AT246923B - Process for the production of the new antibiotic R-451 and its components R-451 A to R-451 E as well as the O-acyl derivatives, ethers and salts with bases of the antibiotic and the components - Google Patents

Description

  

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  Process for the preparation of the new antibiotic R-451 and its
Components R-451 A to R-451 E as well as the 0-acyl derivatives, ethers and salts with bases of the antibiotic and the components
The invention relates to a process for the production of the new antibiotic R-451 and its components R-451 A-R-451 E and the 0-acyl derivatives, ethers and salts with bases of the antibiotic and the components.



   The method according to the invention is carried out in such a way that the new species Micromonospora carbonacea, deposited under registration number NRRL 2972, or one of its mutants *) or variants with essentially the same biological activity under usual conditions, preferably aerobically in an aqueous nutrient medium, the assimilable carbon and nitrogen contains, incubated, the antibiotic formed is isolated from the culture and optionally purified or broken down into its components and then the antibiotic or at least one of its components R-451 A, R-451 B, R-451 C , R-451 D and R-451 E optionally converted into one of their 0-acyl derivatives, into ethers or into salts with bases.



   Cultures of the type of microorganisms used according to the invention have been deposited in the culture collection of the United States Department of Agriculture, Northern Utilization Research and Development Division, Peoria, Illinois.



   The new species Micromonospora carbonacea, which will be referred to as M. carbonacea for short in the following, has received the NRRL number 2972 and was originally obtained from a soil sample obtained from Olean, NY, USA. M. carbonacea is characterized when it is cultivated under incubation for 20 days at 28 ° C in a medium made from 0.5% yeast extract, 10/0 dextrose, 0.1% calcium carbonate and 1.510 agar, through jet-black spots in an otherwise orange-colored vegetative mycelium.

   The spores of M. carbonacea are usually elongated to ellipsoidal, and the dimensions of mature spores are on average zo. The fully ripe spores are darkly colored when viewed microscopically in the light passing through them. Sporophores do not appear statistically evenly distributed ("randomly") over the entire mycelium, but rather concentrated in certain areas of the vegetative mycelium. This normally does not seem to split into polymorphic elements (fragments), but remains a whole until it is possibly autolysed. The vegetative mycelium has an average diameter of 0.6 p, is not acid-fast, gram-positive.

   The organism feeds on certain types of protein and starch, is aerobic and does well in the range of 16 to 370C, but not at 500C or above. The shape and orange color (with brownish black spots) are typical of fresh soil isolates, but these characteristics can be temporarily or permanently lost if the strains are repeatedly isolated and transferred.



  *) "Mutants" are understood to mean both natural and artificial mutants, in particular mutants which are formed from the microorganisms in question with the aid of mutation agents such as high-frequency radiation (X-ray or ultraviolet radiation), actinophages and nitrogen monsters.

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   To isolate the microorganism mentioned, some of the soil sample in question is shaken in sterile distilled water and, after making suitable dilutions, the suspension is diluted
 EMI2.1
 0, o calcium propionate and 2, 00/0 agar; everything in distilled water.



   The cultures are tested for antibiotic activity by first growing them for up to 60 days at 260C in the following medium: ouzo beef extract, 0.50/0 tryptose, 0.1% dextrose, 2.4% soluble starch, 0, 5% yeast extract, 1.5% agar, all in tap water. The whole aqueous agar is then extracted with butanol and the butanol-water extract is concentrated. The butanol-water mixture extracts enough antibiotic to deliver a concentrate that prevents the growth of Staphylococcus aureus and Bacillus subtilis in a disc test. Antibiotic activity against the same organisms can be observed if one incubates submerged for 96 hours in an aqueous medium which contains suitable nutrients, e.g.

   B. 0.50/0 yeast extract, 0.1% soluble fish ingredients, 0.1%) calcium carbonate, 0.1%; by spraying dried corn steep liquor and 3, 00/0 lactose.



   Tables I-V compare the new Micromonospora species described above with certain known Micromonospora species. If there are dashes in individual table fields, this means that the relevant data is either missing or not included in the tables.



   The colony observations recorded in Table I were made after the media concerned had been incubated for 14 days at 24-260C. The chosen color names refer to the following
 EMI2.2
    The first color name consists of a color name according to the "Descriptive Color Name Dictionary" by Taylor, Knoche and Granville, published by the Container Corporation of America [1950], in conjunction with a number corresponding to this name, which is taken from the "Color Harmony Manual", 4th edition [1958 ], also published by the Container Corporation of America, the second designation consists of a color name and a number with the same meaning, according to the National Bureau of Standards (USA), Circular 553 of November 1, 1955.



   Table H compares the characteristic properties of colonies of the preferred according to the invention and some known species of the genus Micromonospora when using different media after 14 days of incubation at 26 C. The data for the known species with which those used according to the invention are compared are from Bergey's "Manual of Determinative Bacteriology "7th Edition [1957] edited by the Williams & Wilkins Company, Baltimore.



   In the tables mA and EB, the growth and colony color of the micromonospora species preferably used according to the invention are compared with the corresponding properties of three available known micromonospora species when using further culture media. The media used here are those commonly used for the determination of Streptomyces species. Colony color is designated according to the system explained in connection with Table I. The above-mentioned microorganisms are able to use various carbon and nitrogen sources.



   In Table IV, their carbon source usage is compared to that of certain known Micromonospora species using visual estimates of their growth on agar plates in a medium,
 EMI2.3
 and 10/0 of the test carbohydrate in question, all in distilled water.



   In Table V, the nitrogen source utilization of the microorganisms of the genus Micromonospora preferred according to the invention is compared with that of other Micromonospora species on the basis of visual estimates of their growth on agar plates in a medium consisting of 10/0 glucose, 1.550 agar and the nitrogen source concerned (in a concentration as indicated ), all in distilled water. The color designations used in Table V are based on the system explained in connection with Table I.



   As stated above, the invention includes the use of mutants and variants of the new Micromonospora species mentioned for the production of antibiotics. An example of such a variant, which differs from the type described above in a subordinate manner, e.g. B. morphologically or biochemically, is M. carbonacea var. Aurantiaca. A culture of this has also been deposited in the United States Department of Agriculture's culture collection, Northern Utilization Research and Development Division, Peoria, Illinois, U.S.A. and is available there under its NRRL number 2997. This variant was also originally derived from one in Olean, N.

   Y., isolated soil sample and is taxonomically identical to M. carbonacea, with the following differences:
M. carbonacea var. Aurantiaca does not reduce nitrate; sometimes forms a yellow diffusible pigment when cultured in a mannose or xylose based carbohydrate medium, and

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 forms spores to a limited extent. The colony color on most agar media is orange, with a tendency to turn orange-brown with age. The orange surface consists mainly of vegetative mycelium and occasionally contains small black fields that consist almost entirely of dark-colored spores. This variant grows well on Bennett's agar; the colony increased. Their color is fiery red-g5NC, intense red-orange-35.

   On tomato paste-oatmeal agar with a 0.1% sodium carbonate content, growth is mediocre; the colony increased. Their color is bitter! 6 colors-g5PC, deep orange-51.



   The variant is equivalent to the type described above with regard to the formation of antibiotics in general and of antibiotics of the R-451 group in particular.



   The above-described microorganisms which can be used according to the invention provide antibiotically active substances with the aid of the fermentation processes described below. After the fermentation incubation) these can be found both in the mycelium as well as in the broth; after the mycelium has been separated from the broth, the broth is preferably used as the source for its recovery.



   Paper chromatographic investigations have shown that the antibiotic substances formed by M. carbonacea and its variants consist of at least five components, which are hereinafter referred to as R-451 A, R-451 B, R-451 C, R-451 D and R. -451 E. Their separation from each other depends on the method used. In a specific partition chromatographic system, the substances with the migration properties corresponding to components A, B, C and D are isolated as a group (antibiotic active) which is referred to below as R-451.



  One of the main components of this group appears to be R-451D.



   To form the antibiotic substances, the microorganism M. carbonacea is cultivated at a temperature of about 25 to 40 ° C. under submerged aerobic conditions in an aqueous nutrient medium which contains an assimilable carbon and nitrogen source. Suitable carbon sources are carbohydrates such as starch, dextrin, certain sugars and the like. The like. Suitable nitrogen sources can be both organic and inorganic in nature, but are preferably organic products such as soybean meal, peptones and the like. like



   Incubation takes place at a pH in the range from about 6 to 8; over a period of about 60 to 72 hours. At the end of this period, maximum antibiotic formation is reached.



   Since most of the antibiotically active substances are in the broth, the mycelium is filtered off and discarded. The antibiotic substances are isolated from the broth by solvent extraction such as described below. They can be separated into their components by partition chromatographic methods, as explained in more detail below.



   The antibiotic substances are first separated from the extract obtained from the broth by evaporating off the solvent in the form of a residue. The residue is paper chromato-
 EMI3.1
 Agar plate sets. After a contact time of 15 minutes, the paper is removed from the plate. mt, which are then incubated for 16-20 hours at 370C. The Rp values of the antibiotically active components can be inferred from the position of the inhibition zones.



   Table VI shows the solvent systems used and the RF values determined for the antibiotic components. Descending solvents were used in all systems.



   In the following examples, suitable methods for incubating the microorganisms preferred according to the invention, for extracting the antibiotic substances from the broth and for separating these substances into their main components are explained. In some of these examples, test values for the antibiotic substances in question, expressed in units per mg, are given as a measure of their activity. The test is carried out microbiologically according to a standardized agar diffusion test method ("cup type"; see Donald C. Grove and William A. Randen. "Assay Methods of Antibiotics - A Laboratory Manual", New York [1955]) using Staphylococcus aureus (ATCC 6538P) as a test organism.

   A curve is plotted by plotting the effect achieved against the dosage of the antibiotic substance in question (diluted in phosphate buffer at PH = 8) in the following medium

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 EMI4.1
 
<tb>
<tb> peptone <SEP> 0, <SEP> 6%
<tb> Pancreatic <SEP> casein hydrolyzate <SEP> 0. <SEP> 41. <SEP>
<tb>



  Yeast extract <SEP> 0. <SEP> alo
<tb> beef extract <SEP> 0, <SEP> 15%
<tb> Dextrose <SEP> 0, <SEP> 15%
<tb> Agar <SEP> 1, <SEP> 5% <SEP>
<tb>. <SEP> PH <SEP> S, <SEP> 6
<tb>
 
The suspension of the test organism (Staphylococcus aureus) used is standardized in such a way that it transmits light with a wavelength of 660 mg to 20 g in a colorimeter. The quantitative effectiveness of the respective antibiotic sample is determined from the reference curve and expressed in units per mg; The unit is such an amount of test substance that is necessary to produce an inhibition zone of 15 mm diameter in a steel cylinder with an outer diameter of 6.5 mm.



   Example 1: Obtaining R-451 by incubating M. carbonacea on a laboratory scale.



   A) Germination:
Aseptically place a lyophilized culture of M. carbonacea in a 300 ml shake flask containing 100 ml of the following sterile medium:
 EMI4.2
 
<tb>
<tb> Bacto beef extract <SEP> 3 <SEP> g <SEP>
<tb> Tryptose <SEP> 5 <SEP> g <SEP>
<tb> Dextrose <SEP> 1g
<tb> Soluble <SEP> Starch <SEP> 24g <SEP>
<tb> yeast extract <SEP> 5 <SEP> g
<tb> tap water <SEP> 1000 <SEP> ml *) <SEP>
<tb>
 
 EMI4.3
 (280 rev / min, 5 cm shaking movement).



   B) Incubation:
 EMI4.4
 ben, each of which contains 500 ml of the following medium:
 EMI4.5
 
<tb>
<tb> yeast <SEP> 5 <SEP> g <SEP>
<tb> Soluble <SEP> fish ingredients <SEP> 1 <SEP> g
<tb> Corn <SEP> steep <SEP> liquor <SEP> (dried) <SEP> 1 <SEP> g
<tb> Calcium carbonate <SEP> 1 <SEP> g <SEP>
<tb> Lactose <SEP> 30 <SEP> g
<tb> tap water <SEP> 1000 <SEP> ml
<tb>
 
The flasks and their contents are incubated for 2-3 days at 260C on a rotating shaker and the flask contents are then combined.



   C) Extraction of the antibiotic substances:
A filter aid (diatomaceous earth) is added to the combined substrate according to part B) of this example, and the filtrate is filtered and washed with ethyl acetate or chloroform, keeping the pH of the aqueous phase at or above 7 (but preferably as close to 7 as possible) . The solvent is evaporated from the extract in vacuo, the oil that remains is dissolved in excess chloroform and clarified by filtration.

   The clear chloroform solution is concentrated to a small volume in vacuo and the concentrated solution thus obtained is added to ten times the volume of petroleum ether (boiling range 30-600 ° C.) with stirring. The resulting precipitate is reduced *) In this and in similar recipes throughout the description, the solvent (mostly water) is specified once as an absolute amount, at other times with the prefix "ad". Although, strictly speaking, these are different statements, these two terms can be interchanged with one another as required, within the scope of the precision required for these recipes.

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 filtered *) and contains most of the antibiotic substances formed.

   He is active against
Staphylococcus aureus, when solutions with a content of only 10 mol are subjected to the disc diffusion test.



   D) Purification of the raw antibiotic substance obtained according to part C) of this example:
Purification by column chromatography on aluminum oxide is used here. The main eluate is designated as R-451 and appears to be free of the R-451 E component. The aluminum oxide cleaning delivers a water-soluble product and thus enables the preparation of aqueous dosage forms or more soluble forms of the antibiotic.



   An aluminum oxide adsorption chromatography column is prepared by pouring a suspension of pure aluminum oxide (for chromatography; commercial product No. 71'707 from Merck & Co.) in chloroform-methanol (3: 1) into a glass column with a clearance of about 51 mm until the aluminum oxide layer has reached a height of about 61 cm.



   10 g of the crude antibiotic substance obtained in step C) of this example are dissolved in 50 ml of chloroform and this solution is slowly applied to the column. This is then eluted first with chloroform-methanol (3: 1), collecting 12 fractions of 500 ml each, and then with methanol-chloroform (3: 1), collecting a further 37 fractions of 500 ml each. The fractions are examined by evaporating small samples in air and testing their residues against Staphylococcus aureus and combined into groups according to their activity against this organism.



  The main amount of eluted substance and the main activity are found in fractions 13-30. These are combined, concentrated in vacuo and the dissolved substance is precipitated by adding to petroleum ether. The precipitate, consisting of 1.7 g of R-451, is filtered off and dried in the air. It shows activity against Staphylococcus aureus (in the disc diffusion test) at concentrations of 1 / 1g / m1 and up.



   Table VII shows how the individual fractions are pooled and how the solid antibiotic substance is isolated from them, together with the results of the disc tests against Staphylococcus aureus and the RF values of the pooled fractions in the relevant system.



