaciens has the following microbiological characteristics: (I) Morphological observations:
1) Aerial mycelium: The aerial mycelium produces open spirals in abundance on glycerine-Czapek agar, glycerine-calcium malate agar, starch-synthetic agar, etc.



   2) Spores: The spores are spherical, oval or elliptical in shape, their surface structure is thorny (relatively thinner and longer thorns) and the size of the spores is 0.5 to 0.7 microns by 0.8 to 1.0 Micron.



   (II) Characteristics of various culture media are shown in Table 3 below.



   Table 3: Culture medium growth aerial mycelium Soluble pigment Sucrose weak growth sparse, cotton wool-like, Czepek agar colorless to cream-colored whitish-gray none Glycerine-white to cream-colored, partly Czapek agar dark brown with formation of grayish colored and cotton wool-like none or very much
End mycelium weakly pink Krainsky glucose - light brown to white with a pink tinge none or weak asparagine agar red brown brown-yellow Ushinsky glucose brown with reddish pink to lavender light brown asparagine agar sting calcium malate - weak growth none none agar cream colored glycerine calcium light brown pink gradually changing into grayish no malate agar (cotton wool-like) starch-synthetic - dark brown with pink with a strong reddish tinge, none shear agar purple tinge gradually changing into grayish (cotton wool-like) bouillon agar yellowish-brown very sparse,

   don't know any G, lucose broth- thick. Good. brown to white to yellowish agar dark brown cream-colored none glucose peptone agar brown sparse, white none tyrosine agar reddish brown white none
Potato thick, good and brown abundant, gray-brown black-brown pistil to black-brown of the colony
Carrot stamp brown white to cream-colored brown on the edge
Framed ring-shaped, sparse, none white
Milk area growth, light brown
Egg light brown none none Kultum1ediulll wax Lultmyccliuln soluble pigment spoon-coagu- light brown none none serum glucose-Czapek- surface and sparse, no white solution soil growth, light brown gelatin (20 "C)

      cream-colored to none none pale brown Bennett brown to black-brown pink with a reddish tinge pale brown cellulose medium no growth
Note: The incubation temperature was generally 28 ° C unless otherwise stated.



   (III) Physiological properties:
Production of hydrogen sulfide: negative
Tyrosinase generation: negative
Generation of nitirite: positive
Coagulation of skimmed milk: positive
Peptonization of skimmed milk: negative
Hydrolysis of starch: positive Loss of gelatin: positive (weak)
Dissolution of coagulated Loeffler 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 rhamnose.



   3) Unsuitable: xylose, sucrose, raffinose, dulcitol, sorbit, mannitol and cellulose.



   (V) Temperature for the culture: 15 to 38 "C.



   The above-mentioned microbiological characteristics of the strain which produces the substances SF-837-A2, SF-837-A3 and SF-837-A4 can be summarized as follows: The conidiophore is spiral-shaped, the spores thorny. Cream- to brown- to reddish-brown colonies are observed on synthetic culture media, with pink-colored aerial mycelium and cotton-like, gray-colored ones
Aerial mycelium are generated. No noticeable formation of soluble pigment is observed, but the
Formation of soluble pigment of light brown color can be observed on a limited number of synthetic culture media. On the other hand, a brown-colored colony is generally observed on organic culture media, which forms white to cream-colored aerial mycelium, but without the production of soluble pigment.

   On a potato stamp, however, an aerial mycelium of greyish brown color is abundantly formed, and the generation of a blackish-colored soluble pigment is observed.



   With regard to the description of Waksman (Waksman The Actinomycetes, Vol. 2, 1961) it can be stated that the above-mentioned cultural characteristics of the new strain are in part closely related to those of the Fradiae series, Ruber series and Flavus series of the Art
Streptomyces. This is discussed in more detail below.



   First, considering that the new strain reacts negatively against melanin formation and the formation of a
Having aerial mycelium of pink tint, the new strain can be compared with the strains of the Fradiae series, namely Streptomyces fradiae, Streptomyces laridus, Streptomy ces roseus and Streptomyces fuscus. However, the new strain differs from the strains of the Fradiae series in that its aerial mycelium of pink color appears temporarily at the beginning of the incubation and in some cases, in the middle and later stages of incubation, is transformed into gray-colored and cotton-like aerial mycelium, while the pink The color of the stems of the Fradiae series is very stable. Furthermore, the aerial mycelium of Streptomyces fradiae is straight and different from the spirals of the new strain.

   Furthermore, Streptomyces fradiae clearly differs from the new strain in that Streptomyces fradiae shows abundant formation of aerial mycelium on sucrose-Czapek agar and colorless growth on starch agar.



   The new strain differs from Streptomyces fuscus and Streptomyces luridus in that Streptomyces fuscus exhibits no formation of spirals and Streptomyces luridus exhibits the formation of spirals only on a limited number of culture media. while the new strain shows copious formation of spirals on many culture media. Streptomyces roseus is still similar to the new strain in that the formation of spirals is also observed, but they clearly differ from each other in that Streptomyces roseus shows colorless growth on starch-synthetic agar, but shows no formation of aerial mycelium on a potato stamp.



   Second, in view of the fact that it reacts negatively to melanin formation and shows growth of red color, the new strain can be compared with Streptomyces albosporeus, Streptomyces erythraeus, Streptomyces niveoruber and Streptomcyces purpurascene, etc., which belong to the Ruber series.



   Streptomcyes niveoruber clearly differs from the new strain in that the surface structure of the spores of the former is smooth, while those of the latter are thorny. Furthermore, Streptomyces albosporeus differs from the new strain in that Streptomyces albosporeus forms an aerial mycelium, which is predominantly straight, clearly showing red growth on sucrose-Czapek agar and on glycerol-calcium-malate agar, but does not produce aerial mycelium on starch agar. Streptomyces erythraeus is different from the new strain in that Streptomyces erythraeus shows a red-colored growth on sucrose-Cza pek agar and on potato stamps and a cream-colored growth on glucose-asparagine agar and starch-synthetic agar, on which the new strain is more reddish tinted growth produced.



   Furthermore, Streptomcyces purpurascens is similar to the new strain due to the thorny structure of the spore surface, but the former differs from the latter in that the former produces noticeably soluble pigment of red color on synthetic culture media, while the latter is devoid of such soluble pigment.



