DE2167020A1 - PROCESS FOR THE PRODUCTION OF ROSAMICIN (ANTIBIOTIC 67-694) - Google Patents

Description

^ s- U

-I.

December 7, 1976 982-12 Germany IK / cf

Scherico Ltd.,

Lucerne, Switzerland

PROCESS FOR THE PRODUCTION OF ROSAMICIN (ANTIBIOUS

TIKUM 67-694)

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The invention relates to a process for the production of new antibiotic products. In particular, the invention relates to the production of rosamicin by incubating previously unknown species of the genus Micromonospora of the order Actinomycetales. This species of the genus Micromonospora is called Micromonospora rosaria in the following. Rosamicin (antibiotic 67-69 * 0 may, if desired, similar in its pharmaceutically acceptable salts and esters can be converted.

The method includes the incubation of microorganisms of the species Micromonospora rosaria in a nutrient medium aerobic conditions, this being continued until there is sufficient antibiotic activity in the medium is detectable. According to the invention, the method for producing this antibiotic also includes the use of natural and artificial variants and mutants j | of the species Micromonospora rosaria, which can also produce the antibiotic 67-694. For example can such mutants from the microorganisms described by using high-frequency radiation (X and UV radiation), from actinophages or from nitrogen mustard.

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The preferred microorganism to be used in the process of making antibiotic 67-69 ^ is from the genus Micromonospora and was called Micromonospora rosaria (M.rosaria). This species was obtained from a soil sample isolated that was pulled from a park (Little Thicket) in San Jacinto County, Texas. A characteristic one The characteristic of this species is the ability to produce the antibiotic 67-69 ^. A culture of this organism was found in of the Department of Agriculture (Department pf Agriculture) of the United States of America (Northern Utilization Research and Development Division, Peoria, Illinois,) where they are deposited under their NRRL number 3718 is generally available.

M.rosaria is aerobic and thrives well on a variety of solid and liquid nutrients. 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 containing 3% NZ amine type A (a peptone obtained by enzymatic treatment of casein., Which in this case as a nitrogen source and which is available from Sheffield Chemical Company, Norwich, New York), 1% dextrose and 1.5 $ agar, moderate growth, lack of air mycelium and

_mm ~ Z _mm

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a slight protrusion of the colonies, which are in the form of granules or are weakly folded. Diffusible Pigments are not observed.

The chosen color designations for the surfaces c: r colonies refer to the following systems: First, the Parb 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 connection with a ¹ number corresponding to this name, which is taken from the "Color Harmony Manual", 4th, 'position, 1958j 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

according to the National Bureau of Standards (USA), ^

Circular 535 dated November 1, 1955. Thereafter is the color of the colony described above maroon-g4ni or medium brown 58. Although the color of the colony surfaces and pigment formation represent general distinguishing features for the individual microorganisms, such as also occur with other microorganisms, that these distinguishing features can change temporarily or permanently in sub-cultures.

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Microscopic examination of the previously described culture shows a branched mycelium with a mean length of 10-20 u and a diameter of approximately 0.6 u. There are numerous chlamydospores that have a diameter of up to 2.0u. Conidia could not in this case to be observed.

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 by methods which are described by Gordon et.al in J. Bacterio.logy 69, 147 (1956) and T5s 15 (1957). Further growth characteristics of Micromonospora rosaria in various media are given in Table I below. Unless otherwise stated, these growth characteristics have been observed on cultures grown for 14 days at temperatures between 24 and 26 ° C.

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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: Mediocre, ge
folds, no dif
well-founded pig
ments observed.
Color: dark brown-g ^ pn;
dark brown-59.

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medium Growth characteristics Glucose
Asparagine agar Glucose yeast
Extract agar Growth: weak to no wax
tum. Czapek's agar
(Glucose) Growth: good, folded; education
colored by claret
diffusible on Pig
ments. Calcium mono-
hydroxysuccinate
Agar Growth: Mediocre, in good shape
of granules., education
from slightly reddish brown
colored diffusing
pigments.
Color: deep brown ₍ #pl; dark -
brown-59 Regular agar
(Water agar) Growth: bad, not good
writable. Nutrient agar Growth: bad, not good
writable. Loffler's serum
Agar (Difco *) Growth: Mediocre, weak he
raised above the agar top
area; formation of red
brown colored diffuse.
dable pigments.
Color: Brick Red-g6NG; mitt
leres red-brown ^. potato Growth: good, substrate strongly decomposed
sets and enlightened.
Color: dark buff
Braun-g4PG;
Intens i vbrown-55 ♦ Peptone glucose
Agar Growth: bad, not good
writable. Growth: medium to good;
Formation of red-brown
diffusible pig
ments.
Color: Maroon-g4NI;
medium brown-58.

