DE2102718A1 - New antibiotic and process for making it - Google Patents

Description

Patent attorneys

Dr.-Ing. by Kreisler Dr.-Ing. Schönwald Dec-28-1970

Dr.-ing.Th.Msyer Dr.Fucs Dipl.-Chsm.Alek von Kreisler 982-FTG-l Dipl.-Chem. Carola Keller Dr.-Ing. Klöpsch CG-Ug

Cologne, Deichmannhaus

SCHERICO LTD. Töpferstrasse Lucerne (Switzerland)

New antibiotic and procedure

for its production

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The invention relates to new antibiotic products, processes for their production and isolation and cleaning and compositions containing such products. In particular, the invention relates to new antibiotic products made by incubating previously unknown species of Genus Micromonospora of the order Actinomycetales can be obtained. This species of the genus Micromonospora is hereinafter referred to as Micromonospora rosaria called.

Part of the invention relates to the new antibiotic products which are called antibiotic 67-694 below or antibiotic products which are contained in this antibiotic ψ or which contain said antibiotic 67-69 ^ ·. These substances can be present in free form or as pharmaceutically usable functional derivatives. The invention also relates to a method for producing the antibiotic product which is referred to as antibiotic 67-69 ^, or an antibiotic product which contains this antibiotic, or which is contained in said antibiotic 67-69 ^.

The process includes the incubation of microorganisms

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of the species Micromonospora rosarxa in a nutrient medium under aerobic conditions, which is continued until a sufficient antibiotic Activity in the medium can be determined. According to the invention The process of making this antibiotic also involves the use of natural ones and artificial variants and mutants of the species Micromonospora rosaria, which also contain the antibiotic 67-694 can produce. For example can such mutants from the microorganisms described by using high frequency Radiation (X and UV radiation) can be produced by actmophages or nitrogen mustards.

The preferred microorganism for the process for the production of the antibiotic 67-69 ^ Is of the genus Micromonospora and was called Mi ^ rormonospora rosaria (M.rosaria). This species was isolated from a soil sample collected from a park (Little ThicRet) in San Jacinto County, Texas. A distinctive feature of this species is that Ability to produce the antibiotic 67-694. One Culture of this organism was in the culture collection;

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of the United States Department of Agriculture (Northern Utilization Research and Development Division, Peoria, Illinois,) where they under their NRRL number: 3718 generally available is.

M. rosaria is aerobic and thrives well on a variety of solid and liquid nutrient media. It grows particularly well in aerobic submersion cultures. M. rosaria can be distinguished from the other Micromonospora species by a large number of characteristics. Thus, a macroscopic examination of M.rosaria shows (NRRL 3718) after fourteen days incubation at 24-26 ⁰ C on an agar medium which J>% NZ amine type A (a peptone obtained by enzymatic treatment of casein, which in this case as a nitrogen source acts, is obtained and which is available from Sheffield Chemfcal Company, Norwich, New York), contains \% dextrose and 1.5 # agar, moderate growth, lack of air mycelium and easy sticking of the

there are colonies which are in the form of granules / or are weakly folded. Diffusible pigments will not observed.

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The color designations chosen for the surfaces of the colonies relate to the following systems: First, the color designation can be derived from 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, position , 1958, also published by the Container Corporation of America, and, secondly, the color designation can consist of a color name and a number with the same meaning the National Bureau of Standards (USA), Circular 535 dated November 1, 1955. According to this, the color of the colony described above is maroon-g4ni or medium brown 58. Although the color of the colony surfaces and the pigment formation represent general distinguishing features for the individual microorganisms, it can still happen, as with other microorganisms, that these distinguishing features are temporarily or also can change constantly with sub-cultures.

Microscopic examination of the previously described

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Culture shows a branched mycelium with an average length of 10-20μ and a diameter of approximately 0.6μ. Numerous chlamydospores with a diameter of up to 2.0 u are formed. Conidia could not be observed in this case.

M. rosaria thrives well in a temperature range of 27-37 C, above 50 C no growth could be observed. M.rosaria hydrolyzes gelatin, milk and starch, but does not reduce nitrates to nitrites. The test results just described were obtained according to the methods described by Gordon et.al in J. Bacterialogy 69, 147 (1956) and 73, 15 (1957). Further % growth characteristics of Micromonospora rosaria

in various media are given in Table I below. Unless otherwise stated, these growth characteristics were observed in cultures that were grown for 14 days at temperatures between 24 and 26 C have grown

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Table I.

medium Growth characteristics Bennett's agar Growth: good, folded,
Formation of wine
red colored dif
fundable pig
ments
Color: dark brown-g5pn;
dark brown-59 to
black. Emerson's agar Growth: medium, flat
until grooved; Bil
dung of claret
colored diffuse
dable pigments
Color: Dark Mahogany
brown-Μβρη;
brownish gray
64 to black. Tomato pulp -
Oatmeal agar Growth: medium, ge
folds, no dif
fundable pig
ments observed.
Color: dark brown-g4pn;
dark brown-59.

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medium

Glucose asparagine agar

Glucose yeast extract agar

Czapek ¹ s agar (glucose)

Calcium monohydroxysuccinate agar

Normal agar (water agar) growth characteristics

Growth: weak to no growth.

Growth: good, folded; Formation of claret colored diffusible pigments.

Nutrient agar

Löffler's Serum Agar (Difco *)

potato

Peptone-Glucose Agar

Growth: Medium, in the form of granules, education of slightly red-brown colored diffusible pigments.

Color: deep brown ^ pl; dark brown-59

Growth: bad, not describable.

Growth: bad ₃ not positive

writable. j

Growth: Mediocre, weakly I

lifted over the agar top!

surface; formation of red

brown colored α if feasible pigments.

Color: Brick Red-g6NG; middle Rothi-fctun-43. ^!

Growth: Good, strong decomposed substrate \

sets and enlightened. j

Color: dark leather / arbenes j brown-g4PG;
Intensi ^ brown-i? 5.

Growth: bad, not

growth

Colour;

Fair to good; Formation of red-brown diffusible pigments.

Maroon-g4NI; medium brown-38.

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medium
Egg agar (Dorset
Egg Medium - Difco * Growth characteristics
Growth: Mediocre, none
Decomposition or on
brightening of the substra
tes; Formation of dif
solid lavender
colored pigments
Color: Two-tone cononies:
1) gray-tinged orange
g4LC,
medium crange-53i
2) dark gray tinted
Purple-g10PL;
very dark purple
24j;
Color of the diffusing
pigment:
orchldeenf arbenes "Pu * ¹ -
pur-glOGE; gray ge
tinted purple-228. Gelatin medium Hydrolysis: positive. Starch agar Growth: good, formation of wine
red diffusible
Pigments.
Color: brick red-g6PN; mitt
leres red-brown-43.
Hydrolysis: positive. Tyrosine agar Growth: weak, no react
tion. Litmus milk
(Difco *) Coagulation and partly pep-
toning with acidic reaction. cellulose
medium The cellulose becomes very long
sam decomposed (3 to 6 months or
long ^ formation of pale pink
red-colored diffusible
Pigments in the medium

* "Difco" is a registered trademark (Difco Laboratories Inc., Detroit, Michigan).