  The RF values were measured in the usual manner by Bioc. utography of the developed paper chromatogram against Staphylococcus aureus determined.



   Example 2: Obtaining R-451 by incubating M. carbonacea var. Aurantiaca on a laboratory scale.



   By replacing the lyophilized culture of M. carbonacea according to part A) of Example 1 with a corresponding culture of M. carbonacea var. Aurantiaca and otherwise essentially following the procedure of the example mentioned, one arrives at the same time in R-451.



   Example 3: Obtaining R-451 by incubating M. carbonacea on an industrial scale.



   A) Germination:
Aseptically place a lyophilized culture of M. carbonacea in a 300 ml volumetric flask containing 100 ml of the following sterile nutrient medium:
 EMI5.1
 
<tb>
<tb> Bacto- <SEP> beef extract <SEP> 3g <SEP>
<tb> Tryptose <SEP> 5 <SEP> g
<tb> Dextrose <SEP> 1 <SEP> g <SEP>
<tb> Soluble <SEP> Starch <SEP> 24 <SEP> g <SEP>
<tb> yeast extract <SEP> 5 <SEP> g
<tb> Calcium carbonate <SEP> 1 <SEP> g <SEP>
<tb> tap water <SEP> 1000 <SEP> ml
<tb>
 
The flask and its contents are incubated for four days at 350 ° C. (or until germination has taken place) on a rotating shaker (280 rev / min, 5 cm movement).



   B) pre-vaccination:
Add 5 ml of the first inoculum obtained from the above germination to three 300 ml sections & oI-
 EMI5.2
 72 h at 300C on the rotating shaker.



   C) Preparation of the inoculum. 25 ml of the second inoculum obtained by the above pre-inoculation are transferred to ten 2 1 flasks, *) It may be necessary to reprecipitate one or more times from chloroform if an oil precipitates instead of a filterable solid.

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 each of which contains 500 ml of the sterile culture medium used for germination, and the flasks and their contents are incubated for 72 h at 300 ° C. on a rotating shaker (280 rev / min, 5 cm shaking movement). The contents of the flask are then combined and aseptically transferred into a sterile inoculum flask with a lateral attachment tube (total volume about 51).



   D) Tank fermentation:
The 5 l of the inoculum obtained according to step C) is aseptically transferred into a fermenter with a capacity of about 130 l and containing 90 l of the sterile nutrient medium used for germination, and incubated aerobically for 20-30 h (until the compact cell volume - determined by 5 min long centrifugation of a 10 ml sample at 2800 rpm - about 2-3 ml, i.e. 20-30, under the following conditions:
 EMI6.1
 
<tb>
<tb> temperature <SEP> 350C
<tb> Supply <SEP> to <SEP> sterile <SEP> air <SEP> about <SEP> 153 <SEP> l / min
<tb> Press <SEP> about <SEP> 0.5 <SEP> off <SEP>
<tb> stirring movement <SEP> 180 <SEP> rev / min
<tb>
 As soon as the compact cell volume has reached at least 2 ml, the contents of the
 EMI6.2
    500 liters of medium contains:

   
 EMI6.3
 
<tb>
<tb> yeast extract <SEP> 8, <SEP> 5 <SEP> kg
<tb> Soluble <SEP> fish ingredients <SEP> 1, <SEP> 7 <SEP> kg
<tb> Corn <SEP> steep <SEP> liquor <SEP> (dried) <SEP> 1.7 <SEP> kg
<tb> Calcium carbonate <SEP> 1, <SEP> 7 <SEP> kg <SEP>
<tb> Lactose <SEP> 51.0 <SEP> kg
<tb> Foam control agent <SEP> (GE <SEP> 60) <SEP> 500 <SEP> ml
<tb> Soft <SEP> water <SEP> 1665 <SEP> 1
<tb>
 
 EMI6.4
 425 1 of air with a pressure of about 0.5 atü. Towards the end of the incubation period, the activity of the antibiotic formed reaches a maximum which remains essentially constant, as can be determined by taking samples and testing against Staphylococcus aureus.



   During the incubation, the pH remains essentially constant in the range from 7.0 to 7.3. The compact cell volume reaches a constant value of about 2.0 ml.
 EMI6.5
 equal volumes of toluene extracted, the toluene extracts combined and evaporated to dryness in vacuo, a dark brown oily residue remaining. This is suspended in 1000 ml of chloroform, filtered and the chloroform solution is added to ten times the volume of petroleum ether with vigorous stirring. Stirring is continued for 5 minutes, the semisolid oily precipitate formed is separated from the supernatant liquid by decantation, redissolved in 600 ml of chloroform and the resulting solution is added to five times the volume of petroleum ether with vigorous stirring.

   The amorphous precipitate which separates out is filtered off, washed with petroleum ether and air-dried.
 EMI6.6
 
F) Alternative method for isolating the antibiotic substances from tank fermentation:
In place of the method of part E) of this example, the extraction method of example 1 C) can also be used. This gives about 125 g of a solid product with an activity comparable to that of the product from Example 1 C).



   G) Separation of the antibiotic substances into components:
In contrast to the method of Example 1 D), a column chromatography on diatomaceous earth is used here, with the aid of which the antibiotic substances are separated and the components R-451 A, R-451 B, R-451 D, R-451 E and the others Component R-451 Cl can be isolated.



   The separation of the accruing according to part E) or - less advantageous - part F) of this example
 EMI6.7
 means system and purified diatomaceous earth (commercial product Chromosorb W from Johns Manville & Co., not washed with acid, grain size 60-100 meshes) as an inert carrier for the heavier

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 Phase.

   First prepare a two-phase solvent system with the following composition:
 EMI7.1
 
<tb>
<tb> Petroleum ether <SEP> (boiling range <SEP> 60-900C) <SEP> 25 <SEP> parts
<tb> Ethyl acetate <SEP> 75 <SEP> parts
<tb> methyl alcohol <SEP> 70 <SEP> parts
<tb> i <SEP> Distilled <SEP> Water <SEP> 30 <SEP> parts
<tb>
 
Then bring 1.9 kg of the inert carrier with 950 ml of the heavier phase of the above two-phase
The solvent mixture equilibrates and the mixture is slowly poured into a 9 cm wide glass column with a fritted disk at the bottom, three quarters of which was previously filled with the lighter phase. The diatomaceous earth is allowed to settle in the column to form a compact sediment and is then compressed further by means of a nitrogen pressure of about 0.07 to 0.15 atm (the height of the column produced in this way is 137 cm).



   7.0 g of the amorphous mixture from part E) or F) of this example are now dissolved in 38 ml of the heavier phase of the two-phase solvent system, 19 g of diatomaceous earth are added to the solution and the mixture thus obtained is placed on top of the filling of the column up and compresses tightly. Elute with the lighter phase of the two-phase solvent system.



   The eluates are withdrawn at a rate of 50 ml / min. The number of individual fractions is arbitrary, but depends on the "hold-up volume" (HUV) of the column. (The HUV is defined as the volume that the lighter phase of the two-phase system takes up within the entire column load.) In the column described above, the HUV is approximately 3500 ml. The first HUV collected is discarded. One sample of each of the following fractions is paper chromatographed using the solvent system I (Table VI) described above and the developed and dried paper chromatogram is tested bioautographically against Staphylococcus aureus.

   The fractions are combined into groups according to the test result, the fraction groups are evaporated to dryness in a vacuum, the residues are weighed and again examined by paper chromatography: sch and bio autograph to determine their relative composition. This leads to the results of table Vin.



   1 g of the product obtained from HUV 3-10 is then chromatographed again, using the inert carrier (Chromosorb W) for the heavier phase of the following two-phase solvent system:
 EMI7.2
 
<tb>
<tb> Petroleum ether <SEP> (boiling range <SEP> 30 <SEP> - <SEP> 600C) <SEP> 10 <SEP> parts
<tb> Toluene <SEP> 200 <SEP> parts
<tb> n- <SEP> butyl alcohol <SEP> 15 <SEP> parts
<tb> Ethyl alcohol <SEP> 15 <SEP> parts
<tb> Distilled <SEP> water <SEP> 70 <SEP> parts
<tb>
 
First, 600 g of the diatomaceous earth carrier are brought into equilibrium with 300 ml of the heavier phase of the last-mentioned two-phase solvent system, the mixture is slowly poured into a glass column with a diameter of 5 cm which is partially filled with the lighter phase of this two-phase solvent system,

   The weight of the carrier allows it to settle into a compact sediment and is further compressed by a nitrogen pressure of around 0.07 to 0.15 atmospheres. The height of the column prepared in this way is 107 cm and its hold-up volume is 900 ml. The product to be chromatographed is then dissolved in 10 ml of the heavier phase, the solution is mixed with 5 g of the inert carrier and mixed. The mixture is then applied to the filling of the column and pressed firmly. It is then eluted with the lighter phase of the two-phase solvent system at a flow rate of 100 ml / min. Fractions of 50 to 900 ml (corresponding to 1/18 - 1/1 of the HUV of the column) are continuously removed.

   The first HUV that occurs immediately after application of the product is discarded, the second and the following are collected.



   A sample of each collected fraction is immediately paper chromatographed using solvent system I (Table VI) and the developed and dried paper chromatogram is bioautographically tested against Staphylococcus aureus. (Solvent system I is preferred because it migrates fastest.) The fractions,. are combined in groups according to their paper chromatographic data and evaporated to dryness in vacuo, the residues are weighed and again subjected to a paper chromatograph and bio: utographic test to confirm their composition.

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   The result of the column chromatographic separation thus carried out is shown in Table IX.



   Example 4: Obtaining R-451 by incubating M. carbonacea var. Aurantiaca on an industrial scale.



   If the lyophilized culture of M. carbonacea, as it is used in part A) of example 3, is replaced by a corresponding culture of M. carbonacea var. Aurantiaca and otherwise the method of example 3 C Telle A) -D) , then E) or F), finally G)], you get to R-451 in the same way.



   Example 5: Separation of component R-451 D from R-451.



   100 mg of the residue from HUV 1, 5-14 according to Table IX, Example 3 G)] or the corresponding fraction from Example 4 are suspended in 40 ml of ether at room temperature with vigorous stirring, and the filtrate is left to stand in the cold overnight. The precipitate that separates out is filtered off, washed with ice-cold ether and air-dried, giving about 81 mg of R-451 D in the form of an almost colorless powder with a test value of 1155 units / mg.



   The R-451 D obtained in this way proves to be biologically homogeneous in a quantitative test against Staphylococcus aureus. If it is chromatographed on a thin layer of silica gel according to Stahl's method using acetone-benzene (1: 1) as the eluent and the developed and dried plate is then treated with sulfuric acid, no other substance than
Discover R-451 D.



   Example 5 a: The product from Example 5 can be further purified as follows: Dissolve in as little hot isopropanol as possible, add activated charcoal (about leo the weight of the substance to be purified), filter and cool in an ice bath. The resulting precipitate is filtered off, washed with cold isopropanol and dried in a high vacuum at room temperature. The product purified in this way has a test value of 1450 units / mg.



   Example 6: Preparation of R-451 D-Acetate.



   25 mg of R-451 D are dissolved in 1 ml of pyridine, 0.4 ml of acetic anhydride is added and the mixture is left to stand overnight. The precipitate is then filtered off, washed with water to remove the pyridine and the acetic acid formed, and dried over phosphorus pentoxide overnight in a high vacuum. Yield: 11 mg of a colorless amorphous powder, melting point = 135-1360C. A second amount can be obtained from the mother liquor of the precipitate mentioned by cooling overnight in a refrigerator (50C), filtering off the precipitate, washing free of pyridine and acetic anhydride and drying, as indicated above. Yield: 6 mg of a colorless, amorphous powder.



   If the product is chromatographed on a thin layer using silica gel (commercial product "Silikagel G" from E. Merck AG., Darmstadt) as the adsorbent and acetone-benzene (1: 1) as the eluting system, and the developed and dried plate with sulfuric acid sprayed, only a single component with the RF value 0.87 can be determined. (Under these conditions, R-451 D has an RF value of 0.99.) On alkaline hydrolysis in methanol, the R-451 D used is reformed from R-451 D acetate.



   Example 6a: The product from Example 6 can be further purified as follows: Dissolve in methylene chloride, add activated carbon, filter and add hexane to the clear filtrate. The precipitate is filtered off and air dried.



   Example 7 Separation of component R-451 E.



   This non-polar component of the antibiotic mixture is usually found in high concentration in the supernatant liquid of the chloroform-petroleum ether precipitation, which is described in part E) of Example 3, and in the corresponding fraction obtained according to Example 4. It is also contained in the precipitated antibiotic substances in a smaller amount and can be isolated from there with the aid of the column chromatography method which is described in part G) of Example 3. To obtain R-451 E, fraction HUV 1-1, 5 (Table IX) is evaporated to dryness in vacuo and air-dried. This gives about 320 mg of an amber-colored substance, from which, by reprecipitation from ether-hexane, 280 mg of an almost colorless amorphous powder, purified R-451 E, is obtained.

   To shoot against Staphylococcus aureus and Bacillus subtilis according to the result of a paper chromatographic analysis in system I (Table VI) and subsequent bioautography. the antibiotic R-451 E produced in this way is biologically homogeneous.



   Example 8: Separation of the components R-451 B, R-451 Cl and R-451 A.



   A) pre-separation:
The separation of the above-mentioned components from the crude antibiotic mixture is initiated by partition chromatography similar to that described in part G) of Example 3, wherein

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 a two-phase solvent system and a purified diatomaceous earth (commercial product Chromosorb W from Johns Manville & Co., not washed with acid, grain size 60 - 100 mesh) are used as the inert carrier for the heavier phase:
First the two-phase system (petroleum ether, ethyl acetate, methanol, water) is prepared as described in Example 3G). 2.5 kg of the inert support are then brought into equilibrium with 1250 ml of the heavier phase of the two-phase solvent mixture and the column is filled as described in Example 3 G).

   The HUV of this column is approximately 8,000 ml.



   7.4 g of the amorphous mixture according to Example 3 E) or the corresponding fraction obtained according to Example 4 are now dissolved in 40 ml of the heavier phase of the two-phase solvent system, 20 g of diatomaceous earth are added, the solid mixture is added to the top of the column filling and compressed tightly. Elute with the lighter phase of the two-phase solvent system. The column chromatographic separation process described in Example 3 G) gives the results in Table X.



  As you can see, the components R-451 B and R-451 Cl are concentrated in the fraction group HUV 1, 9-2, 8, the component R-451 A in the fraction group HUV 4, 0-5, 0.