   The comparison of the new strain was also carried out with Streptomyces microflavus and Streptomyces roseoflavus, which belong to the Flavus series but are considered to be closely related to the new strain. In terms of their cultural characteristics, Streptomyces microflavus and Streptomyces roseoflavus are closely related to the new strain in certain respects, but these two strains produce spores with smooth surfaces (H. D.



  Tresner et al Journal of Bacteriology, Vol. 91, pages 1998 to 2005, 1966), which is clearly different from the thorny structure of the spores of the new strain.



   Consequently, no strain can be found among the known species of the genus Streptomyces which matches the new strain.



   On the other hand, the new antibiotics SF-837-A2, SF-837-A3 and SF-837-A4 substances produced by the new strain are typical of the macrolid antibiotics. Among the novel antibiotic substances produced according to the present invention, the substance SF-837-A2 has an absorption maximum at a wavelength of 232 mu in ultraviolet light, and hence this substance can be used as the antibiotic of the same series as leucomycin, josamycin.

   Spiramycin, Miamycin and Tertiomycin are considered.In addition, the substances SF-837-A3 and SF-837-A4 have a high absorption maximum near the wavelength of 280 mp in the ultraviolet spectrum and as a result, these two substances can be used as antibiotics of the same series, such as niddamycin, Carbomycin B, Tylosin, Relomycin and Macrocin can be considered.



   The microbiological properties of the new strain are therefore compared in the following with those of those microorganisms which produce the above-mentioned known antibiotics.

 

   It is easily seen that the new strain is different from the leucomycin producing microorganism Streptomyces kitasatoensis, the josamycin producing microorganism Streptomyces narbonensis var. Josamyceticus, the spiramycin producing microorganism Streptomyces ambofaciens, the Miamycin producing microorganism Streptomyces ambofaciens. the tertiomycin producing microorganism Streptomyces eurocidicus, the niddamycin producing microorganism Streptomyces albire ticuli, the carbomycin B producing microorganism
Streptomyces halstedii. the tylosin and macrocin producing microorganism Streptomyces fradiae and the relomy cin producing microorganism Streptomyces hygroscopi cus, there are clear differences between the morphological ones
 



     Ultraviolet rays, X-rays, high frequency electromagnetic waves, radioactive rays, chemicals, etc. All natural and artificial variants and mutations of the new strain can be used in the method according to the invention as long as they retain their ability to produce one of the substances SF-837-A2, SF-837-A3 and SF-837-A4.



   According to the method of the present invention, the new strain or its variants or mutants can be cultured in a known manner in a culture medium which contains the nutrients which are usually required by the microorganisms. All known nutrients which have conventionally been used for the cultivation of Streptomyces can be used as nutrient sources. For example, glucose, starch, glycerin, dextrin, sucrose, saccharified starch, molasses and the like are suitable as a carbon source. Soybean meal, wheat embryo, meat extract, peptone, corn steep liquor, soluble vegetable protein, dried yeast, ammonium sulfate, sodium nitrate and the like can be used as the nitrogen source.

   If necessary, inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, phosphates and the like can be added to the culture medium. In addition, organic and inorganic materials can be added which promote the growth of the new strain and support the production of at least one of the substances SF-837-A2, SF-837-A3 and SF-837-A4.



   As a method for culturing the new strain, liquid cultivation, and particularly liquid cultivation under submerged aerobic conditions, is particularly preferable, similar to the general methods for the production of the known antibiotics. The cultivation takes place under aerobic conditions, and the suitable fermentation temperature is in the range of 20 to 30 C. However, for the production of the substances SF-837-A2, SF-837-A3 and SF-837-A4 in industry or in the laboratory often preferred to carry out the cultivation at a temperature in the vicinity of 28 ° C.

   Under these conditions, the concentration of substances SF-837-A2, SF-837-A3 and SF-837-AA in the culture broth reaches a maximum after 2 to 5 days of fermentation, both with the shake culture method and with the tank culture method.



   Substance SF-837 is the main metabolic product of Streptomyces mycarofaciens, and Substances SF-837-A2, SF-837-A3 and SF-837-A4 are normally produced in smaller amounts, which total in the range of about 5 to 20 Percent by weight of the produced SF-837 substance.



   The substances SF-837, SF-837-A2, SF-837-A3 and SF-837-A4 can be produced at the same time by cultivating the new strain and have similar physicochemical properties as already mentioned. They can be obtained, purified and isolated from the culture broth, and in general any known method for obtaining, purifying and isolating known basic antibiotics can be used.



   The substances SF-837-A2, SF-837-A3 and SF-837-A4 can be obtained from the culture broth in various ways.



   The mixture of substances SF-837, SF-837-A2, SF-837-A3 and SF-837-A4 produced by culturing the new strain is present in the solid phase, which contains the mycelium cake, as well as in the liquid phase of the Culture. The culture can therefore be filtered in a known manner to give the filter cake (i.e. the solid phase containing the mycelium cake) and separately therefrom the filtrate (i.e. the liquid phase of the fermentation broth).



  The active substances present in the filtrate can be mixed with a suitable, water-immiscible organic solvent, such as. B. ethyl acetate, butyl acetate, chloroform, benzene, ethyl ether, methyl isobutyl ketone, butanol, etc.



  extracted under neutral or weakly alkaline conditions. The active substances present in the filter cake can also be extracted with acidified water or with a suitable water-miscible organic solvent such as methanol, ethanol, acetone, etc. The culture can, however, also be extracted directly with a water-immiscible organic solvent tel can be extracted without first filtering the Myce liumkuchens, so that the active substances immediately from the
Culture can be transferred into the organic solvent used.



   The active substances carried over in solution into the organic solvent can then be extracted again with acidified water. The extract obtained in acidified water can then be neutralized or made weakly alkaline by adding alkaline substances in sodium hydroxide, potassium hydroxide, sodium carbonate or sodium bicarbonate and then shaken together with a suitable organic solvent, so that the active substances again pass into the organic solvent phase. By repeating these mutual extraction operations, the impurities can be largely removed from the active substances.

   The extract in the organic solvent can then be evaporated to dryness under reduced pressure, so that a crude powder is obtained which contains the substances SF-837, SF-837-A2, SF-837-A3 and SF-837-A4. However, the solution of the active substances in unacidified water can either be freeze-dried as such to give the acid addition salts of the active substances, or the aqueous solution can be neutralized or made weakly alkaline by adding a suitable alkaline material to give the active substances in shape to precipitate the free base.