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S.

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
g ^ LC,
middle range-53,
2) dark gray tinted
Purple-glOPL;
very dark purple;
Color of the diffusing
pigment:
or chideenf arbtnes "Puüt-
pur-glOGE; gray ge
tinted purple-228. Gelatin medium Hydrolysis: positive. Starch agar Growth: good, formation of wine
red diffusible
Pigments.
Color: brick red-göPN; 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 |
I.

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

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

Table II shows the growth characteristics of M.rosaria on various carbohydrate media. Each medium consists of $ 0.5 yeast extract, $ 1.5 agar and Vfo of the relevant test carbohydrate, all in distilled water.

Table II

used test
Carbohydrate Growth characteristics D-arabinose ++ black, even, formation of
dark red diffusible
Pigments. L-arabinose ++ black, just, formation of
dark red diffusible
Pigments. D-glucose +++ grooved, black brown, bil
dung of claret diffuse
dable pigments. D-galactose + even, brown, formation of wine
red diffusible pig
ments. / 3-lactose + brown, formation of weak
reddish colored diffusing
pigments. I. D-Laevulose ++ even to granularly dark
brown, formation of claret
colored diffusible
Pigments.

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Used test
Carbohydrate Growth character eristica -γ-
Ι D-ribose ++ dark brown, even, formation of
claret diffusible
Pigments. a-melibose + D-melicitis + Formation of pale pink ge
colored diffusible pig
ments, L-rhamnose ++ dark brown, even, formation of
claret diffusible
Pi SH! en ten. strength ++ b ^ ur.jizoj.iivs ZiMunf: thDr ^ dif. *
detectable pigments. Sucrose +++ black, granular, formation of
dark claret colored
diffusible pigments. D-xylose ++ brown, granular, formation of
claret colored diffusing
pigments. D-mannitol ++ dark brown, even, formation of
dark red colored diffuse
dable pigments. Growth on media that is 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 wax turn

++ = good growth

+ = mediocre V / axis turn

+ = bad growth

= no growth

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The following Table III describes the growth characteristics of Micromonospora rosaria using a nitrogen source in a medium which contains a specific nitrogen source. In each case the medium consists of the specified amount of the nitrogen source, Vfo glucose, 1.5 $ agar and the remainder of distilled water.

Table III

Used stick
source of material Growth characteristics $ 0.5 yeast extract ■
(Difco *) Growth good; Formation of wine
red colored, diffusible
Pigments $ 1.0 NZ amine
Type A Growth good; no formation of
diffusible pigments. \% Asparagine Growth poor; formation of
pale pink colored diffuse
dable pigments. $ 1 glutamine
acid Growth poor; formation of
pink colored diffusible
Pigments. \% Sodium nitrate Growth poor; formation of
pale pink colored diffuse
dable pigments. 1 $ ammonium
nitrate Growth poor; no picture
diffusible pig
ments observed.

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

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The antibiotic active substance known as Antibiotic 67-69 ^ is made 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 incubation /. For the sake of simplicity, see below

the description of the manufacture of the antibiotic J

active substance referred to Äa.s antibiotic 67-694.

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 can be a surface culture '1

be used in bottles or Sch'ittalflp.scheri, 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, A combination of carbon and nitrogen sources is used in the case of the microorganism grows particularly well. Preferred carbon sources are e.g. glucose, mannitol, levulose, sucrose, starch and ribose and preferred nitrogen sources are e.g. corn steep liquor, yeast extract, soybean meal, animal peptones, hydrolysates of Casein and meat extract. Other carbon and nitrogen sources can also be used The usefulness of certain media can be determined by observing the growth characteristics to be assessed by M. rosaria.

The compositions of the Culture media A, B and C are examples used in the preparation of the 67-694 antibiotic can be.