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Micromonospora rosaria can be incubated on various carbon and nitrogen sources.

Table II lists the growth characteristics of M.rosaria on various carbohydrate media reproduced. Each medium consists of $ 0.5 yeast extract, 1.5Jg agar and $ 1 of the test carbohydrate concerned, everything in distilled water.

Table II

test carbohydrate used

D-Arabinos "; L-arabinose D-glucose

D-galactose ß-lactose

D-Laevulose growth characteristics

++ black, even, formation of

dark red diffusible Pigments. _ _

++ black, even, formation of

dark red diffusible, pigments. _.

+++ grooved, black brown, formation of wine-red diffusible Pigments.

+ even, brown, formation of wine-red diffusible pigments.

brown, formation of pale reddish colored diffusible Pigments.

++ even to granular dark brown, formation of wine-red colored diffusible ones Pigments.

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Used Ttst-
Carbon hydrate VJ growth characteristics + D-ribose ++ dark brown, even, formation of
claret diffusible
Pigments. a-melibose + D-melicitis + Formation of pale pink ge
colored diffusible pig
ment en. L-rhamnose ++ dark brown., even, formation of
claret diffusible
Pigments. strength ++ brr ".:", ;;. *.!:.: ■ · -. r'-l ^ ur ^ tfO- ^ dif- *
fund i CI-bar gh pigments. Sucrose +++ black granules. ¹ ?, Formation of
dark claret colored
diffusible pigments. D-xylose ++ brown, granular, formation of
claret colored diffusing
pigments. D-mannitol ++ dur.kslbraun ^. just, B "'. Invitation of
dark clear red colored diffuse
dable pigments. The growth on median, as the only carbohydrate
source glycerine, raffinose, inositol, dulcite or salicin
included ,, is irregular and in general
bad. for control
$ 0.5 yeast
extract

to Table II

+++ = very good growth

++ = good growth

+ = moderate growth

+ = bad growth

= no growth

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Table III below describes the growth characteristics of Micromonospora rosaria using a nitrogen source in a medium containing a specific nitrogen source. In each case the medium consists of the specified amount of nitrogen source ^ consisting of \% glucose, 1> 5 $ agar and the remainder of distilled water.

Table III

Nitrogen source used

0.5 $ yeast extract (Difco *)

$ 1.0 NZ amine Type A

\% Asparagine

1% glutamic acid

Sodium nitrate

1% ammonium nitrate

Growth characteristics

Growth good; Formation of claret colored, diffusible Pigments

Growth good; no formation of diffusible pigments.

Growth poor; Formation of pale pink colored diffusible Pigments.

Growth poor; Formation of pink colored diffusible pigment.

Growth poor; Formation of pale pink colored diffusible Pigments.

Growth poor; no formation of diffusible pigments observed.

* "Difco" is a registered trademark (Difco Laboratories Inc., Detroit, Michigan.)

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The antibiotic active substance, which is known as Antibiotic 67-694, is produced by incubation formed by Micromonospora rosaria in a nutrient medium under aerobic conditions. The education of other antibiotic active substances

by M. rosaria

■ was observed as by-products during the incubation /. For the sake of simplicity, see below the description of the preparation of the antibiotic active substance on the antibiotic 67-694 Referenced »

The manufacture of the antibiotic 67-694 can be done by Incubation of the strain Micromonospora rosaria in an aqueous nutrient medium under aerobic conditions, preferably occur in submerged aerobic conditions. For the production of small quantities (micrograms to Milligrams) of the same, a surface culture in flasks or shaker flasks can be used, a submersion culture is found for larger quantities of the microorganism as favorable.

The nutrient medium used to make this Antibiotic 67-694 can be a liquid be, the assimilable carbon sources,

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such as carbohydrates, and assimilable nitrogen

sources,

such as proteinaceous material.

In order to promote the growth of the microorganism and thus also to increase the yield of the antibiotic, will be a combination of carbon and Nitrogen sources used where the microorganism grows particularly well. Preferred carbon sources are e.g. glucose, mannitol, levulose, sucrose, starch and ribose and preferred stick

Substance sources are e.g. corn steep liquor, yeast extract, Soybean Meal, Animal Peptones, Casein Hydrolysates and Meat Extract. Other carbon and Nitrogen sources can also be used, the usefulness of certain media can be assessed by observing the growth characteristics of M. rosaria.

"The compositions of the

Culture Media A, B, and C are examples that can be used in making antibiotic 67-694.
Medium A

Meat extract 3 g

Tryptose 5 g

Yeast extract 5 g

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Dextrose
Potato starch calcium carbonate tap water

1 g • 24 g

2 g 1000 ml

Medium B

Yeast extract

Residues of maize

Potato starch tap water

5 g

spring water 1 g strength 30 E. calcium carbonate 1 g tap water 1000 ml Medium C Yeast extract 7 g Fish extract 1 g Residues of corn spring water 1 g

30 g '

1000 ml

The manufacture of the antibiotic 67-69 ^ is at Temperatures carried out that are favorable for the growth of the microorganism. Very cheap are temperatures between 20 and 40 C and preferred

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wisely between 27 and 37 ° C. During the growth of the microorganisms and the fermentation, the pH of the medium should be between 6.0 and 8.5, preferably between 7 * 0 and 8.0. The pH of the fermentation broth can be adjusted before sterilization and inoculation and periodically during fermentation whenever necessary ψ . Buffer substances such as calcium carbonate can also be added to the fermentation broth in order to maintain the desired pH value. Usually a growth period of 2 to 7 days is necessary to achieve optimal yields of Antibiotic 67-694 ..

It can, especially when working on a technical scale, such as in a Fermen-

"tation in a tank, be desirable for the in

position of the antibiotic / two-stage process to use. In the first stage, germination takes place, with a vegetative inoculum of M-rosaria will be produced. This is done by inocolating a suitable medium, e.g. with a slant culture or a lyophilized culture of M. rosaria and by incubating the medium over a period of time,

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that is enough to produce the vegetative inoculum. This tent space usually varies between 24 and 120 hours, 72 to 96 hours is one preferred period. In the second stage, fermentation, what is obtained from germination is used Vegetative inoculum in a suitable nutrient medium in amounts up to 5VoI. $ based on volume of the nutrient medium used in the fermentation stage.