   B) Separation of R-451 B and R-451 Cl from one another:
1.8 g of the residue of the fraction group HUV 1, 9-2, 8 obtained in the above chromatographic separation are chromatographed on 1800 g of cellulose powder (Whatman brand, not washed with acid, fine), which is in a 7.5 cm column Diameter and 150 cm height, using the following single-phase solvent mixture:
 EMI9.1
 
<tb>
<tb> Acetone <SEP> 1 <SEP> part
<tb> Petroleum ether <SEP> (boiling range <SEP> 30-600C) <SEP> 25 <SEP> parts
<tb> Benzene <SEP> 50 <SEP> parts
<tb>
 
The substance to be chromatographed is dissolved in 68 ml of the solvent mixture, 50 g of cellulose are added and the mixture is applied to the top of the column.

   The elution is then carried out at a flow rate of 50 ml / min, with 100 ml fractions being continuously taken. The HUV of this column is approximately 6,000 ml. The eluates are pooled in groups as shown in Table XI. As can be seen, the component R-451 B is concentrated in the fraction group HUV 17, 7-27, 7, the component R-451 Cl in the fraction group HUV 1, 7-17, 6.



   C) Cleaning of R-451 B:
200 mg of the residue obtained from fraction group HUV 17, 7-27, 7 (Table XI) are dissolved in 5 ml of acetone and the acetone solution is poured into 50 ml of hexane with vigorous shaking. The resulting precipitate is filtered off, washed with hexane and dried over phosphorus pentoxide at room temperature in vacuo, giving 120 mg of an almost white powder. The R-451 B thus obtained may be contaminated with traces of other components; However, if it is chromatographed by Stahl's method on a thin layer of silica gel using acetone-benzene (1: 1) as the eluent and the developed, dried plate is then treated with sulfuric acid, only one substance can be detected, so that one further cleaning is not necessary.



   D) Purification of R-451 Cl:
100 mg of the residue obtained from fraction group HUV 1, 7-17, 6 (Table XI) are dissolved in 5 ml of acetone and the solution is poured into 50 ml of isopropyl ether with vigorous shaking. The resulting precipitate is filtered off, washed with isopropyl ether and dried over phosphorus pentoxide at room temperature in vacuo. This leads to about 70 mg of an almost white powder consisting of R-451 Cl. The product thus obtained is, as can be seen from quantitative testing against Staphylococcus aureus, essentially biologically homogeneous.



   E) Purification of R-451 A:
 EMI9.2
 (Table X) obtained residue in 10 ml of dichloromethane, filtered, the solid residue was washed with dichloromethane and dried at room temperature in vacuo over phosphorus pentoxide. This gives 70 mg of a whitish powder consisting of R-451 A.



   Example 9: Instead of following the method described in Example 8 [Parts A) -C)], R-451 B can also be isolated as follows: A 64 cm high chromatography column with a diameter of about 50 mm is prepared by placing a slurry in Methylene chloride of 500 g of Florisil (60 to 100 mesh), which was activated for 18 h at 1050C, is poured into a glass column - the hold-up volume of the column prepared in this way is 1100 ml. 50 g of the amorphous crude antibiotic is dissolved as you would from it

 <Desc / Clms Page number 10>

 Example 3 [Part E)] is obtained in 300 ml of methylene chloride and the clear solution obtained is applied to the Florisil column.

   For elution, send first 4 HUV methylene chloride, then 8 HUV methylene chloride-acetone (95: 5), then 8 HUV methylene chloride-acetone (90:10), then 8 HUV methylene chloride-acetone (80:20), 8 HUV methylene chloride-acetone (50:50) and finally 16 HUV of pure acetone through the column.



  Table XIA shows the result of this separation, the letters in brackets in the right-hand column of the table designating the R-451 components present in the relevant fraction.



   That according to the previous paragraph or example. 8 parts A) -C)] obtained product can be further purified as follows. The amorphous powder is dissolved in hot isopropanol, activated carbon (about 1010 of the weight of the substance to be purified) is added, the mixture is stirred for 10 minutes, filtered and the filtrate is cooled to 5 C. The precipitate which separates out is collected on a filter, washed with cold isopropanol and dried in a high vacuum at room temperature for 24 hours. The product has a test value of 760 units / mg.



     Example 10: Preparation of the methyl ether of R-451 D.



   500 mg of R-451 D are dissolved in 11 ml of ethyl acetate and 11 ml of ethyl ether, an excess of an ethereal diazomethane solution is added and the yellow solution is left to stand at 50 ° C. for 72 hours. It is then evaporated to dryness in a stream of nitrogen, the residue is dissolved in 5 ml of isopropanol, cooled and the precipitate formed is filtered off.

   It is washed with cold isopropanol and the product obtained, which consists of 260 mg of the desired methyl ether in the form of colorless prisms with the following physical properties, is dried
 EMI10.1
 
Raw R-451, produced according to Example 1 D), is a colorless amorphous substance with the following physical and chemical properties:
 EMI10.2
 
<tb>
<tb> I. <SEP> Melting point <SEP> 200-204 C
<tb> n. <SEP> analysis values <SEP> (qualitative <SEP> group determination):
<tb> 1. <SEP> Ninhydrin test <SEP> negative
<tb> 2. <SEP> ferric chloride <SEP> negative
<tb> II. <SEP> Optical <SEP> rotation <SEP>: <SEP>
<tb> [α] 25D <SEP> = <SEP> -16 <SEP> (0.5% <SEP> in <SEP> chloroform)
<tb>
 
IV. Solubility:
Soluble in water; insoluble in petroleum ether; soluble in most organic solvents; dialyzable through a cellophane membrane in a methanol-methanol system.



   V. Ultraviolet spectrum:
Fig. 1 of the drawings shows the absorption spectrum for a concentration in methanol of 5 g / 100 ml (X means the wavelength in mali, D the optical density) - max at 288 ma L (E1% = 50).



   VI. Infrared spectrum:
Fig. 2 of the drawings shows the spectrum in Nujol *), where X is the wavelength in ju, y is the frequency in cm * and A is the absorption. The absorption maxima (schw = weak, m = medium, m-st = medium to strong, st = strong, sst = very strong, Sch = shoulder) are at the following wavelengths:

   
 EMI10.3
 
<tb>
<tb> \ <SEP> (jLi) <SEP> Intensité <SEP>
<tb> 2.92 <SEP> st
<tb> 5.84 <SEP> m
<tb> 6.00 <SEP> m <SEP> (wide)
<tb> 6. <SEP> 12 <SEP>
<tb> 6.45 <SEP> m-st
<tb> 8, <SEP> 00 <SEP> Sch
<tb> 8.07 <SEP> st
<tb> 8.33 <SEP> m-st
<tb> 8, <SEP> 5fi <SEP> Sch
<tb> 9, <SEP> 10 <SEP> st <SEP> (wide)
<tb>
 *) Nujol is a trademark for a hydrocarbon-based mineral oil, sufficiently pure for infrared spectroscopy, available from Plow Inc., Memphis, Tennessee.

 <Desc / Clms Page number 11>

 
 EMI11.1
 
<tb>
<tb> À <SEP> (Il) <SEP> Intensity <SEP>
<tb> 9, <SEP> 60 <SEP> st <SEP> (wide)
<tb> 10, <SEP> 26 <SEP> m-st
<tb> 10, <SEP> 55 <SEP> m-st
<tb> 11, <SEP> 02 <SEP> black
<tb>
 
VII. Stability:
At PH <7 already unstable at 200C.

   At pH> 7, the activity is retained even after heating at 1000C for 30 minutes.
 EMI11.2
 : A paper chromatographic analysis, as detailed below, and bioautographic determination of the distribution of stains (against Staphylococcus aureus, as described above) lead to the following RF values for R-451:
 EMI11.3
 
<tb>
<tb> Applied <SEP> solvent system <SEP> RF values
<tb> A: <SEP> 80% <SEP> aqueous <SEP> methanol <SEP> + 3%
<tb> sodium chloride <SEP>; <SEP> ascending <SEP>; <SEP> paper
<tb> before <SEP> with <SEP> sulfate-bisulfate <SEP> on <SEP> PH <SEP> = <SEP> 2, <SEP> 3
<tb> buffered <SEP> 0.80
<tb> B <SEP>: <SEP> n-propanol-pyridine-water-acetic acid
<tb> (15 <SEP>: <SEP> 10 <SEP>: <SEP> 3 <SEP>: <SEP> 12) <SEP>; <SEP> in ascending order <SEP> 1.0
<tb> C <SEP>:

   <SEP> n-propanol-water-acetic acid <SEP>
<tb> (50 <SEP>: <SEP> 40 <SEP>: <SEP> 5); <SEP> in ascending order <SEP> 0, <SEP> 93
<tb> D <SEP>: <SEP> SOoiges <SEP> aqueous <SEP> phenol <SEP>; <SEP> in ascending order <SEP> 0, <SEP> 95
<tb> E <SEP>: <SEP> benzene-methanol <SEP> (9 <SEP>: <SEP> 1) <SEP>; <SEP> in ascending order <SEP> 1, <SEP> 0 <SEP>
<tb> F <SEP>: <SEP> Benzene-Chloroform <SEP> (95 <SEP>: <SEP> 5) <SEP>; <SEP> descending <SEP>; <SEP>
<tb> paper <SEP> first <SEP> with <SEP> formamide-methanol
<tb> (1 <SEP>: <SEP> 1) <SEP> saturated <SEP> and <SEP> before <SEP> use
<tb> (for <SEP> removal <SEP> of the <SEP> methanol) <SEP> to <SEP> the
<tb> air <SEP> dried <SEP> 0, <SEP> 0
<tb> G <SEP>: <SEP> Benzene petroleum ether <SEP> (boiling range
<tb> 30 <SEP> - <SEP> 600C) <SEP> -acetone <SEP> (10 <SEP>: <SEP> 2, <SEP> 5 <SEP>: <SEP> 5) <SEP>;

   <SEP>
<tb> descending <SEP> 0, <SEP> 0 <SEP>; <SEP> 0. <SEP> 14 <SEP>; <SEP>
<tb> 0. <SEP> 28 <SEP>; <SEP> 0, <SEP> 64. <SEP>
<tb>
 



   Of the individual components of R-451, component R-451 A, obtained according to the process of Example 8 E), is a colorless amorphous substance with the following properties:
I. Ultraviolet spectrum:
Transparent in the range from 220 to 360 mil.



     H. Infrared spectrum: (spectrum in Nujol, see Fig. 3 of the drawings)
Absorption maxima (all broad and strongly pronounced) are at the following wavelengths: 2, 90, 6.25, 8.25-10.00.



   R-451 B, produced according to Example 8 C), is a colorless amorphous substance with the following properties:
I. Ultraviolet spectrum:
Transparent in the range from 220 to 360 mu.



     H. Infrared spectrum: (spectrum in Nujol, see Fig. 4 of the drawings)
Absorption maxima (with intensities as indicated) are at the following wavelengths:

 <Desc / Clms Page number 12>

 
 EMI12.1
 
<tb>
<tb> X <SEP> (fi) <SEP> intensity
<tb> 2.94 <SEP> m-st
<tb> 5, <SEP> 72 <SEP> st
<tb> 5.80 <SEP> m-st
<tb> 7, <SEP> 92 <SEP> m-st
<tb> 8.60 <SEP> m-st
<tb> 9, <SEP> 10 <SEP> sr <SEP> (wide) <SEP>
<tb> 9.64 <SEP> st <SEP> (wide)
<tb> 12.50 <SEP> m-st <SEP> (wide)
<tb> 13, <SEP> 90 <SEP> m-st <SEP> (wide)
<tb>
 
 EMI12.2
 : IV. Analysis values: a) Elemental analysis:
 EMI12.3
 
Methoxy groups = 12, 80%, C-methyl groups = 7. 15%, N-methyl groups = 2, 33%.



  V. Infrared spectrum (in Nujol): absorption maxima (with intensities as indicated) are at the following wavelengths:
 EMI12.4
 
<tb>
<tb> # <SEP> () <SEP> intensity
<tb> 2, <SEP> 88 <SEP> st
<tb> 3. <SEP> 35-3, <SEP> 48 <SEP> st <SEP> (wide)
<tb> 3, <SEP> 65 <SEP> black
<tb> 4, <SEP> 54 <SEP> black
<tb> 4, <SEP> 65 <SEP> black
<tb> 5.74 <SEP> st
<tb> 5, <SEP> 82 <SEP> Sch
<tb> 6.02 <SEP> Sch
<tb> 6, <SEP> 10 <SEP> m
<tb> 6, <SEP> 35 <SEP> - <SEP> 6.39 <SEP> m <SEP> (doublet)
<tb> 6.45 <SEP> st
<tb> 6, <SEP> 78 <SEP> - <SEP> 6, <SEP> 87 <SEP> st <SEP> (wide)
<tb> 7, <SEP> 24 <SEP> st
<tb> 7.30 <SEP> Sch
<tb> 7.40 <SEP> Sch
<tb> 7, <SEP> 67- <SEP> 7, <SEP> 80 <SEP> Sch
<tb> 7, <SEP> 92 <SEP> Sch
<tb> 7, <SEP> 98 <SEP> st
<tb> 8,12 <SEP> Sch
<tb> 8, <SEP> 30 <SEP> - <SEP> 8.

   <SEP> 35 <SEP> st <SEP> (wide)
<tb> 8, <SEP> 55 <SEP> Sch
<tb> 8, <SEP> 75 <SEP> - <SEP> 9, <SEP> 80 <SEP> st <SEP> (wide)
<tb> 10, <SEP> 20 <SEP> - <SEP> 10, <SEP> 26 <SEP> st <SEP> (wide)
<tb> 10, <SEP> 30 <SEP> Sch
<tb> 10, <SEP> 33 <SEP> Sch
<tb>
 

 <Desc / Clms Page number 13>

 
 EMI13.1
 
<tb>
<tb> # <SEP> (fi) <SEP> intensity
<tb> 10, <SEP> 61 <SEP> st
<tb> 10, <SEP> 85 <SEP> Sch
<tb> 11, <SEP> 03 <SEP> m
<tb> 11, <SEP> 52 <SEP> m
<tb> 11, <SEP> 70 <SEP> Sch
<tb> 11, <SEP> 85-12, <SEP> 00 <SEP> Sch
<tb> 12, <SEP> 75-12, <SEP> 81 <SEP> black <SEP> (wide)
<tb> 12, <SEP> 98 <SEP> black
<tb> 13, <SEP> 54-13, <SEP> 60 <SEP> m <SEP> (wide)
<tb> 13, <SEP> 85-13, <SEP> 90 <SEP> m <SEP> (wide)
<tb>
 
 EMI13.2
 : Soluble in chlorinated hydrocarbons (methylene chloride, chloroform), alcohol, acetone, pyridine and dilute alkali; sparingly soluble in benzene;

   insoluble in ether, hexane and water. i R-451 D, manufactured as described above, is a white amorphous powder with the following physical and chemical properties:
 EMI13.3
 
<tb>
<tb> I. <SEP> Melting point <SEP> (in the <SEP> Kofler block) <SEP>: <SEP>
<tb> Product <SEP> according to <SEP> example <SEP> 5 <SEP> 138 <SEP> - <SEP> 1400C <SEP>; <SEP>
<tb> Product <SEP> according to <SEP> example <SEP> 5 <SEP> A) <SEP> 160-1610C.
<tb>
 



  Analysis values: a) Elemental analysis of the product according to Example 5:
 EMI13.4
 
 EMI13.5
 
<tb>
<tb> e) <SEP> Qualitative <SEP> group definition <SEP>: <SEP>
<tb> 1. <SEP> Ninhydrin test <SEP> negative
<tb> 2. <SEP> Elson Morgan test <SEP> positive
<tb> 3. <SEP> test <SEP> with <SEP> alkaline <SEP> KMnO <SEP> positive
<tb> 4th <SEP> Anthrone test <SEP> gray-blue
<tb> 5th <SEP> 2,4-dinitro-phenylhydrazine test <SEP> positive
<tb> 6. <SEP> Ehrlich test <SEP> (diphenylamine) <SEP> negative
<tb> 7. <SEP> Triphenyl-tetrazolium test <SEP> negative
<tb> m. <SEP> Optical <SEP> rotation:
<tb> Product <SEP> according to <SEP> example <SEP> 5 <SEP> [α] 25D <SEP> = <SEP> +29.2 <SEP> (1% <SEP> in <SEP> dioxane)
<tb> Product <SEP> according to <SEP> example <SEP> 5 <SEP> A) <SEP> [α] 25D <SEP> = <SEP> -25.3 <SEP> (1% <SEP> in <SEP> methanol)
<tb> -37.7 <SEP> (1% <SEP> in <SEP> pyridine)
<tb>
 
IV.