   The substances SF-837, SF-837-A2, SF-837-A3 and SF-837-A4 are basic in nature, as already mentioned above, and the culture or its filtrate or solution of the active substances can also be mixed with ion exchangers such as ion exchange resins, z. B. Amberlite IRC-50, etc. and ion exchange cellulose are treated so that the active substances are absorbed by the ion exchanger and can then be eluted therefrom using a suitable solvent.



   The obtained raw powder containing the active substances mixed can be further purified by extraction with a suitable organic solvent, e.g. B. Benzene, which can dissolve the active substances but not the impurities, by washing with a suitable organic solvent such as petroleum ether, ligroin or n-hexane, which practically cannot dissolve the active substances, but dissolves the impurities well, and / or by adding petroleum ether, ligroin or n-hexane to a solution of the active substances in a solvent, such as. B. benzene.



   In this way, a purified mixture of SF-837 substance with substances SF-837-A2, SF-837-A3 and SF-837-A4 can be obtained, in which the total amount of SF-837-A2, SF-837 -A3 and SF-837-A4 substances is usually in the range of about 5 to 20 percent by weight of the content of SF-837 substance, depending on the fermentation conditions, the type of production, the purification methods used and various other factors. As such, this purified mixture is ready for processing into medicaments and for administration for therapeutic purposes without isolating the individual components.



   If desired, the purified mixture of
SF-837 substance with small amounts of the substances
SF-837-A2, SF-837-A3 and SF-837-A4 can be converted directly into a mixture of the acid addition salts of the individual components by converting the mixture of the free bases in an organic solvent, e.g. B. Ethyl ether. dissolves the solution with a solution of an organic or inorganic acid, z. B. hydrochloric acid, sulfuric acid, tartaric acid, Zi tronic acid, dissolved in a suitable organic solvent medium, z. B. ethyl ether, treated and in this way the
Acid addition salts precipitate in the form of a mixture, which can be filtered and dried and then as such is ready for further processing into medicaments and for administration for therapeutic purposes.

   The cleaned mixture of the free bases can also tion in a Ge mixture of acetyl derivatives in the usual way by Acetylie, z. B. by dissolving in pyridine, treating the solution with acetic anhydride at room temperature or slightly elevated temperature for a sufficient period
Time to get the diacetyl derivatives of the substances SF-837 and
SF-837-A2 and the monoacetyl derivatives of the substances SF-837-A3 and SF-837-A4, and precipitation of the acetylation products by adding ice water to the reaction mixture, are converted. The mixture of acetylated derivatives can then be filtered and washed with water in order to be cleaned of the impurities, and this mixture can then be used as such for further processing and administration for therapeutic purposes.



   In order to achieve separation of the active substances from one another, it is effective to use chromatographic methods using a suitable adsorbent, e.g. Activated alumina or silica gel and the like, and a suitable developing solvent, as well as various types of countercurrent distribution methods, either alone or in combination. In a purified mixture of the active free bases, which was obtained by the method described above, the substances SF-837-A2, SF-837-A3 and SF-837-A4 usually the quantitatively smaller components and are present in smaller quantities than the SF-837 substance.

   To isolate the components, it is therefore advisable to first subject the purified mixture of the free bases to a countercurrent distribution by an
SF-837 substance-rich fraction from a fraction which is rich in a mixture of substances SF-837-A2,
SF-837-A3 and SF-837-A4 to separate, and these two fractions are then separately subjected to chromatography on silica gel or aluminum oxide in order to obtain a complete
To achieve isolation of the components from each other.



   In general, the separation of the active Substan zen can be carried out with advantage such that one z. B.



   a solution of the above-mentioned crude powder, which contains the active substances mixed, in a suitable organic solvent of chromatography over a column of activated alumina or silica gel using a mixture of ethyl acetate
Benzene (1: 1) or benzene-acetone (4: 1) as developer solvent and, if necessary, this chromatographic solvent
Procedure repeated several times. Usually, the active substances can be eluted from the alumina column one after the other, in that order
SF-837-A4, SF-837-A3, SF-837-A2 and finally SF-837 substance when developing with ethyl acetate-benzene (1: 1). A small portion of each of the fractions of the eluate is examined simultaneously using aluminum thin layer chromatography.

   Fractions which result in a single spot which is characteristic of one of the active substances. and which therefore can be confirmed to contain only a single one of the active substances, are combined and then evaporated to dryness under reduced pressure. and these operations are repeated with each of the fractions containing one of the active substances alone.



   In this way, the pure free bases of the substances SF-837-A2, SF-837-AJ and SF-837-A4 can be separated from one another and obtained in the form of white-colored powders.



   For cleaning and separating the substances SF-837-A2.



  SF-837-A3 and SF-837-A4 can also use any of the known methods that have been used for the purification and separation of macrolid antibiotics, since the new substances are also members of the macrolid antibiotics. However, the production, or isolation, purification and separation of the substances SF-837-A .. is preferred.



  SF-837-A3 and SF-837-A4 were carried out from the culture broth of the new strain and from each other through the following steps. Growing the new strain at a temperature of 20-30 "C in a deeply aerated liquid medium containing known assimilable nitrogen and carbon sources until significant antibacterial activity is achieved in the culture broth, filtering the culture, extracting the culture filtrate at pH from 7 to 9, with a water-immiscible organic solvent which dissolves the active substances, e.g. ethyl acetate, butyl acetate, chloroform, ethyl ether, methyl isobutyl ketone and butanol, then re-extracting the organic extract obtained which contains the active substances contains, with dilute aqueous acid (at pH 2 to 3), e.g.

   B. aqueous hydrochloric acid, aqueous sulfuric acid and the like, alkalizing the aqueous extract obtained (preferably to pH 7 to 9) by adding alkali, re-extraction of the aqueous extract with one of the water-immiscible organic solvents in order to transfer the active substances into the latter organic solvents, at least one repetition of this transfer of the active substances from an organic extract into a water-immiscible organic solvent into a dilute aqueous acid and then into a further portion of the water-immiscible solvent, concentrating the finally obtained organic solution of the active substances partially purified in this way to dryness in a vacuum,

   Dissolving the obtained raw powder of the mixed active substances with ethyl acetate or butyl acetate, passing the solution through - a column of activated carbon, developing with ethyl acetate or - butyl acetate, collecting the active fractions of the eluate, concentrating the active fractions to a smaller volume, extracting the concentrate with aqueous dilute hydrochloric acid, making the aqueous dilute extracts alkaline by adding an alkali, separating the precipitate formed, which consists essentially of the mixtures of active substances, dissolving the precipitate in benzene, chromatographing the benzene solution over a silica gel column using a Mixture of benzene-acetone as developer solvent, collecting the eluate in fractions,