Medium A

Meat extract 3 g

Tryptose 5 g

Yeast extract 5 g

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Dextrose
Potato starch
calcium carbonate
tap water

ι g

24 g 2-g 1000 ml

Medium B

Yeast extract 5 g Residues of maize spring water 1 g strength 30 g calcium carbonate 1 g tap water 1000 ml Medium C Yeast extract 7 g Fish extract 1 fi

Residues of the corn steep water

Potato starch
tap water

1 g

'' 30 g 1000 ml

The manufacture of the antibiotic 67-694 is carried out at Temperatures carried out that are favorable for the growth of the microorganism. Very cheap are temperatures between 20 and 40 ° C and preferably

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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 _a 0. The pH of the fermentation broth can be adjusted prior to sterilization and inoculation and periodically during fermentation whenever necessary. Buffer substances, such as calcium carbonate, can also be added to the fermentation broth in order to maintain the desired pH value. Normally, a growth period of 2 to 7 days is necessary to achieve ojjtimalo yields of antibiotic 67-694 ..

It can, especially if on a technical scale work, such as fermentation in a tank, is desirable for the In

position of the antibiotic / two-stage process to be used. Germination takes place in the first stage instead, in which a vegetative inoculum of M.rosaria is 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 period usually varies between 24 and 120 hours, 72 to 96 hours is a preferred period. In the second stage, fermentation, the vegetative inoculum obtained during germination is poured into a suitable nutrient medium in amounts of up to 5 vol. %, based on the volume

of the nutrient medium that is used in the fermentation stage *

used is given.

The medium used for germination and the conditions maintained for it are generally similar to those on which fermentation is based. In general, any suitable medium can be used and all conditions which lead to good growth of the ilikr or organism can be adhered to. If during germination or \ foaming occur the fermentation, a suitable defoamer, wia, for example, such a trade name Dow Corning B (The Dow Chemical Company, Midland, Michigan) were added v / ground.

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

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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 a. Reached a period of 24 - 100 hours.

As already said., The incubation of M.rosaria and the simultaneous production of antibiotic 67-694 was 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 living air through it the nutrient mediura.

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 Ras ^ n,

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such as alkali or alkaline earth metal hydroxides, preferably Sodium hydroxide. Typical organic solvents useful for the extraction are e.g. benzene, toluene, n-butanol, methylene chloride, chloroform, ethyl acetate and amyl acetate, where Ethyl acetate is a preferred solvent. The total amount of solvent depends on the relative Solubility of the antibiotic, the amount of antibiotic extracted and the the type of extraction itself. If ethyl acetate is used as a solvent, it is 4-10 times the volume of the fermentation broth necessary, with the solvent successively in amounts that are twice as much correspond to the volume of the permentation broth, is used for the extraction.

The organic extract becomes the antibiotic A

effective product e.g. in the fo] send way separately = The organic solvent is preferably im Vacuum to a volume, which is preferably 1/50 of the volume of the fermentation broth, evaporated, and is then extracted with aqueous 0.IN sulfuric acid solution. That way will too

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a separation of the antibiotic active substance achieved by the other compounds incidentally produced during fermentation. The effectiveness this separation can be achieved by determining the relative proportions of the incidentally produced compounds followed by chromatography and the extraction repeated if necessary until an extensive separation is achieved. If the extraction is repeated, then the initially prepared sulfuric acid extract made alkaline, the resulting mixture with extracted from the organic water-immiscible solvent, and the organic extract in turn extracted with O, IN sulfuric acid, is the separation of the antibiotic active substance from Once its by-products are reached, 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 subjected to vigorous

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Stirring to a diethyl ether-hexane mixture, which is present in ten times the amount, added, this Mixture consists of 6 parts by volume of diethyl ether and 4 parts by volume of hexane. The resulting precipitate is filtered off, with small * amounts of diethyl ether-hexane mixture washed and discarded.

Subsequently, the comparable with the washing liquid M

evaporated some piltrate to dryness, the residue is dissolved in an organic solvent immiscible with water, the solution becomes washed a few times with water and then over a suitable drying agent, such as sodium sulfate, dried. After filtration, the solution is evaporated to dryness and the residue dissolved in small amounts of diethyl ether. The solution

is filtered and then with stirring to petroleum ether "

with a boiling range of; 50-60 ^O C. The resulting precipitate is the antibiotic 67-69 ^ u ¹¹ ^ usually has a color that varies between white and light brown. The precipitate is filtered off, washed with a little petroleum ether and dried in vacuo at about 40.degree. 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 met ho de., By means of which the relative Activity of the antibiotic active substance is determined, a cylinder shell sample is under 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 Inc., 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 is made using the following concentrations of antibiotics 0 ig / mg) in a 0.1 molar phosphate buffer (pH 8.0) 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 17j8 + 1.0 mm against the test organism B. subtilis ATCC 6633 results.