   Solubility: Very soluble in chloroform, methylene chloride. Acetone and methanol and 0.1 n-sodium hydroxide; little soluble in ether; insoluble in petroleum ether, toluene, benzene, water and aqueous sodium bicarbonate or carbonate solution.



   V. Ultraviolet spectrum:
Fig. 1 of the drawings shows the spectrum in methanol of the product according to Example 5 at a con
 EMI13.6
 

 <Desc / Clms Page number 14>

 
VI. Infrared spectrum:
5 of the drawings shows the spectrum in Nujol of the product according to Example 5. The absorption maxima are at the following wavelengths:

   
 EMI14.1
 
<tb>
<tb> À <SEP> (/ l) <SEP> Intensity <SEP>
<tb> 2, <SEP> 88 <SEP> st
<tb> 5, <SEP> 73 <SEP> st
<tb> 6, <SEP> 13 <SEP> m-st
<tb> 6.33 <SEP> m-st
<tb> 6.45 <SEP> st
<tb> 7, <SEP> 98 <SEP> st
<tb> 8, <SEP> 32 <SEP> st
<tb> 8 <SEP> 501 <SEP> sot <SEP>
<tb> 10, <SEP> 00
<tb> 10, <SEP> 22 <SEP> st
<tb> 10.

   <SEP> 55 <SEP> st
<tb> 11, <SEP> 00 <SEP> m-st
<tb> 11.55 <SEP> m-st
<tb> 12, <SEP> 30 <SEP> black
<tb> 12, <SEP> 98 <SEP> black
<tb> 13, <SEP> 55 <SEP> m-st
<tb> 13, <SEP> 87 <SEP> m-st
<tb>
 
 EMI14.2
 
 EMI14.3
 
<tb>
<tb> 5À <SEP> (j) <SEP> intensity
<tb> 2.89 <SEP> st
<tb> 3, <SEP> 35- <SEP> 3, <SEP> 48 <SEP> st <SEP> (wide)
<tb> 5.73 <SEP> st
<tb> 6.12 <SEP> black
<tb> 6.35 <SEP> Sch
<tb> 6.45 <SEP> st
<tb> 6, <SEP> 77- <SEP> 6, <SEP> 82 <SEP> st
<tb> 7,10 <SEP> Sch
<tb> 7.24 <SEP> st
<tb> 7.30 <SEP> Sch
<tb> 7.42 <SEP> st
<tb> 7, <SEP> 68-7, <SEP> 84 <SEP> st <SEP> (wide)
<tb> 7.99 <SEP> st
<tb> 8.33 <SEP> st
<tb> 8, <SEP> 48- <SEP> 8, <SEP> 70 <SEP> Sch
<tb> 8, <SEP> 84- <SEP> 9.17 <SEP> st <SEP> (wide)
<tb> 9, <SEP> 57 <SEP> st <SEP> (wide)
<tb> 10.00 <SEP> Sch
<tb> 10, <SEP> 22-10, <SEP> 26 <SEP> st <SEP> (doublet)
<tb> 10.55 <SEP> st
<tb> 10,

   <SEP> 91 <SEP> Sch
<tb> 11, <SEP> 00 <SEP> m
<tb> 11, <SEP> 50-11, <SEP> 60 <SEP> m <SEP> (wide)
<tb> 11, <SEP> 66 <SEP> Sch
<tb> 11, <SEP> 90-12, <SEP> 00 <SEP> m <SEP> (wide)
<tb> 12, <SEP> 25-12, <SEP> 35 <SEP> black <SEP> (wide)
<tb> 12, <SEP> 70-12, <SEP> 85 <SEP> black <SEP> (wide)
<tb> 13, <SEP> 00 <SEP> black
<tb> 13, <SEP> 52-13, <SEP> 60 <SEP> black <SEP> (wide)
<tb> 13, <SEP> 85-13, <SEP> 90 <SEP> black <SEP> (wide)
<tb> 14, <SEP> 42-14, <SEP> 58 <SEP> black <SEP> (wide)
<tb>
 

 <Desc / Clms Page number 15>

 VII. Formation of derivatives: a) Acetate, prepared according to Example 6:
M.p. = 135-1360C; after cleaning according to Example 6 A) mp = 151-160 C.



   Ultraviolet absorption maximum at 285 or 286 mbt (E1% = 9.2).



   Elemental analysis: C 53.08%, H 7.08%, N 0.77%;
Infrared absorption bands (in Nujol) at the following wavelengths:
 EMI15.1
 
<tb>
<tb> X <SEP> () <SEP> intensity
<tb> 2.85 <SEP> black <SEP> (wide) <SEP>
<tb> 5.57 <SEP> m-st
<tb> 5.71 <SEP> st
<tb> 6.14 <SEP> black
<tb> 6.45 <SEP> m-st
<tb> 8.02 <SEP> st
<tb> 8.38 <SEP> m-st
<tb> 8.88 <SEP> st
<tb> 9.17 <SEP> st
<tb> 9.55 <SEP> st
<tb>
 
VIII. Stability:
R-451 D is stable at 00C and in the dark. At room temperature in direct sunlight, the activity slowly decreases after 24 hours and disappears completely after about three weeks. When dissolved in methanol, R-451 D is stable for at least two weeks. If the pH is below 5.5, the activity is quickly lost, whereas if the pH is greater than 7 and up to 14, the activity is retained for several days.

   On treatment with 0.1 N-methanolic hydrochloric acid at room temperature, the antibiotic activity is lost within 16 hours; if the acidic mixture is treated with a stoichiometric amount of 2% aqueous sodium bicarbonate solution, then the methanol is evaporated off in vacuo and extracted with chloroform, this is how thin-layer chromatography on silica gel with benzene-acetone (75:25) as the solvent and then extracts Spraying with sulfuric acid reveals a mixture of five components, all of which are less polar than R-451D. When treated for 16 hours with 1.0N NaOH solution at room temperature, the activity is completely retained.



   R-451 E, isolated and purified as described in Example 7, is an almost colorless amorphous powder with the following physical and chemical properties:
I. Melting point: 89-960C
II. Analysis results: a) Elemental analysis:
C 65.17%, H 7.54qu, N1, zo 0.24.2eo. b) Qualitative group regulations:
 EMI15.2
 
<tb>
<tb> 1. <SEP> Ninhydrin test <SEP> negative
<tb> 2. <SEP> Elson Morgan test <SEP> positive
<tb> 3. <SEP> test <SEP> with <SEP> alkaline <SEP> KMnO, <SEP> positive
<tb> 4th <SEP> Anthrone test <SEP> positive
<tb> 5.2, <SEP> 4-dinitro-phenylhydrazine test <SEP> positive
<tb> 6. <SEP> Ehrlich test <SEP> (with <SEP> diphenylamine) <SEP> negative
<tb> 7. <SEP> triphenyl-tetrazolium test <SEP> positive
<tb>
 
 EMI15.3
 
IV.

   Solubility:
Soluble in chloroform, methylene chloride, acetone, methanol and ether; poorly soluble in benzene, toluene and hexane; insoluble in water.



   V. Ultraviolet spectrum:
 EMI15.4
 

 <Desc / Clms Page number 16>

 



   : VI. Red spectrum: (Spectrum in Nujol, see fig. Of the drawings).



  The absorption maxima (intensities as indicated) are at the following wavelengths:
 EMI16.1
 
<tb>
<tb> X <SEP> intensity
<tb> 2.94 <SEP> m-st
<tb> 5.68 <SEP> st
<tb> 5.76 <SEP> st
<tb> 5, <SEP> 83 <SEP> st
<tb> 6, <SEP> 0 <SEP> sst
<tb> 6.16 <SEP> m-st
<tb> 6.22 <SEP> m-st
<tb> 7, <SEP> 87 <SEP> sst
<tb> 8.31 <SEP> black
<tb> 8.55 <SEP> black
<tb> 8.96 <SEP> black
<tb> 9, <SEP> 33 <SEP> black
<tb> 9.55 <SEP> black
<tb> 9, <SEP> 77 <SEP> m-st
<tb> 10.50 <SEP> black
<tb> 10.90 <SEP> black
<tb> 11.26 <SEP> m-st
<tb> 11.42 <SEP> black
<tb> 12.22 <SEP> black
<tb> 12.62 <SEP> m-st
<tb>
 
Biological properties of the antibiotics obtained according to the above examples.



   The antibiotics of the R-451 group, especially R-451 and R-451 D, have a wide range of effects against gram-positive organisms. They are of particular value for the treatment of diseases caused by penicillin-resistant microorganisms (see Tables xn and XVI). They are also useful in the treatment of Staphylococcus aureus, Diplococcus pneumoniae and the like. a., diseases caused by pathogenic microorganisms useful.



   The antibiotics of the R-451 group are also effective against certain microorganisms that can be attacked by penicillin; however, it has been shown that they do not belong to the penicillins, as can be seen from the fact that neither R-451 nor R-451 D are inactivated by penicillinase. By the way, they can also be used in patients who show a sensitivity caused by treatment with penicillin or synthetic penicillin derivatives - a relatively common phenomenon, according to statistics it occurs at around zoo z. B. the population of the United States of America.



   The crude R-451, prepared according to Example 1, can be used, inter alia, as an anti-infectious agent for preventing certain symptoms caused by Staphylococcus aureus, Streptococcus pyogenes, Diplococcus pneumoniae and the like. a. pathogenic organisms are caused. Its in vitro activity against various microorganisms follows from Table xn. The susceptibility of the microorganisms to the antibiotics was determined according to standardized pipe dilution methods: Dilutions to 10-5 of 24 broth cultures were used as inoculum, the endpoints being recorded after incubation for 24 hours at 37 ° C. The nutrient medium was based on yeast extract and beef extra! t "glucose produced.

   Its in vivo activity against certain bacterial infections was also determined. u. between 18-20 g heavy mice. The mice were infected with an inoculum of the bacteria in question by intraperitoneal injection and were then treated twice daily with subcutaneous injections of R-451 dissolved in water. Table xm shows which dose is necessary in each case to protect a certain percentage of animals treated in this way. When 400 total dose (20 mg / kg mouse) are administered orally in two equal doses, the first half an hour before infection, protection against Streptococcus pyogenes is also achieved.

   The acute toxicity of R-451 was tested on mice weighing 18 to 20 g according to various standardized methods, with the following results:

 <Desc / Clms Page number 17>

 
 EMI17.1
 
<tb>
<tb> Method of administration <SEP> LD <SEP> (mg / kg <SEP> mouse)
<tb> subcutaneous <SEP> 1000
<tb> intraperitoneal <SEP> 1000
<tb> intravenous <SEP> 850
<tb> oral <SEP>> 2500
<tb>
 
R-451 A and R-451 B, produced according to Example 8 E) or 8 C), show according to the disk test method (disk diameter 6.3 mm), the inhibition zone being measured after a disk impregnated with the antibiotic was applied to a disk with Agar plate inoculated with the test organism had been applied and incubated for 24 hours at 37 ° C., an in vitro activity against certain microorganisms, as shown in Tables XIV and XV, respectively.

   Further data relating to the in vitro activity of R-451 B (determined on material purified according to Example 9) can be found in Table XVA. (The behavior of the test organisms towards the antibiotic was determined using standardized dilution methods. In each case, dilutions to 10-5 of 24-hour-old cultures were used as the inoculum, and the endpoints were determined after incubation for 24 hours at 370C in a "Difco Pen-assay" - Broth medium added. The table shows the activity of the antibiotic in units per ml, the unit being as defined above.)
The in vivo activity of R-451 B, produced according to Example 8 C), was specified in the case of R-451
 EMI17.2
 



  Further data relating to the in vivo activity of R-451 B (determined on material purified according to Example 18 A) can be found in Table XVB. [The data in this table were determined pharmacologically on mice weighing 18-20 g as test animals against certain pathogenic microorganisms, u. According to the following standardized procedure: 30 mice were infected with an inoculum of the organism in question by intraperitoneal injections. Then 20 of the mice were treated subcutaneously with a solution of the antibiotic in 2 parts of ethanol, 0.5 parts of Tween 80 and 8.5 parts of peanut oil, the injections being given in two equal daily doses; 10 mice were left untreated as control animals. All control animals died within 18 hours.

   The dose that is required so that 500/0 of the treated animals are still alive after 48 h (PD 50) was shown in Table XV B.



   The acute toxicity of R-451 B was determined as follows:
 EMI17.3
 
<tb>
<tb> Method of administration <SEP> ID <SEP> (mg / kg <SEP> mouse)
<tb> subcutaneous <SEP> 2500
<tb> intraperitoneal <SEP> 750
<tb>
 
R-451 D, prepared according to Example 5, shows in vitro activity against various gram-positive organisms, as can be seen from Table XVI, determined as indicated for R-451. Furthermore, the in vivo activity of the R-451 D prepared according to Example 5 was investigated in the manner indicated for R-451. Table XVII shows the results. Further data relating to the in vitro and in vivo activity of R-451 D (determined on material purified according to Example 5 A) can be found in Tables XV A and XV B; the determination of this data is explained in more detail in connection with R-451 B.