The medium used for germination and the conditions observed for them are general similar to those on which fermentation is based. In general, any suitable medium can be used, and any conditions that result in a lead to good growth of the microorganism. Should be at germination or at foaming occur during fermentation, a suitable defoamer, such as e.g. Tradename Dow Corning B (The Dow Chemical Company, Midland, Michigan).

In the fermentation stage, the microorganism will normally continue to grow for as long as

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until optimal amounts of the 67-694 antibiotic are obtained will. The preparation of the antibiotic can be done by periodically sampling and analyzing these samples are followed. Optimal yields of the antibiotic active substance are usually in air. Period from 24 - 100 Hours reached.

As already said, the incubation of M.rosaria and the simultaneous production of the antibiotic 67-694 carried out under aerobic conditions. These Conditions can be achieved in any number of ways, e.g. fermentation in a tank, in the submerged growth of the microorganism is required by passing air through it Jas nutrient medium.

The isolation of the antibiotic active substance from the fermentation broth can be done by extraction of the medium under alkaline conditions, preferably at a pH of about 9.5, with a with Water-immiscible organic solvents can be carried out. The necessary alkalinity of the Medium can by adding suitable rarities,

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such as alkali or Eraalkali metal hydroxides, preferably sodium hydroxide. Typical organic solvents that can be used for the extraction are, for example, benzene, toluene, η butanols Methylene chloride, chloroform, ethyl acetate and Amyl acetate, ethyl acetate being a preferred one Is solvent. The total amount of solvent depends on the relative solubility of the Antibiotic, on the amount of antibiotic, polyglot is extracted, and on the type of Extraction itself. If ethyl acetate is used as a solvent, 4-10 times the volume is the Permentation broth necessary, with the solvent in succession in amounts that are twice as much correspond to the volume of the fermentation broth, is used for the extraction.

The antibiotic product is separated from the organic extract in the following manner, for example. The organic solvent is preferably in vacuo to a volume that is preferably 1/50 of the volume of the fermentation broth is, evaporated, and is then with 0.1N aqueous sulfuric acid solution extracted. That way will too

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a separation of the antibiotic active substance from the others incidentally during fermentation generated connections. The effectiveness of this separation can be determined by determining the relative Proportions of the compounds produced in the process are tracked by chcomatographic route, and the extraction can, if necessary, be repeated until a substantial separation is achieved will. If the extraction is repeated, the sulfuric acid extract initially produced is obtained made alkaline, the resulting mixture with the organic solvent immiscible with water extracted, and the organic extract extracted again with 0.1 IN sulfuric acid. Is the Separation of the antibiotic active substance from its by-products once achieved, so is the sulfuric acid extract is made alkaline, again with the organic water-immiscible solvent extracted, and in vacuo preferably to a volume that is approximately 1/1000 of the volume corresponds to the original fermentation broth, concentrated,

The concentrated extract is then taken under vigorous

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Stirring to a diethyl ether-hexane mixture, which is present in ten times the amount _{3 is} added, this mixture consisting of 6 parts by volume of diethyl ether and 4 parts by volume of hexane. The resulting precipitate is filtered off, washed with small amounts of diethyl ether-hexane mixture and discarded. The piltrate combined with the washing liquid is then evaporated to dryness, the residue is dissolved in an organic solvent immiscible with water, the solution is washed several times with water and then dried over a suitable drying agent such as sodium sulfate. After filtration, the solution is evaporated to dryness and the residue in low. Dissolved amounts of diethyl ether. The solution is filtered and then added to petroleum ether with a boiling range of 3-6 ° C. with stirring. The resulting precipitate is the antibiotic 67-69 ^ and usually has a color that varies between white and light brown. The precipitate is filtered off, washed with a little petroleum ether and dried under vacuum at about 4O ⁰ C. If you work according to the procedure just described, you get an antibiotic 67-694,

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which according to the method described below has an activity of approximately 700 µg / mg.

This test method, by means of the aie relative Activity of the antibiotic active substance is determined, a cylinder dish sample is below Use of Bacillus subtilis ATCC 6633 as Test organism.

The conditions that are maintained for this sample are similar to those of the cylinder plate sample for erythromycin (Grove and Randall, Medical Encyclopedia Tnc, New York (1955), pages 96-IO3), with 21 ml for the base layer and 4 ml for the brood layer of antibiotic medium No. 5 be used. A standard curve "using the following antibiotic concentrations."

(jug / mg) in a 0.1 molar phosphate buffer (pH-8, O) recorded: 0.64, 0.8, 1.0, 1.25 and 1.56. An activity of 1000 ^ ug / mg is such Attributed to material at which µg in 1.0 ml of a 0.1 molar phosphate buffer has an inhibition zone of 17 * 8 + 1.0 mm against the test organism B. subtilis ATCQ 6633 results.

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The antibiotic 67-694, which has a test value of about 700 ^ g / mg, can, for example, on the following Way to be further cleaned. D ^ s antibiotic is in a mixture consisting of nine parts by volume of halogenated, lower alkanes, such as chloroform, and one part by volume of an alcohol such as methanol is dissolved. The solution will be placed on a column containing a solid adsorbent such as silica, aluminum oxide, magnesium silicate, kieselguhr or cellulose, and then the column is filled with a mixture that from 4 parts by volume of a chlorinated, lower alkane, such as chloroform, and one part by volume a lower alcohol such as methanol is eluted. The individual fractions of the eluate are collected and the presence of antibiotic active material in the fractions is indicated by Determined with Staphylococcus aureus. In this way the output from the column can be monitored weruen.

The fractions containing the antibiotically active material can be based on thin-layer chromatography Silica gel plates are further tested, being

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a solvent is used which corresponds to that used in column chromatography. The plate on which a first sample has been chromatographed is sprayed with a suitable agent, such as a mixture of methanol and sulfuric acid, and heated to. to locate the organic substances on the plate as permanent points. A second sample is tested against ι arcina lutea on another plate in order to determine the presence and the R _f value of the antibiotic active material. In this test, a mobile solvent consisting of chloroform and methanol in an alcohol ratio of 4: 1 is used, the running time is one hour and an R value between 0.4 and 0.5 was determined for gas antibiotic 67-694. Chromatographs of the non-purified antibiotile <x? N67-694 resulting from the production process described show that it contains two antibiotics. In a chloroform-methanol mobile phase (volume ratio 4: 1) these antibiotics have R _f values of 0.359 and 0.58.

Fractions containing the active material a.ntibiotisch r.iit älm !!! 'fro .: R ₁₁ value -. Are pooled nthalten and Vakuur!

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evaporated to a residue. The residue, which has been obtained from those fractions which _{showed an R f} value of 0.4-0.5, is dissolved in acetone and then added to petroleum ether. The precipitate obtained is separated by filtration or centrifugation and discarded, while the filtrate or the exiting liquid from the centrifuge is evaporated to dryness _t. The residue represents the antibiotic 67-694, which has an activity of 1000 ^ g / mg according to the test method described above.