   Examine each of the fractions by means of aluminum oxide thin layer chromatography, pooling and concentrating to dryness in vacuo only depending on the fractions which give a single point which is characteristic of the SF-837-A4 substance, the free base of the SF-837-A4 substance is obtained in the form of a white powder, combine and concentrate to dry kene in a vacuum depending on the fractions, which result in a single point that is characteristic of the SF-837 substance, the free base of the SF-837 substance in the form of pure powder is obtained, combine and concentrate to dryness in a vacuum the fractions which are eluted from the silica gel column after the above SF-837-A4 fractions but before the above SF-837 fractions, and which are in solution, both the substance SF -837-A2,

   such as the substance SF-837-A3 together with small amounts of the substance SF-837, being a powder of a mixture of SF-837-A, substance and SF-837-A3 substance together with a small amount of the SF-837 -Substance is obtained, dissolving this powder in benzene, chromatographing the resulting solution of the powder in benzene over a column of activated aluminum oxide using a mixture of ethyl acetate-benzene and then a mixture of ethyl acetate-acetone as developer solvent, collecting the eluate in fractions, Examine each of these fractions by aluminum thin layer chromatography.

   Combine and concentrate in vacuo only those fractions to dryness which give a single spot characteristic of SF-837-A3 substance. whereby the free base of the SF-837-A3 substance is obtained in the form of pure powder, also combining and concentrating to dryness in vacuo the remaining fractions of the eluate to obtain a powder mainly containing SF-837-A2 substance , redissolving this powder in benzene, chromatographing this benzene solution over a column of activated aluminum oxide using a mixture of ethyl acetate-benzene as the developer solvent, collecting the eluate in fractions, examining each of the fractions by aluminum oxide thin-layer chromatography, combining and concentrating to dryness in vacuo only those factions which have a single spot

   which is characteristic of the SF-837-A2 substance, with the free base of the SF-837-A, substance being obtained in the form of pure powder.



   The substances SF-837-A2, SF-837-A3 and SF-837-A4, which have been separated from one another in the pure state as described above, can each subsequently be converted into their acid addition salt or into the above-mentioned acetyl derivative, if desired. The conversion into the acid addition salt can be carried out, for example, by dissolving the free base in a suitable organic solvent, such as ethyl ether, etc. and adding a saturated solution of an organic or inorganic acid, such as tartaric acid, citric acid, acetic acid, hydrochloric acid, sulfuric acid and the like in ethyl ether, respectively.

   The conversion into the diacetyl derivative of the substance SF-837-A2 or into the monoacetyl derivative of the substances SF-837-A3 and SF-837-A4 can take place, for example, by dissolving the free base in pyridine and then adding a large excess of acetic anhydride to this solution as already described above.



   The substances SF-837-A2, SF-837-A3 and SF-837-A4 belong to the class of macrolid antibiotics. The substance SF-837-A2 has an absorption maximum at a wavelength of 232 mIt in the ultraviolet range of the spectrum and can therefore be compared with some of the known macrolid antibiotics, namely with the spiramycin group (1, II and 111), the leucomycin group (A, to Ag and B, to B4), Josamycin, Tertiomycin (A and B) and Miamycin.



  The differences between the substance SF-837-A2 and these known macrolid antibiotics are explained below:
First, spiramycin differs from substance SF-837-A2 by the differences in its Rf values in the silica gel thin layer chromatography, which are summarized in Table 5 below, and also by comparing the optical rotation values and the pKa 'values of spiramycin , which are mentioned on pages 304 to 317 of Flelvetica Chimica Acta, Vol. 39., since spiramycin I has a value [x] n -960 and pKa '7.7, spiramycin II [cr], 800 and pKa' 7, 6 and spiramycin III [a] D -79 "and pKa '7.6.



   Table 5:
Rl value
Antibiotic developer solvent consisting of n-butanol: acetic acid: water (3: 1: 1)
SF-837-A, substance 0.68
Spiramycin 1 0.11
Spiramycin II 0.13
Spiramycin III 0.19
Tertiomycin A 0.78
Tertiomycin B 0.76
Second, miamycin, leucomycin-B group (B1, B2, B3 and B4) and leucomycin-A2 differ from the substance SF-837-A2 in that miamycin has a zero value for optical rotation [Antibioties and Chemotherapy, pages 37 bis 39 (1957)] by leucomycin-B ,, B2. B3 and B4 have a melting point of 214 to 215 "C, and 214 to 216" C, and



  216 to 217 "C, or 221 to 223 C (Gazette of Japanese Patent Publication No. Sho-35-18750), and in that Leu comycin-A2 has a value of es Er" = 158 for the absorption maximum im Ultraviolet portion of the spectrum and a molecular weight of 1220 to 1250 (Gazette of Japanese Patent Publication No. Sho-35-18750).



   Third, tertiomycin-A and -B clearly differ from the substance SF-837-A2 by the differences in their melting points, optical rotations and Rf values in silica gel thin layer chromatography, as shown in Table 5 and Table 6. Teriomycin-A has a melting point of 202 to 204 "C and an optical rotation [a] D -44" (Journal of Antibiotics, pp. 105-109 (1955)] and tertiomycin-B has a melting point of 97 to 99 "C and a optical rotation of [a] D -55 "(Journal of Antibiotics, pp. 161 to 163 (1955)].



   Fourth, leucomycin-As, A3, A4, A5, A6, A7, A8 and Ag and josamycin differ from the substance SF-837-A2 by direct comparison of their Rf values in alumina thin-layer chromatography (using ethyl acetate as the developing solvent), such as from Table 6 below, and by the known properties of Leucomycin-A ,, as described in the Gazette of Japanese Patent Publication No.

   Sho-36-98, and further in that leucomycin-A3 has a melting point of 120 to 121 "C and an optical rotation of [a] D -55.4 (Antimicrobial Agents and Chemotherapy, pages 631 to 636 (1967) ], by the known properties of leucomycin-A4, A5, A6, A7, A8 and Ag, as described in the Journal of Antibiotics, pages 272 to 278 (1968) and by the fact that josamycin has a melting point of 130 to 133 ° C and an optical rotation of [a] D -70 "C (Gazette of Japanese Patent Publication No.



  Sho-41-21759).