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-to.

The antibiotic 67-69 ^, which has a test expression of about 700 ^ ug / mg, can be further agreed, for example, in the following way. The antibiotic is dissolved in a mixture consisting of nine parts by volume of halogenate, lower alkanes, such as chloroform, and one part by volume of an alcohol, such as, for example, methanol. The solution is applied to a column which contains a solid adsorbent such as silica, aluminum oxide, magnesium silicate, kieselguhr or cellulose, and the column is then filled with a mixture consisting of h parts by volume of a chlorinated, lower alkane such as chloroform. and a part by volume of 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 determined by determination with Staphylococcus aureus. In this way the output from the column can be monitored.

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 in order to localize the organic substances on the plate as dark spots. A second sample is tested on another plate against Tareina lutea, in order to determine the presence and the R _f value of the antibiotic active material. In this test, a solvent consisting of chloroform and methanol in a volume ratio of 4: 1 is used, the running time is one hour and an R n value between 0.4 and 0.5 was determined for the antibiotic 67-694. Chromatograms of the non-purified antibiotic 67-694 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 0.39 and 0.58.

Fractions containing antibiotically active material with a similar R ₊ value are combined in a vacuum

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evaporated to a residue. The residue ,, the has been obtained from those fractions which showed an R ^ value of 0.4-0.5 in acetone dissolved and added to petroleum ether. The precipitate obtained is by filtration or separated by centrifugation and discarded,., during the filtrate or the one emerging from the centrifuge Liquid evaporated to dryness. Of the The residue represents the antibiotic 67-694, which has an activity of 1000 μg / tng according to the test method described above.

E_, ie alternative method to isolate and purify the antibiotic 67-694, is given by evaporating the first extract "obtained by extracting the fermentation broth with the water-immiscible organic solvent," evaporating the residue in takes up a suitable solvent and chromatographed. Fractions., Containing the antibiotic are, _s then treated according to the method described above to win the pure antibiotic.

The cleaning of the antibiotic 67-69 *! · Can also by preparing a suitable derivative of

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Antibiotic like s.B, -aines Eaters 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., This To produce esters is to react with the corresponding acid anhydride or acid chlorides given.

The following cternal and physical properties were determined for the antibiotic 67-69 ^ which occupies an activity of 1000 ugy4ng, found:

1. Chromatography, the R _r values: The measured R fourths of the antibiotic 67-69 ^ 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 where 250 Micron thick silica gel sheets were used, under the trade name "Uniplate" (Analtech Inc. Wilmington, Delaware) using agar incubated with S. lutea found.

Table IV

solvent
(in parts by volume) R "values of the inhibition
zone against S.lutea Chloroform: methanol: Y
Ammonia 2: 1: 1 J%
O.98 Butanol: acetic acid:
Water 3: 1: 1 0O7 Chloroform: methanol
4: 1 0.45 Chloroform: methanol 0.48 II. Chemical and physical data

1. Elemental analysis found. Calculated for C H .N0 ₍ carbon 63.18 64.00

Hydrogen 8.86 8.84

Nitrogen 2.29 2.41

Oxygen (difference) 25. 67 24.75

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2. Empirical formula

3 Molecular weight:

4. Optical rotation:

5- melting point:

6. pK: ^ a

7 Neutralization:

equivalent to :

8. Ultraviolet adsorption: absorption in methanol

581 (determined in the mass spectrometer _o )

[α]? - -33- ^ ° (OO * in

Ethanol)

¹ D

110-114 ° C

p'T

599

learn

- 238

240 mu)

III. Infra-red adsorption spectrum.

The infrared adsorption spectrum of the antibiotic 67-694 in mineral oil is shown in FIG. Herein, λ means the wavelength in microns., V "means 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 figures are given in microns and those peaks attributable to the mineral oil have been omitted.