An alternative method, the antibiotic 67-694 to isolate and purify is given by the fact that one extracts the first, which by extraction the fermentation broth was obtained with the organic solvent immiscible with water, evaporated, the residue is taken up in a suitable solvent and chromatographed. The factions that containing the antibiotic are then treated according to the method described above to get the pure Win antibiotic.

The cleaning of the antibacterial 67-694 can also by preparing a suitable derivative of

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Antibiotic such as an ester and a salt, and can be achieved by isolating this derivative with further purification thereof. Is this If free antibiotic is desired, the derivative must be returned to its free form. Examples of suitable salts are those with hydrochloric acid, sulfuric acid, phosphoric acid, Acetic acid or succinic acid can be obtained. Acetates, butyrates, octanoates, benzoates and succinates are examples of suitable esters which can also be used. The appropriate way to do this To produce esters is to react with the appropriate acid anhydrides or acid chlorides given.

The following chemical and physical properties were for the antibiotic 67-694, which has an activity of 1000 ig '/ mg, found:

1. Chromatography, the R _f value e; The measured R values of the antibiotic 67-694 in various solvents and against Sl-utea can be found in Table 4. They were by means of

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Thin layer chromatography determined using 250 micron silica gel plates under the trade name "Uniplate" (Analtech Inc. Wilmington, Delaware) using agar incubated with S. lutea found.

Table IV

solvent
(in parts by volume) R _f values of inhibition
zone against S.lutea Chloroform: methanol: $ 17
Ammonia 2: 1: 1 O.98 Butanol: acetic acid:
Water 3: 1 ^ 1 O.37 Chloroform: methanol
4: 1 O.45 Chloroform: methanol
3: 2 0.48 II. Chemical and physical data

1. Elemental analysis found. Calculated for carbon 63.18
Hydrogen 8.86 nitrogen 2.29
Oxygen (difference) 23 67

H ^ NO ^ -51-9

64.00 8.84 2.41

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2. Empirical Formula 3 · Molecular Weight 4. Optical Rotation

581 (determined in the mass spectrometer _O P)

5 · Melting point: 110-114 ⁰ C. Ethanol) 6th pK _a 8.7 7th Neutralization:
equivalent to : 599 8th. Ultraviolet Ad-:
sorption in methanol E ^ = 238 (b
learn , el 240 mu)

III. Infra-red adsorption spectrum.

The infrared adsorption spectrum of the antibiotic 67-694 in mineral oil is shown in FIG. Here, λ means the wavelength in microns, y-the Frequency in cm "and T the percentage of permeability.

The largest peaks and bands are shown in Table 5, using the following abbreviations Find:

S - strong, M - medium, W - weak and brd. wide. The information is given in microns and, those peaks that attributable to mineral oil have been omitted.

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Tabel V (S) 2.87 (M) (S) 3.70 (W) 8.57 (S) 5.80 (S) 9.OO (S) 5.92 (M-S) 9.32 (M) 6.17 (M-S) 9-55 (M-S) 9-73 (M) 10.17 (W-M) 7.11 (W) 10.37 (W) 7.60 (M) 11.15 (W-M) 7.82 (M) 11.48 (W) 11.87 (W) 7.98 (W) 11.95 (W, br 8.44 (S) 12.62 13.95 IV. NMR spectrum

The NMR spectrum of the antibiotic 67-694 is in Figure 2 shown. The spectrum was measured on a Varian A-60-A spectrometer (Varian Associates, Cali fornia) obtained, a solution of 40 mg of the antibiotic 67-694 in 0.4 ml of deuterated chloroform, which additionally contained small amounts of deuterium oxide, was used. The δ and T values are

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given in ppm. Tetramethylsilane was used as the standard used. In FIG. 2, a partial spectrum is visible over the entire spectrum, that in the entire spectrum appear at 9.8 ppm and therefore lie outside the part of the spectrum shown. the The intensity of this peak gives rise to the assumption that this is important for the interpretation of the spectrum of P seems to be important.

V. Solubility Solvent solubility

Very sparingly soluble in water

Methanol readily soluble

Acetone readily soluble

Chloroform readily soluble

Benzene is readily soluble

* Diethyl ether is sparingly soluble

The details for the solubility are taken from n., Q. rharmacopea, 17th edition, (196: 3) page 8, taken.

VI. pH stability

In a pH range of 2-10, no significant change in activity could be determined when

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the antibiotic 67-694 in ethanol, which with a suitable buffer was diluted, dissolved and heated to temperatures of 100 C for 30 minutes was heated.

VII. Stability to Enzymes

The stability of the antibiotic 67-694 was tested against trypsin, chymotrypsin, pepsin and α-amylase. Solutions of enzymes with a concentration of 1 mg / ml in Mcllvaines buffer with maximum pH values for the enzymes were prepared. The maximum pH values for the enzymes are 8 for trypsin and chymotrypsin, 2.2 for pepsin and 4. Two hundred (200) ^ g of the antibiotic was dissolved in 0.3 ml of water with 0.5 ml of the Enzyme solution mixed and incubated at yj C for 24 hours. The solution was tested against Escherichia coli and Staphylococcus aureus after 0, 1, 2.5 and 24 hours. The enzymes could not cause any significant decrease in the activity of the antibiotic active substances during this period.

VIII. Color test

The antibiotic 67-694 gives a positive result

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in the "Molisch Starch ~ Kl" and "Elson Morgan" test and a negative reaction with biuret, ninhydrin and in the Sakaguchi test.

The IR adsorption spectrum, the NMR spectrum and _fc the chemical and physical properties

suggest, that the antibiotic 67-694 a Is a macromolecule with a tertiary amino group. Furthermore it has at least two esterifiable hydroxyl groups, where one is selectively esterifiable. That The NMR spectrum also reveals an aldehyde group.

The present invention also encompasses pharmaceutical usable (non-toxic) derivatives

of the antibiotic 67-69 ^, such as the pharmaceutical usable salts and esters. Typical salts are acid addition salts made with inorganic ones Acids such as hydrochloric acid, sulfuric acid or phosphoric acid or reactive derivatives of them, such as the salts of these acids, or organic acids, such as acetic acid, succinic acid, Citric acid, lactic acid, monohydroxysuccinic acid, malonic acid, maleic acid, dihydroxy

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succinic acid, glutaric acid, lauric acid, adamantanic acid, cinnamic acid, camphoric acid, palmitic acid, stearic acid or undecylenic acid *. These salts can be prepared by reacting the antibiotic 67-694 or an antibiotic active substance which contains this antibiotic or which is contained in the antibiotic 67-69 ² J- or reactive derivatives thereof with the acid or reactive derivatives thereof leaves and isolates the resulting salts. Typical pharmaceutically usable esters are those of carboxylic acids such as acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, octanoic acid, stearic acid, succinic acid, benzoic acid, phthalic acid, phenylacetic acid, phenoxyacetic acid or adamantanoic acid. Esters of isomeric acids thereof, such as, for example, isobutyric acid, are also possible. The esters can be obtained by esterifying the antibiotic 67-694 or aiiMMntisoh active substances which contain this or are contained in the antibiotic 67-694 or reactive derivatives thereof with suitable acid anhydrides, acid chlorides or other equivalent compounds. The pharmaceutically acceptable esters

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can be in the form of monoesters or diesters, or as mixed esters.