   Table 6: Antibiotics Rf values SF-837-A2 substance 0.84 Josamycin 0.65 Leucomycin-A, 0.08 Leucomycin-A5 0.05 Leucomycin-A7 0.01 Leucomyein-A9 0.02 Leucomycin-A3 0.65 Leucomycin-A4 0 , 60 Leucomycin-A6 Leucomycin-A8 0.53 Tertiomycin-A 0.61 Tertiomycin-B 0.64
From the above explanations, it is clear that the substance SF-837-A2 does not match any of the known antibiotics and is a new substance.



   The substances SF-837-A3 and SF-837-A4 have an absorption maximum at the wavelength 280 myt in the ultraviolet range of the spectrum, which is different from that of the substances SF-837 and SF-837-A2, which in turn has an absorption maximum at a Have a wavelength of 232 tnu in the ultraviolet part of the spectrum. However, in view of the above-mentioned physicochemical properties of the substances SF-837-A3 and SF-837-A4, it is clear. that these two substances also belong to the group of macrolid antibiotics.



   Among the known group of macrolid antibiotics, carbomycin-B, niddmaycin. Tylosin, relomycin and macrocin are compared with the substances SF-837-As and SF-837-A4 in view of the fact that these known antibiotics also have a remarkable absorption at a wavelength of about 280 m, tt in the ultraviolet region of the spectrum. For comparison, the physico-chemical properties of these antibiotics are summarized in Table 7 below.



   Table 7:
Maximum absorption Optical antibiotics Melting point in the ultraviolet ab- Gross formula Molecular rotation Sorptlonssp'ktrum at weight (degrees) Carbomycin B 141-144 C 278 mu (EIGzcom 276) C42H6O5N 825-35 Niddamycin 132-134 C 279 mu (E 275) C40H76O, 4N 783 -43 tylosin 128-132 C 284 with (E, m 245) C45H77O, 7N 907 -46 relomycin 172-175 C 282 mu (E1rn 245) C45H79O, 7N 909 -44 macrocin 134-136 C 283 with (E, 244 ) C45H29O17N 921 -52.5
Annotation:

   The data presented in Table 7 are based on the Index of Antibiotics froin Actinomycetes by Um ezawa et al, published in 1967.



   First, tylosin, relomycin and macrosin clearly differ from the substances SF-837-A3 and SF-837-A4 by their gross formula and their molecular weights, and in the case of tylosin by the Rf values, as can be seen from Table 8.



   Secondly, carbomycin-B and niddamycin can hardly be distinguished from the substances SF-837-A3 and SF-837-A4 with regard to their general physicochemical properties, but obviously they are among themselves due to the difference in their Rf values in aluminum thin-layer chromatography, as can be seen from Table 8, distinguishable.



   Table 8:
Rf values antibiotics aluminum oxide thin silica gel thin layer chromatography layer chromatography with athylac- phy with benzene tat benzene (2: 1) acetone (2: 1) developed develops SF-837-A3 substance 0.45 0.50 SF-837-A4 substance 0.52 0.55 Carbomycin-B 0.46 0.56 Niddamycin 0.05 0.42 Tylosin 0.02 0.18
When hydrolysed with alkali, one molecule of substance SF-837-A3 gives two molecules of propionic acid and one molecule of substance SF-837-A4 gives one molecule of propionic acid and one molecule of n-butyric acid, while one molecule of carbomycin-B gives acetic acid and isovaleric acid ( Angewandte Chemie, pages 50 to 58, 1957) and one molecule of niddamycin, one molecule of isovaleric acid results (Drugs Research, Vol 12, 1191-5, 1962). This clearly shows

   that the substances SF-837-A3 and SF-837-A4 are different from Carbomycin-B and Niddamycin.



   In view of the fact that no ester linkage other than the lactone linkage occurs in tylosin (Tetrahedron Letters, pages 2339-2345, 1964), nor in relomycin, which is the reduced derivative of tylocin (Antimicrobial Agent and Chemotherapy, pages 45 to 48, 1963), tylosin and relomycin differ from the substances SF-837-A3 and SF-837-A4.

 

   From the above it is clear that the substances SF-837-A2, SF-837-A3 and SF-837-A4 can be regarded as new antibiotic substances which do not correspond to any of the known macrolid antibiotics.



  Example 1:
Streptomyces mycarofaciens identified as ATCC No.



  21454 was inoculated into 300 liters of a liquid culture medium containing 2.0% glucose, 1% peptone, 0.50 / 0 meat extract, 0.40 / 0 corn steep liquor, 0.2% NaCl, 0.3% CaCO3 and 0.2 % Pork fat and had a pH of 7.0.



  and with stirring and with aeration at 28 C for 70
Hours of cultivation.



   The fermented broth was filtered off directly and the filter cake, which contained the mycelium cake, washed with dilute hydrochloric acid. The culture filtrate was combined with the washing liquids, a total volume of 280 liters being obtained (potency 320 mcg / ml). The Fil entered was extracted with 70 liters of ethyl acetate, and 71 liters of the resulting ethyl acetate phase was reduced under reduced
Pressure reduced to about 20 liters. The concentrate was with
Dilute 10 liters of water, bring it to pH 2 by adding 5N hydrochloric acid and shake vigorously. The aqueous phase was separated from the organic phase by adding
3N sodium hydroxide brought to pH 9 and then extracted with 4 liters of ethyl acetate.

   The resulting extract of ethyl acetate was then similarly shaken with two liters of aqueous
Hydrochloric acid, in order to transfer the active substances into the latter, and this hydrochloric acid extract in turn is extracted with one liter of ethyl acetate at pH 8. This ethyl acetate extract was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 92 g of a crude yellow colored powder.



   90 g of this raw powder were dissolved in one liter of ethyl acetate and the solution passed through a column of 1.5 liters of carbon powder which was impregnated with ethyl acetate. Development was carried out using ethyl acetate as the solvent, and the active fractions of the eluate were collected to a total volume of 5.5 liters, which was then reduced under reduced pressure
Pressure were evaporated to dryness and gave 55 g of a white colored powder (potency 700 mcg / mg).



   This powder was having a countercurrent distribution
Benzene and 0.3M phosphate buffer (pH 4.4), (2.5 liters of each solvent was used and the migration of the lower mobile layer was performed ten times). The SF-837 substance was the first through third
Tube distributed and predominantly in the second tube, and larger amounts (about 800/0) of the substances SF-837-A7, SF-837-A3 and SF-837-A4 were retained in the first tube.