   The acute toxicity of R-451 D was determined as follows;
 EMI17.4
 
<tb>
<tb> Method of administration <SEP> ID50 <SEP> (mg / kg <SEP> mouse)
<tb> subcutaneous <SEP> 1000
<tb> intrapci.'itonca.). <SEP> 500
<tb> intravenous <SEP> 150
<tb>
 
For R-451 E, prepared according to Example 7, the in vitro activity listed in Table XVI1I was determined in the manner indicated for R-451 A and R-451 B.



   The antibiotics obtainable according to the invention are brought into one of the dosage forms customary for antibiotics for administration to the patient, see Sect. B. Injection solutions, salts, eye drops. Here
 EMI17.5
 

 <Desc / Clms Page number 18>

 Table 1
 EMI18.1
 
<tb>
<tb> Comparative <SEP> taxonomy <SEP> of <SEP> Micromonospora species
<tb> Part <SEP> A <SEP>: <SEP> Medium <SEP> contains <SEP> 0, <SEP> 1% <SEP> NZ-Amin <SEP> Type <SEP> A, <SEP> 10 / 0 <SEP> dextrose, <SEP> 1, <SEP> so <SEP> agar
<tb> <SEP> microorganism used <SEP> Observed <SEP> properties
<tb> Macroscopic <SEP> Microscopic
<tb> M. <SEP> carbonacea <SEP> No <SEP> aerial mycelium <SEP>; <SEP> colony <SEP> flat, <SEP> moist <SEP>; <SEP> growth <SEP> mycelium <SEP> long, <SEP> branched, <SEP> regular, <SEP> wallless,
<tb> NRRL <SEP> 2972 <SEP> moderate <SEP>;

   <SEP> no <SEP> diffusible <SEP> pigment <SEP> diameter <SEP> 0.6 <SEP> 11. <SEP>; <SEP> ample <SEP> education <SEP> long-educated. <SEP> cher <SEP> to <SEP> ellipsoidal <SEP> spores, <SEP> 1.0 <SEP>: <SEP> 1.5 <SEP>,
<tb> Color <SEP>: <SEP> carried <SEP> by <SEP> in clusters <SEP> occurring <SEP> sporo surface <SEP>: <SEP> periphery <SEP> light orange-g4NA, <SEP> lively <SEP> phores, <SEP> dark <SEP> colored.
<tb> orange-48, <SEP> center <SEP>; <SEP> brown-black <SEP>; <SEP>
<tb> Back <SEP>: <SEP> same
<tb> M. <SEP> chalcea <SEP> No <SEP> aerial mycelium <SEP>; <SEP> Colony <SEP> slightly <SEP> raised, <SEP> furrowed, <SEP> mycelium <SEP> long, <SEP> branched, <SEP> regular, <SEP> wallless,
<tb> ATCC <SEP> 12452 <SEP> waxy <SEP>; <SEP> moderate <SEP> growth <SEP>;

   <SEP> no <SEP> diffuse-very <SEP> fine, <SEP> diameter <SEP> smaller <SEP> than <SEP> 0, <SEP> 5 <SEP> 11. <SEP>; <SEP>
<tb> dable <SEP> pigment <SEP> formed. <SEP> abundant <SEP> formation <SEP> spherical <SEP> to <SEP> ellipsoidal color <SEP>: <SEP> ler <SEP> spores, <SEP> brown, <SEP> 1, <SEP> 0 <SEP> <SEP> in the <SEP> diameter,
<tb> Surface <SEP>: <SEP> coffee brown-g3PN, <SEP> dark olive brown-96; <SEP> mostly <SEP> free, <SEP> but <SEP> some <SEP> individually <SEP> on <SEP>
<tb> Back <SEP>: <SEP> as well as <SEP> sporophores.
<tb>



  M. <SEP> fusca <SEP> no <SEP> aerial mycelium <SEP>: <SEP> colony <SEP> slightly <SEP> increased, <SEP> moist, <SEP> granular <SEP> mycelium <SEP> short, <SEP> irregular, <SEP> forms <SEP> many <SEP> polyNRRL <SEP> B-943 <SEP> nig <SEP> to <SEP> furrowed; <SEP> moderate <SEP> growth; <SEP> completely <SEP> weak <SEP> morphic <SEP> elements, <SEP> diameter <SEP> 0, <SEP> 8-2, <SEP> 0 <SEP> fi <SEP>; <SEP>
<tb> yellowish brown <SEP> diffusible <SEP> pigment <SEP> formed. <SEP> spores <SEP> 1, <SEP> 0 <SEP> <SEP> in <SEP> diameter, <SEP> difficult <SEP> find color: <SEP> bar <SEP> under <SEP> numerous <SEP> dark-walled <SEP> (olive surface <SEP>; <SEP> brown-red-orange-g4PC, <SEP> deep orange-51; <SEP> brown) <SEP> polymorphic <SEP> forms.
<tb>



  Back: <SEP> case brown-g4NE, <SEP> intensive brown-55.
<tb>



  M. <SEP> sp. <SEP> No <SEP> aerial mycelium <SEP>; <SEP> Colony <SEP> slightly <SEP> increased, <SEP> dry. <SEP> granular <SEP> mycelium <SEP> long, <SEP> branched, <SEP> regular, <SEP> wallless,
<tb> ATCC <SEP> 10026 <SEP> nig <SEP> to <SEP> furrowed <SEP>; <SEP> no <SEP> diffusible <SEP> pigment <SEP> formed. <SEP> 0, <SEP> 5 <SEP> 11. <SEP> in the <SEP> diameter <SEP>; <SEP> Spores <SEP> extremely <SEP> rich Color <SEP>: <SEP> lit, <SEP> in <SEP> clusters <SEP> and <SEP> individually, <SEP> 1, <SEP> 0 <SEP> <SEP> in
<tb> surface <SEP>: <SEP> periphery <SEP> coffee brown-g3PN, <SEP> dark olive- <SEP> diameter; <SEP> some <SEP> polymorphic <SEP> forms <SEP> pre-brown-96, <SEP> center red-brown-orange-g4PC, <SEP> deep-orange-51; <SEP> lying; <SEP> both <SEP> spores <SEP> as <SEP> as well as <SEP> polymorphic
<tb> rear side <SEP>:

   <SEP> same as <SEP> forms <SEP> dark-walled.
<tb>
 

 <Desc / Clms Page number 19>

 



  Table 1 (continued)
 EMI19.1
 
<tb>
<tb> Part <SEP> B <SEP>: <SEP> medium <SEP> contains <SEP> 3% <SEP> NZ-Amin <SEP> type <SEP> A, <SEP> 10/9 <SEP> dextrose , <SEP> 1, <SEP> 5% <SEP> agar
<tb> <SEP> microorganism used <SEP> Observed <SEP> properties
<tb> Macroscopic <SEP> Microscopic
<tb> M. <SEP> carbonacea <SEP> Keln <SEP> Inftmycal; <SEP> Colony <SEP> raised, <SEP> furrowed <SEP>; <SEP> growth <SEP> mycelium <SEP> long, <SEP> branched, <SEP> regular, <SEP> wallless,
<tb> NRRL <SEP> 2972 <SEP> good <SEP>; <SEP> no <SEP> diffusible <SEP> pigment <SEP> formed. <SEP> 0.6 <SEP> pt <SEP> in the <SEP> diameter <SEP>; <SEP> spores <SEP> single, <SEP> on
<tb> Color <SEP>: <SEP> individual <SEP> sporophores <SEP> carried, <SEP> elongated <SEP> to
<tb> surface <SEP>; <SEP> teracotta-colored-g5PE. <SEP> interval vlxaun-55;

   <SEP> elliproid, <SEP> 1.0; <SEP> 1.5 <SEP>, <SEP> dark <SEP> colored.
<tb>



  Backward <SEP>: <SEP> same
<tb> M. <SEP> chalcea <SEP> No <SEP> aerial mycelium; <SEP> Colony <SEP> increased, <SEP> incorrect, <SEP> waxy; <SEP> mycelium <SEP> long, <SEP> branched, <SEP> regular, <SEP> wallless,
<tb> ATCC <SEP> 12452 <SEP> moderate <SEP> growth <SEP>; <SEP> no <SEP> diffusible <SEP> 0.5 <SEP> <SEP> in <SEP> diameter <SEP>; <SEP> spores <SEP> abundant, <SEP> a pigment <SEP> formed. <SEP> tufts <SEP> and <SEP> carried in clusters <SEP>, <SEP> spherical
<tb> Color <SEP>: <SEP> to <SEP> ellipsoidal, <SEP> 1.0 <SEP> <SEP> in the <SEP> diameter.
<tb>



  Surface <SEP>; <SEP> copper brown-g5PI, <SEP> intensly brown-55;
<tb> Rückuefte <SEP>; <SEP> red-brown <SEP> to <SEP> beam-g4PI, <SEP> internal brown-56.
<tb>



  M. <SEP> fusca <SEP> No <SEP> aerial mycelium; <SEP> Colony <SEP> increased, <SEP> coarse-grained, <SEP> matt <SEP> mycelium <SEP> irregular, <SEP> many <SEP> polymorphic <SEP> EleNRRL <SEP> B-943 <SEP> (" dull ") <SEP>; <SEP> good <SEP> growth <SEP>; <SEP> whole <SEP> weak <SEP> yellowish <SEP> elements <SEP> present, <SEP> 0, <SEP> 5-2, <SEP> 0 <SEP> P. <SEP> in <SEP> Formed through brown <SEP> diffusible <SEP> pigment <SEP>. <SEP> ser, <SEP> olive brown <SEP> colored <SEP>; <SEP> real <SEP> spores, <SEP> 1.0 <SEP> left
<tb> Color <SEP>: <SEP> in the <SEP> diameter, <SEP> gradual <SEP> transition <SEP> in
<tb> Surface <SEP>: <SEP> terracotta-colored-g5PE, <SEP> intensive brown-55; <SEP> polymorphic <SEP> forms <SEP> with <SEP> diameter
<tb> Back side <SEP>: <SEP> red brown-g5PG, <SEP> intensive brown-55.0, <SEP> 5-2, <SEP> 0 <SEP> J. <SEP> L. <SEP>
<tb>



  M. <SEP> sp. <SEP> No <SEP> aerial mycelium <SEP>; <SEP> Colony <SEP> increased, <SEP> densely grained, <SEP> dry <SEP>; <SEP> Two <SEP> types <SEP> mycelium <SEP>: <SEP> long <SEP> and <SEP> regularly
<tb> ATCC <SEP> 10026 <SEP> good <SEP> growth. <SEP> on the one hand, <SEP> short <SEP> and <SEP> irregular <SEP> on the other <SEP>: <SEP> on the one hand, <SEP> forms <SEP> many <SEP> polymorphic <SEP> elements,
<tb> Surface <SEP>: <SEP> periphery <SEP> red-brown-orange-g4PC, <SEP> intensive- <SEP> 8-2, <SEP> 0 <SEP> lui <SEP> in <SEP> diameter <SEP> ; <SEP> plenty
<tb> orange-50, <SEP> center <SEP> chocolate brown-g4PN, <SEP> dark- <SEP> real <SEP> spores, <SEP> in clusters <SEP> carried, <SEP> 1, <SEP> 0 <SEP> bu <SEP>
<tb> gray-brown-62 <SEP>; <SEP> in <SEP> diameter, <SEP> spherical <SEP> to <SEP> elliptical back <SEP>:

   <SEP> dark cinnamon brown <SEP> ("dark <SEP> spice <SEP> brown") - g4PI. <SEP> soidal.
<tb> dark brown-59.
<tb>
 

 <Desc / Clms Page number 20>

 



  Table II
 EMI20.1
 
<tb>
<tb> <SEP> Medt. <SEP> m <SEP> M. <SEP> carbonacea <SEP> M. <SEP> chalcea <SEP> M. <SEP> fusca <SEP> M. <SEP> parva <SEP> M. <SEP> globosa < SEP> M. <SEP> coerulea
<tb> NRRL <SEP> 2972
<tb> Glucose-asparagine growth <SEP>: <SEP> bad <SEP> No <SEP> aerial mycelium. <SEP> growth <SEP>: <SEP> growth <SEP>: <SEP> bad <SEP> growth- <SEP> bad <SEP> growth <SEP>: <SEP> bad <SEP>
<tb> Agar <SEP> Growth <SEP>: <SEP> extremely <SEP> extremely <SEP> good. <SEP> Bilgut. <SEP> Forms <SEP> no <SEP> soluble <SEP> det <SEP> soluble <SEP> brownish <SEP> pigment. <SEP> nes <SEP> pigment.
<tb>



  Color <SEP>: <SEP> pale pink <SEP> to <SEP> deep- <SEP> color <SEP>: <SEP> changes <SEP>
<tb> orange <SEP> with <SEP> brownish- <SEP> from <SEP> orange <SEP> to <SEP> deep to <SEP> greenish black <SEP> brown, <SEP> with <SEP> gray spore layer <SEP> black <SEP> to <SEP> brown-black <SEP> spore layer
<tb> Gelatin <SEP> Liquefaction <SEP> Liquefaction <SEP> Weak <SEP> Liquefaction- <SEP> Liquefaction <SEP> Liquefaction <SEP> Rapid <SEP> Liquefaction- <SEP>
<tb> gung
<tb> Milk <SEP> If <SEP> slowly <SEP> peptone build-up, <SEP> gel- <SEP> If <SEP> slowly <SEP> <SEP> remains <SEP> unchanged <SEP> coagulation <SEP> and <SEP> Hardly <SEP> coagulated
<tb> digested <SEP> occasionally <SEP> coagulation <SEP> there, <SEP> not <SEP> peptone buildup <SEP>.

   <SEP> Coagulates <SEP> duration <SEP> completely
<tb> weak
<tb> Sucrose <SEP> If <SEP> is used <SEP> If <SEP> is inverted <SEP> If <SEP> is inverted <SEP> If <SEP> is not <SEP> If <SEP> is inverted <SEP> If <SEP > inverted
<tb> inverted
<tb> Starch <SEP> Is <SEP> hydrolysed <SEP> Is <SEP> hydrolysed <SEP> Is <SEP> hydrolysed <SEP> Is <SEP> hydrolysed <SEP> Is <SEP> hydrolysed <SEP> Is <SEP > hydrolyzed
<tb> Cellulose-Will <SEP> rapidly <SEP> decompose <SEP> Will <SEP> weak <SEP> Will <SEP> not <SEP> Will <SEP> not <SEP> Will <SEP> not
<tb> attacked <SEP> decomposed <SEP> decomposed <SEP> decomposed
<tb>
 

 <Desc / Clms Page number 21>

 Table II (continued)
 EMI21.1
 
<tb>
<tb> <SEP> M. <SEP> carbonacea <SEP> M. <SEP> chalcea <SEP> M. <SEP> fusca <SEP> M. <SEP> parva <SEP> M. <SEP> globosa < SEP> M.