In vitro test

The in vitro tests for the antibiotic 67-694 with an activity of 775 µg / mg was determined by the usual dilution method using a yeast-broth medium carried out with a pH of 7 * 4. The samples were stored for 18 hours at 37 ° C incubated and then evaluated. The results can be found in Table VI and show that the Antibiotic in vitro a broad antibacterial spectrum with a particularly good effectiveness against gram-positive bacteria.

Tabel VI 6th Minimal inhibition Organisms experiments 7th Concentration ^ g / mg) 2 0.03-3.0 Staphylococcus aureus 3 0.75-3.0 Streptococcus pyogenes 3 0.03-0.08 Enterococcus sp. 4th O.O3-O.O8 Diplococcus pneumoniae 3 3.0 Escherichia coli 3 O.3-3.O Klebsiella pneumoniae 4th 0.75-7.5 Aerobacter aerogenes 0.3 Proteus sp. O.75-3.O Pseudomonas aeruginosa 10 98 # t-M> 5 3 9 Salmonella schottmuelleri -34-

Dec-28-1970

982-FTG-35 CG-ug

In these screening tests, the antibiotic was 67-694 with an activity of 775 / ig / mg used and the Results were converted to an activity of 1000 µg / mg.

In vivo test

This was carried out with the antibiotic 67-694 with an activity of 1000 Wg / mg on male CP-I mice with an average weight of 20 g. The antibiotic was administered as a suspension or solution in an aqueous vehicle containing 0.5% carboxymethyl cellulose in two doses, one just before and one 4 hours after the intraperitoneal infection by the bacteria. Infected, untreated mice (control experiments ) died within 18 hours. The survival rate in the treated animals was determined 48 hours after infection. The results of the in vivo test, in which representative gram-positive and gram-negative microorganisms were used, are recorded in Table VII.

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organism

Staphylococcus aureus gray

Streptococcus
pyogenes C

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Table VII

Activity against lethal infections in mice

Administration PD, ..- (mg / kg)

Subcutaneous Oral

Subcutaneous Oral

200

50 200

III. Lethal dose

The lethal dose, expressed as the LD ₁ .- of antibiotic 67-694, is given in Table VIII.

Table VIII

LD _r " (mg / kg )

-5) 0

350
625

Administration route

Intraperitoneally

Subcutaneous

Intravenously

The antibiotic 67-694 and the phai-pharmaceutical Usable derivatives thereof can be used alone or in combination with other antibiotically active Compounds used to increase the growth or number of organisms, especially those of the gram-positive organisms as shown in Table VI. The antibiotic 67-694

1 0 9 8 3 1/1 B 3 9

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982-FTG-37 2 ι η j 71 ο

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and the pharmaceutically acceptable derivative thereof, can e.g. in washing solutions for sanitary purposes, for cleaning laboratory objects and for other cleaning purposes, such as bacteriostatic. Dish soap can be used for laboratory laundry. The antibiotic 67-69 ^ and the pharmaceutical Usable derivatives thereof can also be used for the treatment of laboratory and domestic animals that are with Organisms are infected.

BA ORIGINAL

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In the following examples which illustrate the invention, the incubation of Micromonospora rosaria, the manufacture of the antibiotic 67-69 ^, its isolation and purification, the preparation of various derivatives of the same and compositions containing this antibiotic.

example 1 Fermentation in shake bottles

A. germination stage ;

In a 300 ml Erlenmeyer flask, the sterile contents of which have the following composition and is 100 ml, approximately 0.5 ml of an oblique or Lyophilized culture of Micromonospora rosaria given:

109831/1539

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982-FTG-39
CG-ws Bacto meat extract (Difco) Bacto-Tryptose (Difco) 5 g. Bacto yeast extract (Difco) 5 g. Dextrose l g. Potato starch 24 g. calcium carbonate ρ ο- tap water 1000 ml

The contents of the bottles are incubated at 35 ° C. for 72 hours with shaking.

B. Fermentation stage :

5 ml each of the inoculum obtained in the germination stage are transferred under aseptic conditions into some 500 ml Erlenmeyer flasks, each Contain 100 ml of a sterile medium of the following composition:

Bacto yeast extract (Difco) 5 g residues of the corn steep water 1 g. Starch 30 g.

Potassium carbonate - ¹ S-

Tap water 1000 ml.

The contents of the bottles are incubated at 28 ° C for 72 to hours with shaking.

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Di3c-23-l970_ _η - _ΠΛΟ 982-FTG-40 210 2 / CG-ws

After the first 24 hours they will turn off periodically the fermentation broth samples are drawn to the antibiotic activity and thus that point in time determine on which the antibiotic activity of the medium has reached its maximum value. Once this is in place, the contents of the bottles will be combined for subsequent isolation. During germination and fermentation, the pH of the ™ Culture medium maintained at the desired level due to the buffering effect of the calcium ka.rbonaf. The fermentation is carried out under submerged aerobic conditions.

Example 2
Fermentation in the tank

A. Rating level:

25 ml of an inoculum, prepared according to Example IA, become 500 ml under aseptic conditions sterile medium, the composition of which is described in Example IA and which is in a 2 1 Erlenmeyer flask is given. The contents of the bottle are kept at 28 ° C for 72 hours with shaking incubated.

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CG-W3

B. Fermentation stage:

2102 / IO

The product obtained at the kneading stage is under aseptic conditions in a 14 l Fermenta transfer vessel, which is 9 * 5 1 of the in example IB contains the medium described.

6 ml of a defoamer (Dow-Corning B) are added and the pH of the mixture is adjusted to 7.0 either with dilute sodium hydroxide solution (In) or dilute sulfuric acid (In). The mixture is ^{fermented at 28 °} C. until maximum antibiotic activity is reached (normally after 72 to 100 hours). The aerobic conditions are achieved by passing air through (approx. 5.5 l / min.).