   The contents (2.4 liters) of the first tube were concentrated under reduced pressure and gave 7.7 g of a white powder (potency 720 mcg / mg). This powder was dissolved in 15 ml of benzene, the insolubles were filtered off, and the filtered solution was then passed through a column of 800 ml of silica gel impregnated with benzene. The absorbed active substances were developed chromatographically using a benzene-acetone mixture (4 l) and the eluate was melted as a whole in fractions of 50 ml each. A small part of each of the fractions obtained was subjected to thin layer chromatography on alumina and developed with ethyl acetate-benzene (2: 1).



   The factions No. 28 to 30, which gave a single spot in the thin layer chromatogram, were combined and concentrated in vacuo to give 140 mg of a white powder with melting point 120 to 122 "C, which in the analysis as the pure free base of the SF-837-A4 substance was identified.



   The factions No. 38 to 57, which gave three spots in thin layer chromatography, were combined and concentrated in vacuo. 1.1 g of a white powder were obtained which, upon analysis, contained
SF-837-A2 and SF-837-A3 substances together with a small amount of the SF-837 substance.



   1 g of this white powder was dissolved in 4 ml of benzene.



   The solution was subjected to chromatographic separation by passing it through a column of 100 ml
Benzene-impregnated alumina led and with a
Solvent mixture of ethyl acetate-benzene (1: 1) developed. The eluate was taken in fractions of 20 ml each and each fraction was examined by means of aluminum oxide thin-layer chromatography (using ethyl acetate-benzene [2: 1] as developer solvent). The factions No. 30 to 48, which gave a single spot in thin-layer chromatography, were combined and concentrated in vacuo to give 230 mg of a white-colored powder with a melting point of 122 to 125 "C, which in the analysis turned out to be the pure free base of SF-837- A3 substance proved.



   After the faction No. 80 was eluted from the column, the developing solvent was changed to a mixture of ethyl acetate-benzene (2 1) and the fractions No. 90 to 125 were collected together and concentrated in vacuo to give 280 mg of a white colored powder, the analysis of which showed a content of about 70 percent by weight of the SF-837-A2 substance. 250 mg of this powder were dissolved in 3 ml of benzene and the solution was again subjected to chromatography through a column of aluminum oxide impregnated with benzene and developed with 300 ml of ethyl acetate-benzene (1: 3) and then with a mixture of ethyl acetate-benzene (2: 1). The eluate was collected in fractions of 5 ml each and each fraction was examined by aluminum oxide thin-layer chromatography.

   The factions No. 36 to 47, which gave a single spot in thin layer chromatography, were combined and concentrated in vacuo to give 80 mg of a white colored powder with a melting point of 125 to 128 "C, which was analyzed as the pure free base of SF-837-A2- Substance has been identified.



  Example 2:
Streptomyces mycarofaciens was inoculated into 30 liters of a liquid culture medium which contained 3.00 / 0 glucose, 5/0 soluble vegetable protein, 0.20 / 0 KCI and 0.30 / 0 CaCO3 and had a pH of 7.0, and Fermented in a fermentation vessel at 30 "C for 85 hours with stirring and aeration. The fermented broth was filtered directly, and the filter cake, which contained the mycelium cake, washed with dilute hydrochloric acid Liters (potency 300 mcg / ml).

   The filtrate (pH 8.4) was extracted with 7 liters of butyl acetate, and the obtained butyl acetate extract was then treated similarly to Example 1 to perform the mutual extraction of active substances, whereby a crude, yellow-colored powder was obtained in a yield of 15.2 g were.



  15 g of this crude powder were dissolved in 200 ml of butyl acetate and the solution was subjected to chromatography on a column of 400 ml of carbon impregnated with butyl acetate and then developed with butyl acetate. The active fractions of the eluate were collected to a total volume of 1.8 liters and then concentrated in vacuo to about 220 ml. The concentrate was diluted with 50 ml of distilled water, adjusted to pH 2 by adding 6N hydrochloric acid and then shaken vigorously. The aqueous phase was separated from the organic phase and this aqueous solution was adjusted to pH 9.0 by the addition of 1N sodium hydroxide in order to precipitate a precipitate. This precipitate was filtered and dried in a desicator to give 6.3 g of a white colored powder (potency 720 mcg / mg).



  This powder was dissolved in 12 ml of benzene and the insolubles were filtered off. The filtered solution was then chromatographed through a 600 ml column of silica gel impregnated with benzene, and the adsorbed active substances were developed with a mixed solvent of benzene-acetone (6: 1). The eluate was in fractions of each
20 ml collected. The substances SF-837-A2, SF-837-Aj and SF-837-A4 were eluted from the column before the substance SF-837. Each of the fractions was analyzed by silica gel thin layer chromatography (using 2: 1 benzene-acetone as the developing solvent). The factions No.



  64 to 82 were combined and concentrated in vacuo to obtain 800 mg of a white colored powder, the analysis of which showed a content of substances SF-837-A2, SF-837-A3 and SF-837-A4 together with a small amount of substance SF -837. This white colored powder was then subjected to chromatographic separation using a silica gel acid. and then subjected to chromatographic separation with an aluminum oxide column, as described in Example 1, 15 mg of the pure SF-837-A2 substance as the free base, 85 mg of the pure SF-837-A3 substance as the free base and 35 mg of the pure SF-837-A4 substance was obtained as a free base in the form of white colored powders.



  Example 3:
200 mg each of the free bases of the substances SF-837-A2, SF-837-A3 and SF-837-A4. which had been isolated in Examples 1 and 2 were added dropwise with a saturated solution of citric acid. The formed precipitates were filtered and dried to give the citrates of substances SF-837-A2, SF-837-A3 and SF-837-A4 in amounts of 210 mg, 220 and 190 mg, respectively.



  Example 4:
300 mg of the SF-837-A3 substance were suspended in 20 ml of distilled water, and an aqueous solution of tartaric acid was added to the suspension. The mixture was shaken well and brought to pH 4. The mixture was then filtered to remove insolubles and the filtrate was freeze-dried to give 340 mg of the tartarate of the SF-837-A3 substance in the form of a white powder with a melting point of 115 ° C.



  Example 5:
300 mg of the SF-837-A, substance were dissolved as free base in 1.5 ml of pyridine, and 1.0 ml of acetic anhydride was added to this solution. The mixture was shaken and then allowed to stand at room temperature for 20 hours.