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Tabel V 8.57 (S) 2.87 (M) 9οΟΟ (S) 3-70 (W) 9-32 (S) 5 * 8θ (S) 9-55 (S) 5-92 (M-S) 9-73 (M) 6.17 (M-S) 10.17 (M-S) 10 = 37 (M) 11.15 (W-M) 7.11 (W) 11 ο 48 (W) 7 * 6θ (M) 11.87 (W-M) 7.82 (M) 11-95 (W) 12.62 (W) ? "· 9δ (W) 13-95 (W, br 8.44 (S) - IV. NMR spectrum

The NMR spectrum of the antibiotic 67-694 is shown in FIG. 2. The spectrum was obtained on a Varian A-OQ-il spectrometer (Varian Associates, Cali fornia), with a solution of 40 mg of the antibiotic 67-694 in 0.4 ml of deuterated chloroform, which also contained small amounts of deuterium oxide. The £ and f. Values are

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given in ppm, the standard was tetramethylsilane used. In FIG. 2, a partial spectrum is visible over the total spectrum, that of the total spectrum appear at 9 · 8 ppm and therefore outside of the shown Part of the spectrum. The intensity of this peak gives rise to the assumption that that this seems to be important for the interpretation of the spectrum.

V. Solubility

Solvent solubility

Very little soluble in water

Methanol readily soluble

Acetone readily soluble.

Chloroform readily soluble

Benaol readily soluble

Biethyl ether sparingly soluble.

The solubility information is from US. Pliarniaeopea, 17th edition, (± 965) page 3, taken from «

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 set to temperature "- ■"> 100 ° C for 30 minutes e r} 1 i t ζ t mird e.

VII. Stability to Enzymes

The stability of the antibiotic 67-694 was tested against trypsin, chymotrypsin, pepsin and α-anylase. Solutions of enzymes with a concentration of 1 mg / ml in Mcllvaine's 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 for α-amylase, two hundred (200) / ig of the antibiotic was dissolved in 0.3 nil water with 0.5 ml dt Mix the enzyme solution and incubate at 37 ° C for 24 hours. The solution was tested against Esc-Aerichia coil and Staphylococcus aureus after 0, 1, 2.5 and 24 hours = the enzymes could not cause any significant decrease in the activity of the antibiotically active substances during this period.

VIII. Color test

The antibiotic 67-694 gives a positive result

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351

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

The IR adsorption spectrum, the NMR spectrum, and the chemical and physical properties suggest that the antibiotic 67-69 ¹ J is a macromolecule with a tertiary amino group. It also has at least two esterifiable hydroxyl groups, one of which is selectively esterifiable. The NMR spectrum also suggests an aldehyde group. Rosamicin (antibiotic 67-694) has the following area formula

22nd

xr

K (CH ₃ )

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According to the invention, those which can be used pharmaceutically can also be used (non-toxic) derivatives of the antibiotic 67-694, such as the pharmaceutically acceptable salts and esters, as well as their salts, are produced. Typical salts are acid addition salts made with inorganic acids such as hydrochloric acid, sulfuric acid or phosphoric acid or reactive derivatives thereof, 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, dihydroxysuccinic acid, glutaric acid, lauric acid, adamantanic acid, cinnamic acid, camphor acid, Palmitic acid, stearic acid or undecylenic acid. These salts can be made by adding the Antibiotic 67-694 or an antibiotic active substance, which contains this antibiotic or which is contained in Antibiotic 67-694, or reactive Derivatives thereof, with the acid or reactive derivatives thereof, can react and the resulting salts isolated. Typical pharmaceutically acceptable 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. Also esters of isomeric acids thereof, e.g. Isobutyric acid, are possible. The esters can be

709826/0815

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December 7, 1976 982-12 (iermaay IK / cf

esterification of the antibiotic 67-694 or antibiotic active substances that contain it, or are contained in antibiotic 67-694, or reactive derivatives obtained therefrom with suitable acid anhydrides, acid chlorides or other equivalent compounds will. The pharmaceutically acceptable esters can be in the form of monoesters or beasts or exist as mixed esters.

In vitro test

The in vitro tests for the antibiotic 67-694 with an activity of 775 µg / ing were determined according to the usual Dilution method carried out using a yeast broth medium 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.

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982 - PTG-3 ⁱ i CG-ug

Organisms Table VI

try

Staphylococcus aureus 6

Streptococcus pyogenes 7

Enterococcus sp. 2

Diplococcus pneumoniae 3

Escherichia coli 3

Klebsiella pneuraoniae 4

Aerobacter aerogenes 3

Proteus sp. 3

Pseudomonas aeruginosa 4 Salmonella schottmuelleri Minimum inhibition concentration ^ /

0.03-3.0

O.75-3.O

O.O3-O.O8

O.O3-O.O8

3.0

0.3-3 * 0

0.75-7.5

0.3

O.75-3.O

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709826/081

98P.-FTG-35
CG-Ug

In these trials the antibiotic was 67-694 with an activity of 775 µg / mg was used and the results were based on an activity of 1000 µg / mg converted.