Example 3 Isolation of the antibiotic 67-694

The pH of the fermentation broth obtained according to Example 2 is adjusted to 9 * 5 with sodium hydroxide solution and then it is extracted with ethyl acetate in individual amounts of 20 1 until the essential Quantities of the antibiotic are removed from the broth. Generally 2 to 5 Ex-

The combined extracts

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are concentrated in vacuo to 200 ml, this concentrate is 0.1 η sulfuric acid with 200 ml back-extracted, the pH of the acidic extract adjusted to 9 * 5 with dilute sodium hydroxide solution and again extracted with ethyl acetate. The combined Extracts are concentrated to 10 ml in vacuo and the concentrate is vigorously stirred to 100 ml Diethyl ether-hexane mixture (6: 4 parts by volume) given. The resulting precipitate is filtered with small amounts of diethyl ether-hexane mixture washed and discarded. The piltrate combined with the washing liquid becomes in a vacuum evaporated to a solid residue. This residue is in .approx. 5 ml of diet ether dissolved the The solution is filtered and added to 50 ml of petroleum ether (boiling range 30-60 ° C) with stirring. The one with it resulting, a shade between white and light brown exhibiting precipitate is filtered off, washed with a little petroleum ether and in vacuo at 40 C dried. The antibiotic active product obtained in this way (impure antibiotic 67-694)

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has an activity of about 700 μg / mg after test method described above.

Example 4 Antibiotic Purification 67-694

The antibiotic 67-694 obtained according to Example 3 is dissolved in a chloroform-methanol mixture (9: 1 by volume) and the solution obtained is applied to a column which contains 250 g of silica. The column is eluted with a chloroform-methanol mixture (8: 1 by volume), with fractions of 5 ml being collected at a dropping rate of 0.5 ml / min. A sample is taken from each fraction and part of it is tested against Staphylococcus aureus. Another portion is chromatographed on silica gel GF plates, the thickness of which is 250 microns (the plates are available under the name "Uniplate" from Analtech Inc., Wilmingten, Delaware, USA). Fractions containing the antibiotic 67-694 show an R _f value of approximately 0.4 to 0.5. These are combined and become one in a vacuum

i, 109831/1539

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982-FTG-44 CG-WS

Evaporated residue. The residue is dissolved in acetone, diethyl ether is added, the resulting precipitate is separated off by filtration or centrifugation and discarded. The filtrate or the liquid emerging from the centrifuge becomes solid in a vacuum Residue evaporates. The antibiotic active product thus obtained is the antibiotic 67-694 with an activity of 1000 jug / mg and the following Key figures:

Melting point = 110-114 ° C

[a [p ⁵ = -33.4 ° (0.3 $ in ethanol)

E ** = 238 (at 240 mu) χ crn *

JR and NMR spectrum as in Figures 1 and 2. The other chemical and physical Properties are identical to those given above.

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982-FTG-45 ο 1 η? 71 ft

CG-ws ^{lx u L} ' '

Vs

Example 5

Alternative isolation and cleaning process

The pH of the fermentation broth obtained according to Example 2 is adjusted to about 9.5 with sodium hydroxide solution, whereupon it is extracted repeatedly with 20 l of ethyl acetate. The combined extracts are in vacuo. evaporated, the residue dissolved in a chloroform-methanol mixture (9: 1 by volume) and the solution applied to a column containing silica. In the following elution with a chloroform-methane mixture (8: 2 parts by volume), fractions of 5 ml each are collected. Some of the samples taken from the individual fractions are used for determination against Staphylococcus aureus and another part for thin-layer chromatographic determinations. After the fractions with similar R _f values have been combined - those containing the antibiotic 67-694 have a value of about 0.4 to 0.5 - the solvent is evaporated in vacuo. The residue will

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982-FTG-46? 1 Π? 7 1

CG-ws ^L '

Vfc

dissolved in acetane and diethyl ether is added. A precipitate is formed, which is separated off by filtration or centrifugation and discarded will. The filtrate or the supernatant liquid obtained during centrifugation becomes evaporated to dryness in vacuo. The antibiotic product obtained in this way is that Antibiotic 67-694 with an activity of

"approx. 1000 ug / mg and has physical characteristics

data as given in Example 4.

Example p

Potassium dihydrogen phosphate salt of the antibiotic 67-694

To 500 mg of antibiotic 67-694 in 1j5 ml of water ljO mg potassium dihydrogen phosphate are added

ben and the mixture is stirred for two hours at room temperature. After adding 50 mg of activated charcoal, the mixture is stirred for a further 15 minutes, the solution is filtered and lyophilized in order to obtain the desired product. Melting point 118-121 ° C, [a] ^ ⁰ = -21.2 ° (0.5 % in water).

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CG

CG-ws

If the method just described is used and equivalent amounts of acids that have a similar solubility in water, such as hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, monohydroxysuccinic acid, acetic acid, succinic acid, citric acid, malonic acid or maleic acid, the corresponding ones are obtained pharmaceutically acceptable acid addition salts. In general, these salts in vitro and in vivo have an antibacterial spectrum similar to Js _{3 and} sometimes even an identical antibacterial spectrum as the free base. If there are differences, these can be attributed to the different solubilities. Because of their good solubility properties in aqueous media, these salts are particularly suitable for aqueous compositions and solutions intended for oral and parenteral administration.

1 0 9 8 3 1/1 B 3 9

Dec-26-1970 ο 1 η 9 982-PTG-48 * ^{! U L} ' ' CO-U ₆

Example 7 Tartr rate of antibiotic 67-694

1 g of antibiotic 67-694 is dissolved in 60 ml of diethyl ether dissolved and a solution / on 250 mg dihydroxysuccinic acid added in 1.5 ml of ethanol. The mixture is stirred for 15 minutes and then the precipitated Filtered off salt. After washing the same, it is dried to obtain the desired product.

Melting point: 129-134 ° C
[α] p ° = -12 ° (0.3 $ in water)

If you use the method just described and use equivalent amounts of acids in a Ether-alcohol mixture have similar solubility, a, such as glutaric acid, lauric acid, adamantanic acid, cinnamic acid, camphoric acid, palmitic acid, Stearic acid or undecylenic acid, the corresponding pharmaceutically acceptable acid addition salts are obtained. Generally these are due to their molecular weights and solubility properties particularly well suited for use in topical compositions such as creams and ointments.

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Example 8 Antibiotic monoacetate 67-694

1.0 g of antibiotic 67-694 is dissolved in 10 ml of anhydrous acetone and 0.17 ml of acetic anhydride is added. After the mixture has been stirred at room temperature for 4 hours, a 1.2% ammonium hydroxide solution is added slowly and with continued stirring to precipitate the product. After filtering, washing with aqueous acetone and water, and drying, the desired product is recrystallized from aqueous acetone.
Melting point: 112-116 ° C
[aJ ~ =? -25 (o, j5 $ in ethanol)

Example 9 Antibiotic monoisovalerate 67-694

2.0 g of the antibiotic 67-694 are in 10.0 ml Dissolved anhydrous acetone and 0.65 ml of isovaleric anhydride are added. The mixture will Stirred at room temperature for 16 hours, then slowly and with continued stirring a 1.2 $ Ammonium hydroxide solution, added to precipitate the product. The precipitate is filtered off with

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dilute, aqueous acetone and washed with water and dried. After recrystallization the desired compound is obtained from aqueous acetone.