  The reaction mixture was then poured into ice water and shaken to give a precipitate. This precipitate was filtered off and dried in a desicator, 330 mg of a white powder being obtained. Recrystallization of this powder from carbon tetrachloride would give 310 mg of the diacetyl derivative of the SF-837-A2 substance in the form of white crystals with a melting point of 130 to 234 "C.



   In a similar way, 300 mg each of the substances SF-837-A3 and SF-837-A4 in the form of their free bases were treated with an excess of acetic anhydride in solution in pyridine, so that the mononeetyl derivatives of the substances SF-837-A3 and SF- 837-A4 in the form of white crystals in amounts of 295 mg (melting point 182 to 185 C), or



  300 mg (melting point 166 to 168 "C) were obtained.



   PATENT CLAIM 1
Process for the preparation of a mixture of SF-837-substance, 5 F-837-A2-substance, SF-837-A3-substance and SF-837-A4-substance or the individual components SF-837-A2-substance, SF- 837-A3 substance and SF-837-A4 substance and acid addition salts thereof, characterized in that a strain of Streptomyces mycarofaciens or its mutants or variants is cultured under aerobic conditions in a culture medium which contains assimilable nitrogen and carbon sources . around a
Mixture of SF-837 substance.

     SF-837-A2 substance. SF-837-A3-
To generate and enrich the substance and SF-837-A4 substance in the culture and then this mixture is obtained from the culture and, if necessary, the SF-837-A, substance, SF-837-A;
Substance and SF-837-A4 substance isolated therefrom.



      SUBCLAIMS
1. The method according to claim 1, characterized in that a strain of Streptomyces mycarofaciens of ATCC No. 2145t is cultivated under aerobic conditions in a liquid culture medium which contains assimilable carbon and nitrogen sources in order to produce the SF-837-A, substance, the SF-837-A3-substance and the SF-837-A4-Sub - to generate and enrich punch in the culture, and then the mixture of active substances is isolated from the culture medium and then the substances SF-837-A2,

         SF-837-AD and SF-837-A4 separate from each other.



   2. The method according to claim 1, characterized in that a strain of Streptomyces myearofaciens of ATCC No. 21454 in a liquid culture medium containing assimilable carbon and nitrogen sources at a temperature of 20 to 30 "C under aerobic conditions until a considerable antibacterial activity is achieved in the culture broth, the culture filters, the
Culture filtrate extracted at a pH of 7 to 9 with a water-immiscible organic solvent for substances SF-837-A2, SF-837-A3 and SF-837-A4, for example ethyl acetate, butyl acetate, chloroform, ethyl ether, methyl isobutyl ketone and butanol, the obtained organic extract containing the active substances, extracted with dilute aqueous acid, for example aqueous hydrochloric acid or aqueous sulfuric acid,

   making the obtained aqueous extract alkaline by adding alkali, preferably to pH 7 to 9, extracting the aqueous extract as well as its with a water-immiscible organic solvent in order to convert the active substances into this organic solvent, at least once this transition of the active substances from an organic extract in a water-insoluble organic solvent into a dilute aqueous acid and then into a further portion of the water-immiscible organic solvent, the finally obtained organic solution of the active substances, which was partially purified in this way, evaporated to dryness in a vacuum, dissolving the obtained raw powder of the mixed active substances in ethyl acetate or butyl acetate,

   the solution is placed on a column of activated charcoal, eluted with ethyl acetate or butyl acetate, the active fractions of the eluate are collected and concentrated to a smaller volume, the concentrate is extracted with aqueous, dilute hydrochloric acid, the aqueous extract is made alkaline by adding alkali, the formed The precipitate, which consists essentially of the mixed active substances, is separated off, the precipitate is dissolved in benzene, the benzene solution is subjected to column chromatography on silica gel using a mixture of benzene-acetone as the eluent and the eluate is collected in fractions, each of these fractions using aluminum oxide - Thin-layer chromatography examined, those fractions combined and evaporated to dryness in vacuo, which result in a single spot in this thin-layer chromatography,

   which is characteristic of SF-837-A4 substance, the free base of SF-837-A4 substance being obtained in the form of a pure powder, those fractions which are obtained from the silica gel column after the
Fractions which give the single spot characteristic of the SF-837-A4 substance are obtained and which contain both the substances SF-837-A3 and SF-837-Aj together

** WARNING ** End of DESC field could overlap beginning of CLMS **.



   

 

Claims (1)