   <SEP> coerulea
<tb> Medium <SEP> NRRL <SEP> 2972 <SEP>
<tb> Nitrate reduction <SEP> From <SEP> Nitrates <SEP> arise <SEP> Off. <SEP> Nitrates <SEP> arise <SEP> Variable <SEP> No <SEP> reduction <SEP> From <SEP> nitrates <SEP> arise <SEP> No <SEP> reduction
<tb> nitrites <SEP> nitrites <SEP> nitrites
<tb> Temperature <SEP> Good <SEP> growth <SEP> with <SEP> Optimal <SEP> growth
<tb> 16-37 C, <SEP> no <SEP> between <SEP> 30 <SEP> and
<tb> Watchfulness <SEP> at <SEP> 50 C <SEP> 35 C
<tb> Growth <SEP> Aerobic <SEP> Aerobic <SEP> Aerobic <SEP> Aerobic <SEP> Aerobic <SEP> Aerobic
<tb> (aerobic <SEP> or
<tb> anaerobic)
<tb>
 

 <Desc / Clms Page number 22>

 Table IIIA
 EMI22.1
 
<tb>
<tb> <SEP> Médium <SEP> M. <SEP> carbonacea <SEP> M. <SEP> chalcea <SEP> M. <SEP> fusca <SEP> M. <SEP> sp.

   <SEP> ATCC <SEP> 10026
<tb> NRRL <SEP> 2972 <SEP> ATCC <SEP> 12452 <SEP> NRRL <SEP> B-943
<tb> Bennett's Agar <SEP> -)
<tb> Growth <SEP>: <SEP> good <SEP>; <SEP> increased <SEP> and <SEP> growth <SEP>: <SEP> good. <SEP> growth <SEP>: <SEP> good. <SEP> growth <SEP>: <SEP> good. <SEP>
<tb> furrowed <SEP> colony. <SEP> color; <SEP> periphery <SEP> terra- <SEP> color; <SEP> terracotta <SEP> color; <SEP> periphery <SEP> terra color <SEP>:

   <SEP> light orange-g5NA, <SEP> cotta-colored-g5PE, <SEP> in- <SEP> g5PE, <SEP> intense brown-55. <SEP> cotta-colored-g5PE, <SEP> inlive orange-48 <SEP> (brown- <SEP> tensivixaun-55 <SEP> m, <SEP> Zen- <SEP> intensive brown-55, <SEP> zen black <SEP> spotted ). <SEP> trum <SEP> chocolate brown- <SEP> trum <SEP> clove- <SEP>
<tb> g4PN, <SEP> dark brown-59. <SEP> brown <SEP> ("clove <SEP> brown") - <SEP>
<tb> 3PL, <SEP> dark yellow brown-78. <SEP>
<tb>



  Emerson agar
<tb> growth; <SEP> moderate: <SEP> - <SEP> growth: <SEP> good. <SEP> growth; <SEP> good. <SEP> growth <SEP>: <SEP> good. <SEP>
<tb> raised <SEP> colony. <SEP> Color <SEP>: <SEP> bittersweet <SEP> Faibe <SEP>: <SEP> tangerine color <SEP>: <SEP> oak brown <SEP>
<tb> color <SEP>: <SEP> terracotta colored- <SEP> ("bitter <SEP> sweet @) - g5PC, <SEP> colors-g5PA, <SEP> lively- <SEP> g4PI, <SEP> intense brown- 55.
<tb> g5PE, <SEP> intense brown-55. <SEP> deep orange-51. <SEP> orange.
<tb>



  Tomato paste <SEP> *) <SEP>
<tb> Oatmeal Agar <SEP> Growth <SEP>: <SEP> good <SEP>; <SEP> furrowed <SEP> growth <SEP>: <SEP> good. <SEP> Growth <SEP>: <SEP> medium. <SEP> Growth <SEP>: <SEP> medium. <SEP>
<tb>



  Colony. <SEP> color: <SEP> periphery <SEP> terra- <SEP> color: <SEP> light peach- <SEP> color: <SEP> light peach color <SEP>: <SEP> periphery <SEP> orange-g5LA, <SEP > cotta-colored-g5PE, <SEP> inten-g5IA, <SEP> medium red-orange-37. <SEP> g5IA, <SEP> medium red orange-3'7. <SEP>
<tb> intense orange-50, <SEP> center <SEP> tivbraun-55, <SEP> center
<tb> brown-black. <SEP> chocolate brown-4PN, <SEP>
<tb> dark brown-59.
<tb>



  Glucose growth <SEP>: <SEP> bad. <SEP> growth <SEP>: <SEP> good. <SEP> Growth <SEP>: <SEP> medium. <SEP> growth <SEP>: <SEP> bad. <SEP>
<tb>



  Asparagine agar <SEP> color <SEP>: <SEP> terracotta color <SEP>: <SEP> bright orange <SEP>
<tb> g5PE, <SEP> intense brown-55. <SEP> ("sun <SEP> organge") - g5LA,
<tb> intense orange-50.
<tb>



  Glucose yeast *)
<tb> extract agar <SEP> growth <SEP>: <SEP> moderate <SEP>; <SEP> ge <SEP> growth <SEP>: <SEP> good. <SEP> growth <SEP>: <SEP> good. <SEP> growth <SEP>: <SEP> good. <SEP>
<tb> fear <SEP> colony. <SEP> color <SEP>: <SEP> red- <SEP> color: <SEP> deep brown-g4PI, <SEP> color: <SEP> terracotta-<SEP> color: <SEP> oak lxa brown-orange-g4NC, <SEP> deep orange-61. <SEP> dark brown-59. <SEP> g5PE, <SEP> intense brown-55. <SEP> g4PI, <SEP> intense brown-55.
<tb>
 
 EMI22.2
 

 <Desc / Clms Page number 23>

 additional table IIIB
 EMI23.1
 
<tb>
<tb> <SEP> medium used <SEP> M. <SEP> carbonacea <SEP> M. <SEP> chalcea <SEP> M. <SEP> fusca <SEP> M. <SEP> sp. <SEP> ATCC <SEP> 10026
<tb> NRRL <SEP> 2972 <SEP> ATCC <SEP> 12452 <SEP> NRRL <SEP> B-943
<tb> Potato <SEP> Growth <SEP>: <SEP> medium. <SEP> growth <SEP>:

   <SEP> good. <SEP> growth <SEP>: <SEP> good. <SEP> growth <SEP>: <SEP> good.
<tb>



  Color <SEP>: <SEP> rust red <SEP> ("barn- <SEP> color <SEP>: <SEP> bittersweet- <SEP> color <SEP>: <SEP> bittersweet- <SEP>
<tb> red ") <SEP> -g6PG, <SEP> medium red- <SEP> g5PC, <SEP> deep orange-51. <SEP> g5PC, <SEP> deep orange-51.
<tb> brown-43.
<tb>



  Carrot <SEP> growth <SEP>: <SEP> bad. <SEP> growth <SEP>: <SEP> good. <SEP> growth <SEP>: <SEP> bad. <SEP> Growth <SEP>: <SEP> medium. <SEP>
<tb>



  Color <SEP>: <SEP> chocolate brown color <SEP>: <SEP> fire red-gSNC, <SEP>
<tb> g4PN, <SEP> dark brown-59. <SEP> intense red orange-35.
<tb>



  Sucrose- <SEP> Nitrite- <SEP> No <SEP> growth. <SEP> No <SEP> growth. <SEP> No <SEP> growth. <SEP> No <SEP> growth.
<tb>



  Agar <SEP> (Czapek agar)
<tb> Tyrosine Agar <SEP> (Be <SEP> growth <SEP>: <SEP> medium. <SEP> growth <SEP>: <SEP> good. <SEP> growth <SEP>: <SEP> medium . <SEP> growth <SEP>: <SEP> bad.
<tb> observations <SEP> according to <SEP> color <SEP>: <SEP> no <SEP> diffusible <SEP> color <SEP>: <SEP> black <SEP> diffuse <SEP> color <SEP>: <SEP> no <SEP> diffusible- <SEP> color <SEP>: <SEP> no <SEP> diffusible- <SEP>
<tb> 2.7 <SEP> and <SEP> 14 <SEP> days <SEP>; <SEP> res <SEP> pigment. <SEP> dable <SEP> pigment. <SEP> res <SEP> pigment. <SEP> clear <SEP> pigment.
<tb> according to <SEP> Gordonu.
<tb>



  Smith, <SEP> J. <SEP> Bac. 69,
<tb> 147)
<tb> Peptone-Iron-Agar <SEP> Growth <SEP>: <SEP> moderate. <SEP> growth <SEP>: <SEP> good. <SEP> Growth <SEP>: <SEP> medium. <SEP> Growth <SEP>: <SEP> medium. <SEP>
<tb>



  (Observations <SEP> color <SEP>: <SEP> no <SEP> reaction. <SEP> color <SEP>: <SEP> no <SEP> reaction. <SEP> color <SEP>: <SEP> no <SEP > Reaction. <SEP> color <SEP>: <SEP> no <SEP> reaction.
<tb> after <SEP> 2, <SEP> 7 <SEP> and <SEP> 14 <SEP>
<tb> days)
<tb> bromocresol purple <SEP> growth <SEP>: <SEP> medium <SEP>; <SEP> growth <SEP>: <SEP> good <SEP>; <SEP> growth <SEP> growth <SEP>: <SEP> moderate <SEP>; <SEP> growth <SEP>: <SEP> good <SEP>; <SEP> VerMilch <SEP> partially <SEP> digestion. <SEP> duration <SEP> takes place <SEP>. <SEP> digestion <SEP> takes place <SEP>. <SEP> duration <SEP> takes place <SEP>.
<tb>



  Color <SEP> none <SEP> PH- <SEP> color <SEP> none <SEP> PH- <SEP> color: <SEP> none <SEP> PH- <SEP>
<tb> change. <SEP> change. <SEP> change. <SEP>
<tb>
 

 <Desc / Clms Page number 24>

 



  Table IV
 EMI24.1
 
<tb>
<tb> <SEP> M. <SEP> carbonacea <SEP> M. <SEP> chalcea <SEP> M. <SEP> fusca <SEP> M. <SEP> sp.
<tb>



  Test carbohydrate <SEP> NRRL <SEP> 2972 <SEP> ATCC <SEP> 12452 <SEP> NRRL <SEP> B-943 <SEP> ATCC <SEP> 10026
<tb> Arabinose <SEP> good <SEP> good <SEP> good <SEP> good
<tb> Glucose <SEP> good <SEP> good <SEP> good <SEP> good
<tb> Galactose <SEP> good <SEP> good <SEP> good <SEP> mediocre
<tb> Lactose <SEP> good <SEP> moderate <SEP> bad <SEP> bad
<tb> Lävulose <SEP> good <SEP> moderate <SEP> bad <SEP> bad
<tb> Mannose <SEP> good <SEP> good <SEP> good <SEP> good
<tb> Raffinose <SEP> bad <SEP> bad <SEP> bad <SEP> bad
<tb> Rhamnose <SEP> bad <SEP> bad <SEP> bad <SEP> bad
<tb> Strength <SEP> good <SEP> good <SEP> good <SEP> good <SEP>
<tb> sucrose <SEP> good <SEP> good <SEP> good <SEP> good
<tb> Xylose <SEP> good <SEP> good <SEP> bad <SEP> bad
<tb> Inositol <SEP> bad <SEP> bad <SEP> bad <SEP> bad
<tb> mannitol

  <SEP> bad <SEP> bad <SEP> good <SEP> good
<tb> Sorbitol <SEP> bad <SEP> bad <SEP> bad <SEP> bad
<tb> For <SEP> control <SEP>: <SEP>
<tb> 0, <SEP> 50/0 <SEP> yeast extract <SEP> bad <SEP> bad <SEP> bad <SEP> bad
<tb>
 

 <Desc / Clms Page number 25>

 TableV
 EMI25.1
 
<tb>
<tb> Used <SEP> nitrogen-M, <SEP> carbonacea <SEP> M. <SEP> chalcea <SEP> M. <SEP> fusca <SEP> M. <SEP> sp. <SEP> ATCC <SEP> 10026
<tb> source <SEP> NRRL <SEP> 2972 <SEP> ATCC <SEP> 12452 <SEP> NRRL <SEP> B-943
<tb> 0.5% <SEP> Difco yeast <SEP> growth: <SEP> medium; <SEP> growth: <SEP> good. <SEP> growth <SEP>: <SEP> good. <SEP> growth <SEP>: <SEP> good. <SEP>
<tb> extract <SEP> colony <SEP> increased <SEP> and <SEP> ge <SEP> color: <SEP> rust-red-g6PG, <SEP> color: red-brown-orange <SEP> color:

   <SEP> bittersweet color fear. <SEP> medium red-brown-43. <SEP> ge-g4PC, <SEP> intensive- <SEP> g5PC, <SEP> deep orange-51.
<tb>



  Color <SEP>: <SEP> periphery <SEP> organe- <SEP> organe-50.
<tb> g5LA. <SEP> intense orange-50,
<tb> center <SEP> brown-black
<tb> licked.
<tb>



  1. <SEP> 0% <SEP> NZ-amine, <SEP> growth <SEP>: <SEP> medium <SEP>; <SEP> j <SEP> growth <SEP>: <SEP> good. <SEP> growth <SEP>: <SEP> good. <SEP> growth <SEP>: <SEP> good. <SEP>
<tb>



  Type <SEP> A <SEP> Colony <SEP> increased <SEP> and <SEP> ge <SEP> Color: <SEP> henna-<SEP> color: <SEP> henna-<SEP> color: <SEP> terracotta fear. <SEP> g5PG, <SEP> intensive- <SEP> g5PG, <SEP> intensive- <SEP> ben-g5PE, <SEP> intensiveColor: <SEP> red-brown-orange- <SEP> brown-55. <SEP> brown-55. <SEP> brown-55.
<tb> g4PC, <SEP> deep orange-51
<tb> (weak <SEP> brown-black
<tb> stained).
<tb>



  1% <SEP> asparagine <SEP> growth; <SEP> bad, <SEP> growth; <SEP> medium <SEP> growth; <SEP> bad. <SEP> growth; <SEP> bad.
<tb> moderate.
<tb>



  Color <SEP>: <SEP> clove- <SEP>
<tb> brown-g3PL. <SEP> dark yellow brown-78.
<tb>



  1% <SEP> Glutarmine <SEP> growth; <SEP> bad. <SEP> wachatum <SEP> medium- <SEP> growth; <SEP> bad. <SEP> watch; <SEP> bad.
<tb> acid <SEP> moderate.
<tb>



  Color <SEP>: <SEP> clove brown-gSPL, <SEP> dark-
<tb>! <SEP> yellow-brown-'78. <SEP>
<tb>



  1% <SEP> sodium nitrate <SEP> No <SEP> growth. <SEP> No <SEP> growth. <SEP> No <SEP> growth. <SEP> No <SEP> growth.
<tb>