In vivo test

This became one with the antibiotic 67-694

Wk activity of 1000 Ug / 'mg in male CP-I mice

with an average weight of 20 g
carried out. The antibiotic was supplied as a suspension or solution in an aqueous vehicle
O ₃ contained 5 % carboxymethyl cellulose in two
Doses entered, one just before and one
4 hours after intraperitoneal infection by the bacteria. Infected untreated mice
(Control tests) 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.

-35-709826 / 0815

organism

Staphylococcus
aureus gray

Streptococcus
pyogenes C

Dec-28-1970 582-iTG -36 CG-ug

Table VII

Verabreich u ng

Subcutaneous Oral

Subcutaneous Oral

Activity against lethal infections in mice

(mgAg )

50 200

50 200

III. Lethal dose

The lethal dose, expressed as the LD of the antibiotic 67-69 ^ is given in Table VIII.

Table VIII Administration route

Intraperitoneally

Subcutaneous

Intravenously

(mg / kg)

350
625
155

The antibiotic 67-694 and the pharmaceutical Usable derivatives thereof can be used alone or in Combination with other antibiotic compounds used to help growth or the number of organisms, particularly those of the gram-positive organisms, as reported in Table VI are to decrease. The antibiotic 67-694

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709826/0815

Dec-28-1970

982-FTG-37

CG-ug

and the pharmaceutically acceptable derivatives thereof can be used, for example, in washing solutions for sanitary purposes, for cleaning laboratory objects and to other cleaning purposes, such as bacteriostatic. Dish soap can be used for laboratory laundry. The antibiotic 67-694 and the pharmaceutical Usable derivatives thereof can also be used for the treatment of laboratory and domestic animals that are with Organisms are infected.

709826/0815

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Pec-28-19T0 S'82-FTG- 33 CG-ws

In the following examples which illustrate the invention, are the incubation of Micromonospora rosaria, the manufacture of the antibiotic 67-694 ,, its isolation and purification, the preparation of various derivatives of the same and compositions which contain this "antibiotic" described.

example 1

Fermentation in shake flasks A. Germination stage :

In a 5OO ml Erlenmeyer flask, the more sterile The content has the composition given below and is 100 ml, are under aseptic Conditions approximately 0.5 ml of a slant or lyophilized culture of Micromonospora rosaria given:

709826/0815 - -58 -

982-FTG-39
CG-vrs G. Bacto meat extract (Difco) G- Bacto-Tryptose (Difco) 5 Bacto yeast extract (Difco) 5 barrel Dextrose 1 G. Potato starch 2h G. Calcium carbonate 2 ml tap water 1000

.0

The contents of the bottles are at 35 c 72 hours incubated with shaking.

B. fermentation stage ;

5 ml each of the inoculum obtained in the germination stage are transferred under aseptic conditions into some 5OO 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. Strength 3 ⁿ S-

Calcium carbonate .1 S *

Tap water 1000 ml.

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

_ 39 _

709826/0815

982-FTG-4O CG-v / s

Make the first 24 hours periodic from 21 © 7020 the fermentation broth samples are drawn to the antibiotic activity and thus that point in time determine at which the antibiotic activity of the medium reaches its maximum value. As soon as this is present, the contents of the bottles are pooled for subsequent isolation- while During germination and fermentation, the pH of the nutrient medium is increased by the buffering effect of calcium karbonaf adhered to at the desired height. The fermentation is under submerged aerobic Conditions carried out.

Example 2

Fermentation in the tank A. Germination stage :

25 ml of an inoculum, prepared according to. example IA, under aseptic conditions, are 500 tnl 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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B. Fermentation stage :

The product obtained at the germination stage will be under aseptic conditions in a 14 l fermentation vessel transferred, which contains 9.5 1 of the medium described in Ball Game IB.

6 ml of a defoamer (Dow-Corning B) are added and the pH value of the mixture decides adjusted to 7.0 with dilute sodium hydroxide solution (In) or dilute sulfuric acid (in). The mixture is fermented at 28 C until maximum antibiotic activity is achieved (usually after 72 to 100 hours). The aerobic conditions are achieved by passing through Air (ca 3.5 l / fain.) Is reached.