** WARNING ** Beginning of CLMS field could overlap end of DESC **. 20 ml collected. The substances SF-837-A2, SF-837-Aj and SF-837-A4 were eluted from the column before the substance SF-837. Each of the fractions was analyzed by silica gel thin layer chromatography (using 2: 1 benzene-acetone as the developing solvent). The factions No. 64 to 82 were combined and concentrated in vacuo to obtain 800 mg of a white colored powder, the analysis of which showed a content of substances SF-837-A2, SF-837-A3 and SF-837-A4 together with a small amount of substance SF -837. This white colored powder was then subjected to chromatographic separation using a silica gel acid. and then subjected to chromatographic separation with an aluminum oxide column, as described in Example 1, 15 mg of the pure SF-837-A2 substance as the free base, 85 mg of the pure SF-837-A3 substance as the free base and 35 mg of the pure SF-837-A4 substance was obtained as a free base in the form of white colored powders. Example 3: 200 mg each of the free bases of the substances SF-837-A2, SF-837-A3 and SF-837-A4. which had been isolated in Examples 1 and 2 were added dropwise with a saturated solution of citric acid. The formed precipitates were filtered and dried to give the citrates of substances SF-837-A2, SF-837-A3 and SF-837-A4 in amounts of 210 mg, 220 and 190 mg, respectively. Example 4: 300 mg of the SF-837-A3 substance were suspended in 20 ml of distilled water, and an aqueous solution of tartaric acid was added to the suspension. The mixture was shaken well and brought to pH 4. The mixture was then filtered to remove insolubles and the filtrate was freeze-dried to give 340 mg of the tartarate of the SF-837-A3 substance in the form of a white powder with a melting point of 115 ° C. Example 5: 300 mg of the SF-837-A, substance were dissolved as free base in 1.5 ml of pyridine, and 1.0 ml of acetic anhydride was added to this solution. The mixture was shaken and then allowed to stand at room temperature for 20 hours. The reaction mixture was then poured into ice water and shaken to give a precipitate. This precipitate was filtered off and dried in a desicator, 330 mg of a white powder being obtained. Recrystallization of this powder from carbon tetrachloride would give 310 mg of the diacetyl derivative of the SF-837-A2 substance in the form of white crystals with a melting point of 130 to 234 "C. In a similar way, 300 mg each of the substances SF-837-A3 and SF-837-A4 in the form of their free bases were treated with an excess of acetic anhydride in solution in pyridine, so that the mononeetyl derivatives of the substances SF-837-A3 and SF- 837-A4 in the form of white crystals in amounts of 295 mg (melting point 182 to 185 C), or 300 mg (melting point 166 to 168 "C) were obtained. PATENT CLAIM 1 Process for the preparation of a mixture of SF-837-substance, 5 F-837-A2-substance, SF-837-A3-substance and SF-837-A4-substance or the individual components SF-837-A2-substance, SF- 837-A3 substance and SF-837-A4 substance and acid addition salts thereof, characterized in that a strain of Streptomyces mycarofaciens or its mutants or variants is cultured under aerobic conditions in a culture medium which contains assimilable nitrogen and carbon sources . around a Mixture of SF-837 substance. SF-837-A2 substance. SF-837-A3- To generate and enrich the substance and SF-837-A4 substance in the culture and then this mixture is obtained from the culture and, if necessary, the SF-837-A, substance, SF-837-A; Substance and SF-837-A4 substance isolated therefrom. SUBCLAIMS 1. The method according to claim 1, characterized in that a strain of Streptomyces mycarofaciens of ATCC No. 2145t is cultivated under aerobic conditions in a liquid culture medium which contains assimilable carbon and nitrogen sources in order to produce the SF-837-A, substance, the SF-837-A3-substance and the SF-837-A4-Sub - to generate and enrich punch in the culture, and then the mixture of active substances is isolated from the culture medium and then the substances SF-837-A2, SF-837-AD and SF-837-A4 separate from each other. 2. The method according to claim 1, characterized in that a strain of Streptomyces myearofaciens of ATCC No. 21454 in a liquid culture medium containing assimilable carbon and nitrogen sources at a temperature of 20 to 30 "C under aerobic conditions until a considerable antibacterial activity is achieved in the culture broth, the culture filters, the Culture filtrate extracted at a pH of 7 to 9 with a water-immiscible organic solvent for substances SF-837-A2, SF-837-A3 and SF-837-A4, for example ethyl acetate, butyl acetate, chloroform, ethyl ether, methyl isobutyl ketone and butanol, the obtained organic extract containing the active substances, extracted with dilute aqueous acid, for example aqueous hydrochloric acid or aqueous sulfuric acid, making the obtained aqueous extract alkaline by adding alkali, preferably to pH 7 to 9, extracting the aqueous extract as well as its with a water-immiscible organic solvent in order to convert the active substances into this organic solvent, at least once this transition of the active substances from an organic extract in a water-insoluble organic solvent into a dilute aqueous acid and then into a further portion of the water-immiscible organic solvent, the finally obtained organic solution of the active substances, which was partially purified in this way, evaporated to dryness in a vacuum, dissolving the obtained raw powder of the mixed active substances in ethyl acetate or butyl acetate, the solution is placed on a column of activated charcoal, eluted with ethyl acetate or butyl acetate, the active fractions of the eluate are collected and concentrated to a smaller volume, the concentrate is extracted with aqueous, dilute hydrochloric acid, the aqueous extract is made alkaline by adding alkali, the formed The precipitate, which consists essentially of the mixed active substances, is separated off, the precipitate is dissolved in benzene, the benzene solution is subjected to column chromatography on silica gel using a mixture of benzene-acetone as the eluent and the eluate is collected in fractions, each of these fractions using aluminum oxide - Thin-layer chromatography examined, those fractions combined and evaporated to dryness in vacuo, which result in a single spot in this thin-layer chromatography, which is characteristic of SF-837-A4 substance, the free base of SF-837-A4 substance being obtained in the form of a pure powder, those fractions which are obtained from the silica gel column after the Fractions which give the single spot characteristic of the SF-837-A4 substance are obtained and which contain both the substances SF-837-A3 and SF-837-Aj together men included. combined and evaporated to dryness, a powder of a mixture of the substances SF-837-A and SF-837-AD being obtained, this powder being dissolved in benzene, the resulting solution of the powder in benzene being subjected to column chromatography on activated aluminum oxide. using a mixture of ethyl acetate-benzene as eluent, the eluate from it collects in fractions, each of these fractions is checked by means of aluminum oxide thin-layer chromatography, those fractions are combined and evaporated to dryness in the va hlumm, which give a single spot, which is characteristic is for the SF-837-A3 substance in the mentioned aluminum oxide thin layer chromatography, the free base of the SF-837-A3 substance being obtained in the form of pure powder, the remaining fractions of the eluate from the above-mentioned column of active aluminum oxide also combined and evaporated to dryness in vacuo. to get a powder. which mainly contains the SF-837-A2 substance, the powder redissolved in benzene, this benzene solution again subjected to column chromatography on activated alumina, using a mixture of ethyl acetate-benzene as the eluent, the eluate therefrom collects in fractions, each of these fractions checked by means of aluminum oxide thin-layer chromatography, those fractions combined and evaporated to dryness in Va kuuno, which result in a single spot, which is characteristic of the SF-837-A2 substance in the aluminum oxide thin-layer chromatogram mentioned. whereby the free base of the SF-837-A2 substance is obtained in the form of pure powder. 3. The method according to claim 1, characterized in that the free bases obtained are converted into their acid addition salts. 4. The method according to claim I and dependent claim 1, characterized in that the SF-837-A2 substance obtained in the form of the free base is acetylated to its diacetyl derivative. 5. The method according to patent claim I and sub-claim 1, characterized in that the SF: 837-A3 substance obtained in the form of the free base is acetylated to its monoacetyl derivative. 6. The method according to claim 1 and dependent claim 1, characterized in that the SF-837-A4 substance obtained in the form of the free base is acetylated to its monoacetyl derivative. PATENT CLAIM II Antibiotic product, produced by the process according to claim 1. SUBCLAIMS 7. Antibiotic product according to claim II, produced by the process according to dependent claim 1. 8. Antibiotic product according to claim 11, produced by the method according to dependent claim 2. 9. Antibiotic product according to claim 11, produced by the method according to dependent claim 4. 10. Antibiotic product according to claim II, produced by the process according to dependent claim 5. 11. Antibiotic product according to claim II, produced by the process according to dependent claim 6.