  1% <SEP> ammonium nitrate <SEP> No <SEP> growth. <SEP> No <SEP> growth. <SEP> No <SEP> growth. <SEP> No <SEP> growth.
<tb>
 

 <Desc / Clms Page number 26>

 Table VI
 EMI26.1
 
<tb>
<tb> Chromatographic <SEP> studies <SEP> on <SEP> the <SEP> formed by <SEP> incubation <SEP> of <SEP> M. <SEP> carbonacea <SEP>
<tb> antibiotic <SEP> substances
<tb> <SEP> solvent systems used <SEP> RF values <SEP> for
<tb> R-451 <SEP> A <SEP> R-451 <SEP> B <SEP> R-451 <SEP> C <SEP> R-451D <SEP> R-451E
<tb> I.
<tb>



  Benzene <SEP> 10 <SEP> V. <SEP> T ..) <SEP>
<tb> petroleum ether
<tb> (boiling range <SEP> 30-60 C) <SEP> 2.5 <SEP> V.T. <SEP> 0 <SEP> 0. <SEP> 14 <SEP> 0.28 <SEP> 0.64 <SEP> 0, <SEP> 74
<tb> Acetone <SEP> 5 <SEP> V. <SEP> T.
<tb>



  Development time <SEP>: <SEP> 1 <SEP> 1/2 <SEP> h
<tb> II.
<tb>



  Toluene <SEP> -20 <SEP> V. <SEP> T.
<tb> n <SEP> - <SEP> butanol <SEP> 1, <SEP> 5 <SEP> V. <SEP> T.
<tb>



  Distilled <SEP> water <SEP> 7 <SEP> V. <SEP> T. <SEP> 0 <SEP> 0.19 <SEP> 0, <SEP> 42 <SEP> 0.61 <SEP> 0.71
<tb> petroleum ether
<tb> (boiling range <SEP> 30-600C) <SEP> 1, <SEP> 5 <SEP> V. <SEP> T.
<tb>



  Development time <SEP>: <SEP> 4 <SEP> 1/2 <SEP> h
<tb> in.
<tb>



  Petroleum ether
<tb> (boiling range <SEP> 60-90 C) <SEP> 5 <SEP> V. <SEP> T. <SEP>
<tb>



  Methanol <SEP> 8 <SEP> V. <SEP> T.
<tb>



  Ethyl acetate <SEP> 5 <SEP> V. <SEP> T. <SEP> 0 <SEP> 0, <SEP> 08 <SEP> 0, <SEP> 26 <SEP> 0.60 <SEP> 0, <SEP > 82 <SEP>
<tb> Distilled <SEP> water <SEP> 2 <SEP> V. <SEP> T. <SEP>
<tb>



  Development time <SEP>: <SEP> 2 <SEP> h
<tb> *) <SEP> V. <SEP> T. <SEP> = <SEP> Volume parts <SEP>; <SEP> in the <SEP> original attempt <SEP>: <SEP> 1 <SEP> V. <SEP> T. <SEP> = <SEP> 1 <SEP> pint
<tb>
 

 <Desc / Clms Page number 27>

 Table VII
 EMI27.1
 
<tb>
<tb> Chromatography <SEP> of <SEP> antibiotics <SEP> of the <SEP> R-451 group <SEP> on <SEP> aluminum oxide
<tb> Tested <SEP> treatment method <SEP> and <SEP> yield <SEP> inhibition zone <SEP> in <SEP> mm <SEP> at <SEP> 6.35 <SEP> mm <SEP> in <SEP> diameter <SEP> paper bioautographic <SEP> distribution
<tb> Fractions <SEP> measuring <SEP> filter paper disks <SEP> and <SEP> concentrations <SEP> (RF values)
<tb> (groups) <SEP> (in). <SEP> t <SEP> g / ml) <SEP> from
<tb> 1000 <SEP> 100 <SEP> 10 <SEP> 1
<tb> Starting material <SEP> - <SEP> 20 <SEP> 14 <SEP> 8 <SEP> 0 <SEP> 0.0; <SEP> 0.15; <SEP> 0.29;

   <SEP> 0, <SEP> 60; <SEP> 0. <SEP> 74
<tb> 1 <SEP> Inactive <SEP> 0000
<tb> 2 <SEP> residue <SEP> dissolved in <SEP> chloroform <SEP>,
<tb> with <SEP> ten times the <SEP> volume <SEP> petroleum ether <SEP> precipitated <SEP>; <SEP> yield <SEP>: <SEP>
<tb> 102 <SEP> mg <SEP> 23 <SEP> 17 <SEP> 10 <SEP> 0 <SEP> 0, <SEP> 0 <SEP>; <SEP> 0.12 <SEP> (track); <SEP> 0.62; <SEP> 0, <SEP> 74
<tb> 3-4 <SEP> Treats <SEP> like <SEP> fraction <SEP> 2 <SEP>; <SEP>
<tb> Yield <SEP>: <SEP> 725 <SEP> mg <SEP> 15 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0.0
<tb> 5 <SEP> - <SEP> 8 <SEP> Treats <SEP> like <SEP> fraction <SEP> 2 <SEP>; <SEP>
<tb> Yield <SEP>: <SEP> 541 <SEP> mg <SEP> 18 <SEP> 11 <SEP> 0 <SEP> 0 <SEP> 0, <SEP> 0 <SEP>; <SEP> 0, <SEP> 18 <SEP> (track) <SEP>;

   <SEP> 0, <SEP> 62 <SEP> (track)
<tb> 9-12 <SEP> inactive <SEP> 0000
<tb> 13 <SEP> - <SEP> 30 <SEP> Treats <SEP> like <SEP> fraction <SEP> 2;
<tb> Yield <SEP>: <SEP> 1, <SEP> 73 <SEP> g <SEP> 26 <SEP> 22 <SEP> 16 <SEP> 8 <SEP> 0, <SEP> 0 <SEP>; <SEP> 0, <SEP> 14 <SEP>; <SEP> 0, <SEP> 28 <SEP>; <SEP> 0, <SEP> 64
<tb> 31 <SEP> - <SEP> 34 <SEP> Treats <SEP> like <SEP> fraction <SEP> 2 <SEP>; <SEP>
<tb> Yield <SEP>: <SEP> 32, <SEP> 5 <SEP> mg <SEP> 25 <SEP> 22 <SEP> 16 <SEP> 8 <SEP> 0, <SEP> 0 <SEP>; <SEP> 0, <SEP> 15 <SEP>; <SEP> 0. <SEP> 29 <SEP>; <SEP> 0, <SEP> 60
<tb> 35 <SEP> - <SEP> 42 <SEP> Dissolved in <SEP> water <SEP>, <SEP> lyophilized <SEP>; <SEP> 0, <SEP> 0 <SEP>; <SEP> 0, <SEP> 13 <SEP> (weak) <SEP>; <SEP>
<tb> Yield <SEP>: <SEP> 111 <SEP> mg <SEP> 22 <SEP> 17 <SEP> 11 <SEP> 0 <SEP> 0.24 <SEP> (weak) <SEP>;

   <SEP> 0. <SEP> 59 <SEP>
<tb> 43 <SEP> - <SEP> 49 <SEP> Treats <SEP> like <SEP> fractions
<tb> 35 <SEP> - <SEP> 42 <SEP>; <SEP> Yield <SEP>: <SEP> 67. <SEP> 5 <SEP> mg <SEP> 22 <SEP> 16 <SEP> 9 <SEP> 8 <SEP> 0, <SEP> 0 <SEP>; <SEP> 0, <SEP> 13 <SEP>; <SEP> 0, <SEP> 25 <SEP>; <SEP> 0, <SEP> 58
<tb>
 
 EMI27.2
 

 <Desc / Clms Page number 28>

 Table VIII
 EMI28.1
 
<tb>
<tb> Chromatography <SEP> of <SEP> antibiotics <SEP> of the <SEP> R-451 group <SEP> on <SEP> diatomaceous earth
<tb> HUV <SEP> ml <SEP> elution <SEP> weight <SEP> of the <SEP> return <SEP> bioautographically <SEP> determine <SEP> level <SEP> averaged in <SEP> mg <SEP> <SEP> RF values
<tb> 1 <SEP> 3500
<tb> 2 <SEP> 3500 <SEP> 4300 <SEP> 0.77
<tb> 2, <SEP> 1 <SEP> - <SEP> 2, <SEP> 9 <SEP> 2800 <SEP> 190 <SEP> 0.79
<tb> 3 <SEP> - <SEP> 10 <SEP> 24500 <SEP> 2200 <SEP> 0, <SEP> 3; <SEP> 0, <SEP> 65 <SEP>;

   <SEP> 0.75
<tb> 11 <SEP> - <SEP> 13 <SEP> 7000 <SEP> 100 <SEP> 0, <SEP> 14 <SEP>; <SEP> 0, <SEP> 28 <SEP>; <SEP> 0, <SEP> 64
<tb> 14-16 <SEP> 7000 <SEP> 100 <SEP> 0
<tb>
 Table IX
 EMI28.2
 
<tb>
<tb> Chriomatographic <SEP> separation <SEP> from <SEP> HUV <SEP> 3 <SEP> - <SEP> 10 <SEP> from <SEP> the <SEP> antibiotics <SEP> of the <SEP> R-451 -Group
<tb> HUV <SEP> ml <SEP> elution <SEP> weight <SEP> of the <SEP> return <SEP> paper chromatography <SEP> medium <SEP> level <SEP> in <SEP> mg <SEP> bioautographic <SEP> determined
<tb> RF values
<tb> 1 <SEP> 900 <SEP> - <SEP> -
<tb> 1 <SEP> - <SEP> 1.5 <SEP> 450 <SEP> 320 <SEP> 0.78
<tb> 1.5-14 <SEP> 11000 <SEP> 680 <SEP> 0.64;

   <SEP> 0.74
<tb>
 Table X
 EMI28.3
 
<tb>
<tb> Pre-separation <SEP> of the <SEP> antibiotics <SEP> of the <SEP> R-451 group <SEP> by <SEP> chromatography <SEP> on <SEP> diatomaceous earth
<tb> HUV <SEP> amount <SEP> elution weight <SEP> of the <SEP> re-bioautographically <SEP> determined <SEP>
<tb> medium <SEP> in <SEP> ml <SEP> stands <SEP> in <SEP> mg <SEP> RF values
<tb> 0, <SEP> 9 <SEP> 7000 <SEP> - <SEP> - <SEP>
<tb> 1, <SEP> 0-1, <SEP> 1 <SEP> 800 <SEP> 186 <SEP> 0, <SEP> 8 <SEP>; <SEP> 0.55
<tb> 1. <SEP> 2-1. <SEP> 8 <SEP> 5000 <SEP> 900 <SEP> 0, <SEP> 56 <SEP>; <SEP> 0, <SEP> 43 <SEP>
<tb> 1, <SEP> 9-2, <SEP> 8 <SEP> 7200 <SEP> 5700 <SEP> 0, <SEP> 16; <SEP> 0.23;
<tb> 0, <SEP> 43 <SEP>; <SEP> 0, <SEP> 55 <SEP>
<tb> 2, <SEP> 9-3, <SEP> 9 <SEP> 8000 *) <SEP> 100 <SEP> 0 <SEP>; <SEP> 0, <SEP> 16 <SEP>; <SEP> 0, <SEP> 23 <SEP>;

   <SEP>
<tb> 0, <SEP> 55 <SEP> (track)
<tb> 4, <SEP> 0-5, <SEP> 0 <SEP> 8000 **) <SEP> 120 <SEP> 0
<tb>
 *) Elution with acetone **) elution with acetone-methanol (1: 1)

 <Desc / Clms Page number 29>

 Table XI
 EMI29.1
 
<tb>
<tb> Chromatographic <SEP> separation <SEP> of the <SEP> fraction group <SEP> HUV <SEP> 1, <SEP> 9-2, <SEP> 8 <SEP> from <SEP> table <SEP> X
<tb> HUV <SEP> <SEP> elution <SEP> weight <SEP> of the <SEP> return <SEP> bioautographic agent <SEP> in <SEP> ml <SEP> status <SEP> in <SEP> mg <SEP> averaged <SEP> RF values
<tb> 0.7 <SEP> 4200
<tb> 0, <SEP> 8 <SEP> - <SEP> 1, <SEP> 6 <SEP> 4800 <SEP> 1100 <SEP> 0, <SEP> 55 <SEP>
<tb> 1, <SEP> 7-17, <SEP> 6 <SEP> 96000 <SEP> 300 <SEP> 0.43
<tb> 17, <SEP> 7-27, <SEP> 7 <SEP> 60 <SEP> 000 <SEP> 200 <SEP> 0, <SEP> 16 <SEP>;

   <SEP> 0.23
<tb> 27.7-28.1 <SEP> 2400 <SEP> 70 <SEP> 0 <SEP>; <SEP> 0.15
<tb>
 Table XI A
 EMI29.2
 
<tb>
<tb> Chromatography <SEP> from <SEP> Antibiotics <SEP> of the <SEP> R-451 group <SEP> to <SEP> Florisil <SEP>
<tb> Elution <SEP> eluate <SEP> weight <SEP> of the <SEP> return <SEP> bioautographically <SEP> determine <SEP> in <SEP> ml <SEP> level <SEP> averaged <SEP> RF -Values
<tb> CH2C12 <SEP> 4400 <SEP> 10, <SEP> 2g <SEP> 0, <SEP> 43 <SEP> (D) <SEP>; <SEP> 0, <SEP> 56 <SEP> (E)
<tb> acetone <SEP> 8800 <SEP> 8, <SEP> 1 <SEP> g <SEP> 0.45 <SEP> (D)
<tb> "+ 10 acetone <SEP> 8800 <SEP> 13, <SEP> 2 <SEP> g <SEP> 0.43 <SEP> (D)
<tb> "+ <SEP> 20% <SEP> acetone <SEP> 8 <SEP> 800 <SEP> 2, <SEP> 3 <SEP> g <SEP> 0, <SEP> 45 <SEP> (D) <SEP>;

   <SEP> 0, <SEP> 24 <SEP> (B)
<tb> zo <SEP> acetone <SEP> 8800 <SEP> 6.3 <SEP> g <SEP> 0, <SEP> 23 <SEP> (B)
<tb> 100tao <SEP> acetone <SEP> 8800 <SEP> 5, <SEP> 4 <SEP> g <SEP> 0.15 <SEP> (3); <SEP> 0.23 <SEP> (B)
<tb>
 Table XII
 EMI29.3
 
<tb>
<tb> In vitro activity <SEP> of <SEP> R-451
<tb> Systematic <SEP> name <SEP> strain name <SEP> Minimum <SEP> inhibition of the <SEP> used <SEP> microorganisms <SEP> concentration <SEP> in <SEP> g / ml
<tb> Bacillus <SEP> cereus <SEP> ATCC <SEP> 7064 <SEP> 0.75
<tb> Bacillus <SEP> cereus <SEP> var.