Example 3 Isolation of the antibiotic

The pH of the fermentation broth obtained according to Example 2 is adjusted to 9.5 with sodium hydroxide solution and then extracted with ethyl acetate in individual amounts of 20 l until the substantial amounts of the antibiotic have been removed from the broth. This generally requires 2 to 5 extractions. The combined extracts

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Dec-28-1970

982-FTG-A2

CG-wö

. 21G7020

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 becomes 100 ml of a vigorously stirred diethyl ether-hexane mixture (6: 4 parts by volume) given. The resulting precipitate is filtered off with small amounts of diethyl ether-Hexari mixture washed and discarded. The filtrate combined with the Viaschiquid becomes in vacuo evaporated to a solid residue. This residue is dissolved in about 5 ml of diethyl ether Solution filtered and with stirring to 50 ml petroleum ether (Boiling range 3Ο-6Ο C) given. The one with it the resulting precipitate, which has a shade between white and light brown, is filtered off, washed with a little petroleum ether and dried in vacuo at 40.degree. The antibiotic thus obtained active product (impure antibiotic 67-694-)

709826/0815

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 5 is dissolved in a chloroform-methanol mixture (9: 1 parts by volume) and the resulting solution placed on a column, the 250 g of silica contains. The column is with a Chlorofonu - Methanot mixture (8: 1 parts by volume) eluted, with fractions of 5 ml at a dripping rate of 0.5 ml / min. A sample is taken from each fraction and part of it tested against Staphylococcus aureus. Another part is chromatographed on silica gel ~ GF plates, the thickness of which is 250 microns (the plates are known as "üniplate" from Analtech Inc., Wilmingten, Delaware, USA). Fractions covering the antibiotic 67-694 contain, show an R value of approx. 0.4 to 0.5 «These are combined and in a vacuum to one

709826/0815

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Dec-28-19Y0 932-FTG-44

Evaporated residue. The residue is dissolved in acetone, dietary 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 ng / mg and the following key figures:

Melting point = 110 [a [^ ⁵ = -33 ^ ° (0.3fo in ethanol) E ^ _cm = 238 (at 240 mu) JR and NiVIR spectrum as in FIGS. 1 and 2. The other chemical and physical properties are identical to those given above.

7 0 9 b "2" ö / 0 8 1 p

Dec-28-1970

982-?

CG-ws

Example 5

Alternative isolation and cleaning process

The pH of the fermentation broth obtained according to Example 2 is adjusted to approx. 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-methanoi-genetic (8: 2 parts by volume) fractions of 5 ml each are collected. Some of the Xtf samples taken from the individual fractions are used for determination against Staphylococcus aureus and a further part for thin-layer chromatographic determinations. After the fractions with similar R values have been combined - those containing the antibiotic 67-69 ² J- have a value of approximately 0.4 to 0.5 - the solvent is evaporated in vacuo. The residue will

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982-FTG-46

CG-ViS

dissolved in acetane and diethyl ether is added. A precipitate is formed, which is obtained by filtration or centrifugation is separated and discarded. The filtrate or the centrifugation supernatant liquid obtained is evaporated to dryness in vacuo. That on this Well-obtained antibiotic product is the antibiotic 67-694 with an activity of approx. 1000 ^ g / mg and has physical characteristics., as given in Example 4.

Example 6

Potassium hydrogen phosphate salt of the antibiotic 67-694

To 5OO mg of antibiotic 67-694 · in Γ3 ml of water, 1250 mg of calcium iodine hydrogenphosphate are added 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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982-F

CG-ViS

If the method just described is used and equivalent amounts of acids are used in Water have a similar solubility, such as hydrochloric acid, sulfuric acid, phosphoric acid, Lactic acid ,, monohydroxysuccinic acid, Acetic acid, succinic acid, citric acid, malonic acid or maleic acid, so obtained the corresponding pharmaceutically acceptable acid addition salts. Generally have these salts in vitro and in vivo have a similar effect, sometimes even an identical antibacterial spectrum as the free base. If there are differences, so can these can be attributed to the different solubilities. Because of their good solubility properties in aqueous media these salts are particularly good for ivaqueous compositions and solutions, intended for oral and parenteral administration are suitable.