If the acid anhydrides used in Examples 8 and 9 are replaced by equivalents "Amounts of an acid anhydride or acid chloride,

such as propionic acid, butyric acid, caproic acid, Octanoic acid, stearic acid, succinic acid, benzoic acid, phthalic acid, phenylacetic acid, phenoxyacetic acid or adamantanoic acid, and you work according to Examples 8 and 9 * so one obtains the corresponding monoester of the antibiotic 67-694.

Example 10 Antibiotic 67-694 diacetate

5.0 g of antibiotic 67-694 are in 50 ml of pyridine dissolved, then added 2.5 ml of acetic anhydride and the resulting solution is allowed to stand at room temperature for 4 days. After that the solution in vacuum at temperatures up to J55 ° C to dryness evaporated, the residue dissolved in acetone and the

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Dfcü-28-1970 ο ι η 9 7 982-FTG-51 ^l ' ^U * -' 'CG-ug

Solution with 5 $ ammonium hydroxide added to the to precipitate the desired product. After filtration and washing with water it becomes

recrystallized from aqueous acetone. Melting point 104-107 ° C.
[α] p ° - -24 ° (0.3Ji in ethanol).

Example 11 Antibiotic dibenzoate 67-694

250 mg of the antibiotic o7-694 are dissolved in 5 ml of pyridine and 0.23 ml of benzotrichloride are added with stirring. The solution is left to stand at room temperature for 5 days, then concentrated in vacuo to 1 ml and diluted with 5 ml of acetone. Then 6% ammonium hydroxide is added, the precipitated product is filtered off, washed with water and dried in order to obtain the dibenzoate of the antibiotic 67-694.
Melting point 116-119 ° C.
[aJ _D ^ = + 20 ° (0.3 $ in ethanol)

It can easily be seen that once a particular monoester of the antibiotic is another Reaction with another acid anhydride or acid

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982-FTG-52

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Is subjected to chloride, a mixed ester can be produced. Such diesters are within the scope of the invention.

Replacing those used in Examples 10 and 11 Acid anhydrides or acid chlorides equivalent amounts of other acid anhydrides or acid chlorides and one works according to these examples, other diesters of antibiotic 67-694 can be obtained. Examples of such esters are the Dipropionate, DivaTerate, Dihexanoate, Distearate, Disuccinates, diphthalates, diphenyl acetates, diphenoxy acetates, diadamantoates and diisopropyl carboxylates.

In the previous examples, which describe the preparation of derivatives, and in the following Examples where. Compositions are illustrated, the antibiotic 67-694 is one such with an activity of 1000 μg / mg.

The following examples 12 to 15 illustrate the diverse forms of the composition in which the antibiotic and its pharmaceutical

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982-FTG- 53 CG-ug

usable salts or esters can be used.

With the exception of the topical, these compositions are intended to contain 5 to 50 mg of the antibiotic to administer active substances as free base per kg body weight and day. It will Considered ^ administering these drugs up to eight times in a 24 hour period. The topical compositions, on the other hand, should be used approximately 2 to 4 times a day infectious site. It goes without saying that the dose amount and the Frequency of administration largely depends on the type of infection and the severity of the Infection and the individual characteristics of the species to be treated is.

Example 12

Capsules

Antibiotic 67-694 250.00 mg

Lactose * 248.75 mg

Magnesium stearate 1.25 mg

-53-

500.00 mg

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Dec-28-1970

982-FTG-54 9 1G2718

CG-ug ^u ' ^w

Manufacturing

1. Mix the antibiotic 67-69 ^ and the Lactiosis.

2. Add the magnesium stearate and mix.

3. You fill the capsules.

Example 13

Oral suspension (with an administration dose of

125 mg / 5 ml)

Antibiotic tartrate 67-694 25.0 g

Magnesium-aluminum-silicate 9 * 5 g

Sodium Carboxymethyl Cellulose USP 2.5 g

Sodium Citrate, USP 25.0 g

Flavorings q.s.

Dyes q.s.

Methyl paraben, USP 0.9 g

"Propyl Parabolic, USP 0.2 g

Polysorbate 80, USP. 1.0 g

Sorbitol solution, USP 500.0 g

Water q.s 1000.0 ml

Manufacturing

1. Heat 200 ml of water to Siedln and löot in that one half of the parabens. One cools down

109831/1539

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about 70 ° C and mixed with the polysorbate 80. The silicate is sprinkled and stirred until an evenly smooth suspension has arisen.

2. In addition, 200 ml of water are heated to the boil and the remaining paraben is dissolved in this. Man does the carboxymethyl cellulose disperse with this until a smooth gel is formed? One mixes with the sorbitol solution and then dissolves the sodium citrate.

3. Add mixture 2 slowly to mixture 1 with constant stirring. The resulting mixture is cooled to 25 ° C. The antibiotic 67-69K _3, the flavoring and the coloring are added and mixed. A sufficient amount of water is then added: to obtain a total volume of 1000 ml.

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Example 14 Cream outside

Antibiotic monoacetate 67-694 10 g

Stearic acid 200 g

Sorbitan monostearate 104 g

Sorbitan monooleate 20 g

Polyoxyethylene sorbitan monolaurate 56 g

Water q.s. 1000 g

Manufacturing

1. Stearic acid, sorbitan monostearate,
Sorbitan monooleate and polyoxyethylene sorbitan monolaurate are heated to 65.degree.

2. Heat about $ 90 of the required water to 70 C, add it to mixture 1 and mix to form the cream base.

J5. The antibiotic 67-694 comes with about $ 10 des Slurried water, fed to a colloid mill and then the milled slurry of the melted Cream base added, it is mixed thoroughly and left to dull.

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5 *

Example 15

External ointment

Antibiotic dibenzoate 67-69 ^ 10g

Petrolatum 990g

1000g

Manufacturing

Melting the petrolatum, slurried in about 10 $ of the same antibiotic on, it leads to a colloid mill and mixed the milled slurry with the rest of the molten petrolatum, after which let cool.