   <SEP> mycoides <SEP> DA <SEP> 39 <SEP> 0.25
<tb> Bacillus <SEP> megatherium <SEP> DA <SEP> 31 <SEP> 0.75
<tb> Bacillus <SEP> subtilis <SEP> ATCC <SEP> 6633 <SEP> 0, <SEP> 25
<tb> Staphylococcus <SEP> albus <SEP> DA <SEP> 2100 <SEP> 0, <SEP> 10
<tb> Staphylococcus <SEP> aureus <SEP> ATCC <SEP> 6538P <SEP> 0.075
<tb> Staphylococcus <SEP> aureus <SEP> ATCC <SEP> 1163 <SEP> 0.25
<tb> Staphylococcus <SEP> aureus <SEP> ATCC <SEP> 12715 <SEP> 0.75
<tb> Staphylococcus <SEP> aureus <SEP> Gray <SEP> 0.50
<tb> Staphylococcus <SEP> aureus <SEP> DA <SEP> 201 *) <SEP> 0, <SEP> 10 <SEP>
<tb> Staphylococcus <SEP> aureus <SEP> DA <SEP> 202 **) <SEP> 0.25
<tb> Staphylococcus <SEP> aureus <SEP> DA <SEP> 203 ***) <SEP> 0, <SEP> 30
<tb> Staphylococcus <SEP> aureus <SEP> DA <SEP> 204 ***) <SEP> 0, <SEP> 15 <SEP>
<tb> Staphylococcus <SEP> aureus <SEP> DA <SEP> 205 ***) <SEP> 0,

  40
<tb> Streptococcus <SEP> fecalis <SEP> DA <SEP> 20 <SEP> 0.75
<tb> Streptococcus <SEP> pyogenes <SEP> ATCC <SEP> 12384 <SEP> 0.175
<tb> Streptococcus <SEP> pyogenes <SEP> DA <SEP> 21 <SEP> 0, <SEP> 375
<tb>
   *) A strain resistant to Novobiocm.



  **) A strain resistant to oleandomycin and erythromycin.



  ***) Strains resistant to penicillin.

 <Desc / Clms Page number 30>

 Table XIII
 EMI30.1
 
<tb>
<tb> In vivo activity <SEP> of <SEP> R-451
<tb> Infecting <SEP> Administered <SEP> dose <SEP> R-451 <SEP> Percentage <SEP> survivors
<tb> Microorganism <SEP> in <SEP> j <SEP> g / mouse <SEP> and <SEP> tag <SEP> mice <SEP> after <SEP> 24 <SEP> h
<tb> Streptococcus <SEP> pyogenes <SEP> 0 <SEP> (control saline solution) <SEP> 0
<tb> 40 <SEP> (2 <SEP> mg / kg <SEP> mouse) <SEP> 50
<tb> 40 <SEP> (2 <SEP> mg / kg <SEP> mouse) <SEP> one
<tb> single <SEP> injection <SEP> 1 <SEP> h
<tb> after <SEP> the <SEP> infection <SEP> 100
<tb> Staphylococcus <SEP> aureus <SEP> 40 <SEP> (2 <SEP> mg / kg <SEP> mouse) <SEP> 50
<tb> Diplococcus <SEP> pneumoniae <SEP> 50 <SEP> (2.5 <SEP> mg / kg <SEP> mouse) <SEP> 100 <SEP> (after <SEP> 2 <SEP> days)

  
<tb>
 Table XIV
 EMI30.2
 
<tb>
<tb> In vitro activity <SEP> of <SEP> R-451 <SEP> A
<tb> concentration <SEP> diameter <SEP> in <SEP> mm <SEP> of the <SEP> inhibition zone <SEP> against
<tb> in <SEP> moved <SEP> R-451 / ml <SEP> Staphylococcus <SEP> aureus <SEP> Streptococcus <SEP> fecalis <SEP> Bacillus <SEP> subtilis
<tb> 1000 <SEP> 18 <SEP> 23 <SEP> 13
<tb> 100 <SEP> 9 <SEP> 15 <SEP> 10
<tb> 10 <SEP> 0 <SEP> 10 <SEP> 0
<tb> 1 <SEP> 0 <SEP> 7 <SEP> 0 <SEP>
<tb>
 Table XV
 EMI30.3
 
<tb>
<tb> In vitro activity <SEP> of <SEP> R-451 <SEP> B
<tb> concentration <SEP> diameter <SEP> in <SEP> mm <SEP> of the <SEP> inhibition zone <SEP> against
<tb> in <SEP> jug <SEP> R-451 <SEP> B / ml <SEP> Staphylococcus <SEP> aureus <SEP> Streptococcus <SEP> fecalis <SEP> Bacillus <SEP> subtilis
<tb> 1000 <SEP> 23 <SEP> 30 <SEP> 17
<tb> 100 <SEP> 20 <SEP> 26 <SEP> 8
<tb> 10 <SEP> 15 <SEP> 22

  <SEP> 0
<tb> 1 <SEP> 8 <SEP> 15 <SEP> 0
<tb>
 

 <Desc / Clms Page number 31>

 Table XV A
 EMI31.1
 
<tb>
<tb> Antibiotic <SEP> spectrum <SEP> (in vitro activity) <SEP> of <SEP> R-451 <SEP> B <SEP> and <SEP> R-451 <SEP> D
<tb> Test organisms <SEP> Minimum <SEP> inhibition concentration
<tb> in <SEP> units / ml
<tb> Systematic <SEP> name <SEP> parent name <SEP> R-451 <SEP> B <SEP> R-451 <SEP> D
<tb> Bacillus <SEP> cereus <SEP> QMCC <SEP> B964 <SEP> 0.3 <SEP> 0, <SEP> 025
<tb> Bacillus <SEP> megatherium <SEP> ATCC <SEP> 5773 <SEP> 1,2 <SEP> 0, <SEP> 15 <SEP>
<tb> Bacillus <SEP> subtilis <SEP> ATCC <SEP> 6633 <SEP> 1,2 <SEP> 0, <SEP> 15 <SEP>
<tb> Sarcina <SEP> lutea <SEP> ATCC <SEP> 9341 <SEP> 1.2 <SEP> 0.3
<tb> Staphylococcus <SEP> aureus <SEP> ATCC <SEP> 6538 <SEP> 0.15 <SEP> 0.025
<tb> Staphylococcus <SEP> aureus <SEP> ATCC <SEP> 6538P <SEP> 0.15 <SEP> 0,

  025
<tb> Staphylococcus <SEP> aureus <SEP> ATCC <SEP> 12715 <SEP> 0.6 <SEP> 0, <SEP> 15
<tb> Staphylococcus <SEP> aureus <SEP> ATCC <SEP> 9996 <SEP> 0, <SEP> 15 <SEP> 0.075
<tb> Staphylococcus <SEP> aureus <SEP> ATCC <SEP> 11G3 <SEP> 0.6 <SEP> 0.3
<tb> Staphylococcus <SEP> aureus <SEP> Gray <SEP> 0.3 <SEP> 0.025
<tb> Staphylococcus <SEP> aureus <SEP> Smith <SEP> 0.6 <SEP> 0.3
<tb> Staphylococcus <SEP> aureus <SEP> DA <SEP> 2027-2036-each <SEP> 0.25
<tb> (10 <SEP> clinical <SEP> isolates, <SEP> the <SEP>
<tb> against <SEP> penicillin, <SEP> streptomycin, <SEP> tetracycline <SEP> and
<tb> Erythromycin <SEP> are resistant <SEP>)
<tb> Diplococcus <SEP> pneumoniae ') <SEP> DA <SEP> 150-0, <SEP> 25
<tb> Streptococcus <SEP> pyogenes *) <SEP> DA <SEP> 21 <SEP> 0, <SEP> 15 <SEP> 0.0075
<tb> Streptococcus <SEP> hacmolyticus <SEP> 0, <SEP> 1 <SEP> 0,

   <SEP> 01
<tb> Streptococcus <SEP> faecalis <SEP> ATCC <SEP> 10541 <SEP> 0.3 <SEP> 0.025
<tb>
 *) Brain-heart infusion broth + 0.51o human serum.



   Table XV B
 EMI31.2
 
<tb>
<tb> In vivo activity <SEP> of <SEP> R-451 <SEP> B <SEP> and <SEP> R-451 <SEP> D
<tb> PD / 50 <SEP> (in <SEP> mg / kg)
<tb> Infecting <SEP> microorganisms <SEP> R-451 <SEP> B <SEP> R-451 <SEP> D
<tb> Streptococcus <SEP> pyogenes <SEP> 1,3 <SEP> 3, <SEP> 75
<tb> Staphylococcus <SEP> aureus <SEP> 2.5 <SEP> 12.5
<tb> Diplococcus <SEP> pneumoniae <SEP> - <SEP> 3.75
<tb>
 

 <Desc / Clms Page number 32>

 Table XVI
 EMI32.1
 
<tb>
<tb> In vitro activity <SEP> of <SEP> R-451 <SEP> D
<tb> Systematic <SEP> name <SEP> of the <SEP> strain name <SEP> minimum <SEP> inhibition concentration
<tb> examined <SEP> microorganisms <SEP> in <SEP> Jlgl <SEP> ml <SEP>
<tb> Bacillus <SEP> cereus <SEP> ATCC <SEP> 7064 <SEP> 0.25
<tb> Bacillus <SEP> cereus <SEP> var.

   <SEP> mycoides <SEP> DA <SEP> 39 <SEP> 0.075
<tb> Bacillus <SEP> megatherium <SEP> DA <SEP> 31 <SEP> 0.25
<tb> Bacillus <SEP> megatherium <SEP> QMCC <SEP> 3773 <SEP> 0.025
<tb> Bacillus <SEP> megatherium <SEP> DA <SEP> 38 <SEP> 0.25
<tb> Bacillus <SEP> sphacricus <SEP> DA <SEP> 32 <SEP> 0.75
<tb> Bacillus <SEP> sphaericus <SEP> DA <SEP> 33 <SEP> 0.25
<tb> Bacillus <SEP> subtilis <SEP> ATCC <SEP> 6633 <SEP> 0.25
<tb> Diplococcus <SEP> pneumoniae <SEP> DA <SEP> 150 <SEP> 0.25
<tb> Sarcina <SEP> lutea <SEP> ATCC <SEP> 9431 <SEP> 0.0075
<tb> Staphylococcus <SEP> albus <SEP> DA <SEP> 2100 <SEP> 0.25
<tb> Staphylococcus <SEP> aureus <SEP> DA <SEP> 2027 <SEP> - <SEP> 2036 <SEP> each <SEP> 0.25
<tb> (1.

   <SEP> 0 <SEP> clinical <SEP> Jsolate, <SEP> the <SEP>
<tb> against penicîllin, <SEP> Strep- <SEP>
<tb> tomycin, <SEP> tetracycline <SEP> and
<tb> Erychromycin <SEP> are resistant <SEP>) <SEP>
<tb> Staphylococcus <SEP> aureus <SEP> Gray <SEP> 0.25
<tb> Streptococcus <SEP> fecalis <SEP> DA <SEP> 20 <SEP> 0.025
<tb> Streptococcus <SEP> pyogenes *) <SEP> DA <SEP> 21 <SEP> 0.25
<tb>
 *) Brain-heart infusion broth + 0.5% human serum.



   Table XVII
 EMI32.2
 
<tb>
<tb> In vivo activity <SEP> of <SEP> R-451 <SEP> D
<tb> Infecting <SEP> dosage <SEP> in <SEP> tig <SEP> R-451 <SEP> D <SEP> Percentage <SEP> of survivors
<tb> Microorganism <SEP> per <SEP> mouse <SEP> and <SEP> day <SEP> mice <SEP> after <SEP> 24 <SEP> h
<tb> Streptococcus <SEP> pyogenes <SEP> 0 <SEP> (control saline solution) <SEP> 0
<tb> 75 <SEP> (3, <SEP> 75 <SEP> mg / kg <SEP> mouse) <SEP> 50
<tb> 100 <SEP> (5.

   <SEP> 0 <SEP> mg / kg <SEP> mouse) <SEP> 100
<tb> Staphylococcus <SEP> aureus <SEP> 250 <SEP> (12, <SEP> 5 <SEP> mg / kg <SEP> mouse) <SEP> 50
<tb> 400 <SEP> (20 <SEP> mg / kg <SEP> mouse) <SEP> 100
<tb> Diplococcus <SEP> pneumoniae <SEP> 75 <SEP> (3, <SEP> 75 <SEP> mg / kg <SEP> mouse) <SEP> 50 <SEP> (after <SEP> 2 <SEP> days )
<tb> 100 <SEP> (5.0 <SEP> mg / kg <SEP> mouse) <SEP> 100 <SEP> (after <SEP> 2 <SEP> days)

  
<tb>
 

 <Desc / Clms Page number 33>

 
 EMI33.1
 
 EMI33.2
 
<tb>
<tb> In vitro activity <SEP> of <SEP> R-451 <SEP> E
<tb> Concentration <SEP> Inhibition zone <SEP> in <SEP> mm <SEP> gcgen
<tb> in <SEP> lig <SEP> R-451 <SEP> U / ml <SEP> Staphylococcus <SEP> aureus <SEP> Streptococcus <SEP> fecalis <SEP> Bacillus <SEP> subtilis
<tb> 1000 <SEP> 20 <SEP> 19 <SEP> 24
<tb> 100 <SEP> 15 <SEP> 13 <SEP> 19
<tb> 10 <SEP> 8 <SEP> 9 <SEP> 15
<tb> 1 <SEP> 0 <SEP> 0 <SEP> 8 <SEP>
<tb>
 
PATENT CLAIMS:
1.

   Process for the production of the new antibiotic R-451 and its components R-451 A to R-451 E and the 0-acyl derivatives, ethers and salts with bases of the antibiotic and the components, characterized in that the new species Micromonospora carbonacea is deposited under registration number NRRL 2972. or one of their mutants or variants with essentially the same biological activity under customary conditions, preferably aerobically in an aqueous nutrient medium. the assimilable carbon and nitrogen contains, incubated, the antibiotic formed isolated from the culture and, if necessary, purified or broken down into its components and then the antibiotic or

   at least one of its components R-451 A, R-451 B, R-451 C, R-451 D and R-451 E optionally converted into one of their 0-acyl derivatives, into ethers or into salts with bases.

 

Claims (1)

2. Embodiment of the method according to claim 1, characterized in that the variant Micromonospora carbonacea var. Aurantiaca, deposited under the registration number NRRL 2997, is used. 3. The method according to claim 1 or 2, characterized in that after the fermentation, the culture is freed from the mycelium, the remaining broth is extracted with a water-immiscible solvent and the desired antibiotic or at least one of its components from the extract thus obtained is isolated by adsorption, partition between different phases, fractional precipitation or a combination of such methods.