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De.c-28-1970 982-FTG-48

Example 7 Tartr rate of the antibiotic 67-69 ^

1 g of antibiotic 67-69 ^ is dissolved in 6O ml of diethyl ether and a solution of 250 mg of dihydroxysuccinic acid in 1.5 ml of ethanol is added. The mixture is stirred for 15 minutes and then the precipitated salt is filtered off. After washing the same, it is dried to obtain the desired product.
Melting point: 129 -

[α] ρ ⁰ = -12 ° (Ο, 35ί in water)

If you use the method just described and use equivalent amounts of acids in a Ether-alcohol mixture have a similar solubility, such as "e.g. glutaric acid, lauric acid, adamantic acid, Cinnamic acid, camphoric acid, palmitic acid, stearic acid or undecylenic acid are obtained in this way the corresponding pharmaceutically acceptable acid addition salts. Generally these are due their molecular weights and solubility properties are particularly good for use in topical compositions, such as creams and ointments.

709826/0815

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Dec-28 Γ / 7 υ 982 FTG-49

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, in order 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
[a] ^ ⁰ = p -25 ° ( 0.3% in ethanol)

Example 9 Monoisovalerate of the antibiotic 67-69 * 1

2.0 g of the antibiotic 67-694 are in 10.0 ml Dissolved anhydrous acetone and 0.65 ml of isovaleric anhydride v / earth admitted. The mixture is stirred at room temperature for 16 hours, then is slowly and with continued stirring a 1,2 ^ Ammonium hydroxide solution added to precipitate the product. The precipitate is filtered off with

709826/0815 -49-

000- ^ 8-1970 982-FTG-50

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

Instead of the acid anhydrides used in Examples 8 and 9, equivalents are used 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 one works according to Examples 8 and 9, the corresponding monoesters of the antibiotic 67-69 ^ ·

Example 10 Diacetate of Antibiotic 67-69 ^

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 4 days at room temperature ditched. The solution then becomes dry in vacuo at temperatures up to 35 ° C evaporated, the residue dissolved in acetone and the

709826/0815

-50-

982-FTG-5I

• Λ.

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 1θ4-107 ° C.
[α] p ° ^ -24 ° (0.3 $ in ethanol).

Example 11 Antibiotic dibenzoate 67-694

250 mg of the antibiotic is dissolved in 67-694> ml of pyridine and ₅ O 25 ml of benzoyl chloride was 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-β94.
Melting point 116-119 ° C.
[a] Jp = + 20 ° (0.3 # in ethanol)

It can easily be seen that ^ as soon as a certain monoesters of the antibiotic react further with another acid anhydride or acid

709826/0815

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DeC'23-1970 982 -FTG-52 IK / cf

ft.

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 by 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, Disuccinate, Diphthalate, Diphenyl acetate, Diphenoxyacetates, 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

709826/0815

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December 7, 1976 982-12 Germany IK / cf

Example 12

50.0 g of the antibiotic 67-69 ¹ I are dissolved in 700 ml of acetone and 13.1 g of propionic anhydride (1.2 m / m) are added. The mixture is stirred for 26 hours at room temperature and then the solvent is removed under reduced pressure and the residue is triturated with dilute ammonium hydroxyl solution for about 10 minutes. The residue is collected by filtration and washed with distilled water. The precipitate is then triturated again with distilled water for about 10 minutes and the residue is then collected by filtration and washed with distilled water. The product is dried at 45 ° C. under reduced pressure for 24 hours. Melting point of the monopropionate of the antibiotic 67-694 128 to 130 ⁰ C Γ «1 -24.2 ° (C = 0.3 I ethanol).

^{f D}
Phosphoric acid addition salt of / V ^ -22 ° (C = 0.3% water),> methanol _{2kq m (£. 13 6oo).}

Dodecyl sulfate T ^^ ³ ^ 240 nm (i 14,000).

709826/0815

- 53 -

Claims (1)

December 7, 197G 9 & 2-12 Germany IK / cf Claim Process for the preparation of rosamicin (antibiotic 67-694) of the formula 22nd H (GH ₃ ); characterized in that _s 3718 3 in a known manner in an aqueous Nährmed.ium under aerobic, preferably submerged incubated conditions and the antibiotic obtained in a pharmaceutically acceptable a microorganism of the species Micromonospora rosaria, particularly of the strain NRRL in free form or in the form Salt or ester isolated. 709826/0815