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Claims (1)

Dec-23-DTO Q82-PTE-5U cG-ug 2 I U ζ / Claims New antibiotic product called Antibiotic 67-694, or a antibiotic product, which this antibiotic contains or which is contained in the Antibiotx, cum 67-694, in its free form or in the form of a pharmaceutically acceptable funick functional derivatives. 2. Product according to claim 1, which is an infrared a'dsorptlion spectrum. in mineral oil that essentially m Figure 1 is shown, and a magnetic nuclear resonance storage in deuterated Has chloroform, which is essentially shown in Figure, in its free form or in Form of a pharmaceutically acceptable functional component Derivatives. 3 * New antibiotic product, which is designated as AntibiotiKum 67-694, which at an activity of 1000 ^ ug / mg by a molecular formula C ₇ ,, H resulting from the elemental analysis. ,. a molecular weight of. ^ 81 ^ determined on a mass spectrometer, a melting point of 110-114 C, - 50 109831/1539 Dec-28-1970 a specific optical rotation of -33 * 4
measured at the sodium density at 2i3 C in
Q, 3% ethanolic solution, a UV adsorption maximum at 240 mu with an E, of 238
Infrared adsorption storage medium mineral oil contained in
Figure 1 is shown, a nuclear magnetic resonance; sspe ± ctrum in deuterated chloroform, the
is shown in Figure 2 and an antibacterial spectrum, which can be seen from Table VI, is characterized in its free iorm or in the form of a pharmaceutically usable functional ion
Derivatives - 4. Product according to one of claims 1 to 3,
in the form of a pharmaceutically / usable functional derivative. 5- Product according to claim 4, wherein the pharmaceutical usable fun.ctäonelle derivative a salt is. 6. Produict according to claim 4, wherein the pharmaceutical Usable functional derivative is an ester is BAD ORIGINAL 109831 / i ^? 9 982-FTG-60 ¹ J. Product according to claim 'ο, wherein the salt is a potassium dihydrogen phosphate salt. 8. The product of claim 5, wherein the salt is a tartrate salt. 9. The product of claim 6, wherein the ester is a monoacetate ester. 10. The product of claim 6, wherein the ester is a diacetate ester. 11. The use of a microorganism of the genus Micromonospora for the production of a antibiotic product called Antibiotic 67-694, or an antibiotic effective product that contains this anti- ™ contains biotiKum, or which one in the antibiotic 67-694 is included. 12. Method of making an anuibiotic effective product that is used as an antibiotic 67-694 or an antibiotic product containing this antibiotic, or - 60 ·· 109831/1539 Dec- / 28-1970 982-PTG-61 which is contained in the antibiotic 6j-6lh , characterized in that a microorganism of the species Micromonospora rosaria is incubated in a nutrient medium under aerobic conditions until substantial antibiotic activity occurs in the medium mentioned. 13 "Method according to claim 12, characterized in that that the microorganism of the species Micromonospora rosaria NRRL 3718 is incubated. 14. The method according to any one of claims 12 or 5, characterized in that the incubation takes place under submerged aerobic conditions. 15. The method according to one of .Ansprüche 12 bis 14, characterized in that the incubation takes place in a temperature range from 20 ° to 40 ° C and in a pH range of 6.0 to 8.3 takes place. 16. The method according to claim Ij, characterized in that the incubation temperature between 27 and 37 ° C and the pH between 7.0 and 8.0 lies. 61 - 109831/1539 Doc-28-1970 982-PTG-62 CG-ug ZIUZ / 17. The method according to any one of claims 12 to 16, characterized in that from the fermented Nutrient medium, an antibiotic product is isolated, which is that antibiotic product represents, which is designated as antibiotic 67-69 ^ · or an antibiotic product which contains this antibiotic or which is contained in the antibiotic 67-694, 18. The method according to claim 17, characterized in that that the isolation of the antibiotic product by means of solvent extraction is carried out. 19. The method according to claim l8, characterized in that that the antibiotic product with an organic, immiscible with water Solvent is extracted from the fermented broth under alkaline conditions and that the antibiotic product is worked up from the organic solvent; will. 20. The method according to claim 19, characterized in that that this is done by extracting the fermentation broth with a water-immiscible organic «62 ■ 1Q9831MS39 Dec-28-1970 982-FTG-63 CG-ug Solvent-obtained extract one or more Male a work-up stage, which involves the extraction of the organic extract with aqueous dilute Acid, adding a base to the aqueous extract and back-extracting the aqueous extract comprises alkaline extracts with a water-immiscible solvent and then the antibiotic product is isolated from the organic solution. 21. The method according to any one of claims 17 to 20, characterized in that the antibiotic effective product-containing solution is divided into fractions by means of chromatographic methods. 22. The method according to any one of claims I7 to 21, characterized by the fact that the antibiotic Effective product is isolated in free form ο 23 · Method according to one of claims 17 to 22, characterized in that the antibiotic product before or after its isolation is converted into a pharmaceutically usable functional derivative. 109831/1539 982-FTG-64 CG-ug 24. The method according to claim 2J>, characterized in that the antibiotic product is converted into an ester, 25 · The method according to claim 2j5, characterized in that that the antibiotic product is converted into a salt - ) 26. The method according to any one of claims 12 to 25 as described herein and in the examples 1 to 11, 27 · Fermented tea · Nutrient medium for Micromonospora rosaria, produced by a process according to any one of claims 12 to 16. 28. Antibiotic active product referred to as P 67-694 antibiotic, or a antibiotic product which contains this antibiotic, or which in the antibiotic 67-69 ^ · is contained in its free form or in the form of a pharmaceutically acceptable one functional derivative produced by a process according to any one of claims 12 to 26. -64- 109831 / D ^ c-28-lOrO 982-FTG-CG-ug 29 »Antibiotic 67-694- in its free form or in the form of a pharmaceutically acceptable functional derivative prepared according to a Method according to one of Claims 12 to 26. 30. Antibi-Otisoh effective product which im chemical structure is identical to one which is claimed in any one of claims 1 to 10, 28 or 29 ο 31. Antibiotically effective therapeutic composition, characterized in that it is an antibiotic product called AntibiotiKum 67-694-, or an antibiotic effective product which contains this antibiotic or which in the antibiotic 67-694- is included, in its free form or in the form of a pharmaceutically acceptable functional derivative and a carrier 32. Antibiotic composition therapeutic reduction according to claim 31, as described herein and set forth in Examples 12 to Vj. 109331/1539 Dec-28-19j2> i 0 27 FTG- 66-θ8: Γ CG-ug 33 »Antibiotically effective therapeutic composition according to one of claims 31 or 32 as shaped unit. 3 ² K method for producing an antibiotic therapeutic composition, characterized in that the antibiotic product, which is referred to as antibiotic 67-69 ^, or an antibiotic product which contains this antibiotic or which in the antibiotic 67-69 ^ is contained, in its free form or in the form of a pharmaceutically / usable functional derivative is brought into a form suitable for therapeutic administration, 33 · The method according to claim 34, characterized in that that the antibiotic effective product or its pharmaceutically usable functional derivative with a suitable pharmaceutical one Carrier is mixed -66- 109831 / 1R39