DE1208449B - Process for the manufacture of the antibiotic ussamycin - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. α .:

C12d

German class: 30 h -6

Number: 1208

File number: C 32458IV a / 30 h

Filing date: March 20, 1964

Opened on: January 5, 1966

The invention relates to a new water-soluble antibiotic, which is described below with ussamycin is called, and its salts.

The antibiotic usamycin is produced in the culture of a new strain of Streptomyces lavendulae, which was obtained by irradiating Streptomyces lavendulae 7 Kl with ultraviolet light and is called Streptomyces lavendulae 7 Kl mutant UV-9. The new strain is kept under this name in Universidade de Recife, ίο Institute) de Antibioticos, Recife (Brazil). The strain S, lavendulae 7 Kl and its mutant UV-9 differ morphologically: According to the classification of P ridham et al. (1958) S. lavendulae 7 Kl belongs to section Rectus-flexibilis (spore chains straight, monopodially branched, diameter 0.3 to 1.1 [X), but the mutant UV-9 belongs to section Spira (spore chains in open spirals from 10 to 20 μ length and 4 to 6 μ diameter). S. lavendulae 7 Kl forms round spores with a diameter of 0.8 to 1.1 μ, mutant UV-9 forms oval spores with a diameter of 1.5 to 1.8 μ. Table I shows a comparison of the growth characteristics of S. lavendulae 7 Kl and its mutant UV-9 on different nutrient media. Process for the manufacture of the antibiotic ussamycin

Applicant:

CIBA Aktiengesellschaft, Basel (Switzerland)

Representative:

Dr.-Ing. Dr. jur. F. Redies, Dr. rer. nat. B. Redies and Dr. rer. nat. D. Turk, Patent Attorneys, Opladen, Rennbaumstr.

Named as inventor:

Oswaldo Goncalves de Lima, Marisa Machado Albuquerque, Lizete Oliveira, Jose Sidney de Barros Coelho, Francisco Decio de Andrade Lyra, Vilneyde Mabel Queiroz, Dipl.-Landw. Carlos Antonio Albert, Ivan Leoncio Albuquerque, Ary Lacerda, Recife (Brazil)

Claimed priority:

Switzerland of March 27, 1963 (3906), of April 24, 1963 (5162)

Table I.

Culture medium

UV-9 7cl

Starch agar

Glycerine asparagine

A at all

Glycerin Peptone Agar

Glycerine Ammonium Agar

Gelatin Aear

Glucose-Asparagine-A gar

Breeding ground for
Melanin formation

White aerial mycelium, soluble pigment, I withered brown

White aerial mycelium, which later turns gray. No soluble pigment

White aerial mycelium is later gray. Soluble pigment

1 Vegetative cream-colored growth. Aerial mycelium is absent. No soluble pigment

Colorless brilliant vegetative growth that deepens in the middle. Lack of Liiftmycels

Colorless vegetative growth. Sparse aerial mycelium, which turns gray. No pigment

No melanin. Soluble pigment that turns brown. Pink aerial mycelium. Not soluble pigment

White aerial mycelium, which turns pink. No soluble pigment

White aerial mycelium. No soluble pigment. White aerial mycelium. No soluble pigment

Colorless, brilliant vegetative growth without deepening in the middle

White aerial mycelium that turns pinkish in color. No soluble pigment

Melanin formation

509 777/408

Table I (continued)

Culture medium UV-9 7cl Glucose agar Cream-colored vegetative growth.
No soluble pigment Light gray aerial mycelium. Pigment stains
the breeding ground Starch Casein Agar White aerial mycelium that later turns gray White aerial mycelium, which later turns pink
colors Potato slices White aerial mycelium that is gray in color
accepts. No soluble pigment Tannin-colored vegetative growth.
Absence of the air mycelium. Soluble
Pigment is missing Nutrient agar Colorless vegetative growth. Aerial mycelium
is missing. Soluble pigment that denotes the
The culture medium turns yellow Light brown vegetative growth.
Aerial mycelium is absent. Soluble brown
pigment Czapek White aerial mycelium, which later turns rat gray
will. Greenish soluble pigment White aerial mycelium, which later
turns pink Glycerine calcium
Agar Sparse white aerial mycelium. No
soluble pigment White aerial mycelium that turns pink Potato glucose
Agar White aerial mycelium, which then turns gray.
No soluble pigment White aerial mycelium, which is brightly cycled
pigment is absent Potato glycerin
Agar White aerial mycelium that later turns gray.
Soluble brownish pigment Aerial mycelium initially gray, then pink.
Pigment light brown

Table II shows the chemical behavior of the two cultures, Table III the utilization of different ones Carbon sources.

Table II

Substrate nitrate Mutant UV-9 S. lavendulae 7 cl gelatin Strong reduction Moderate reduction Litmus milk Superficial cuticle-like growth.
No aerial mycelium. No pigment formation.
Total liquefaction on the 15th day Developing in the form of a brown
Ring. No aerial mycelium. Soluble
brown pigment. Total liquefaction
on the 15th day strength Development on the surface with whiter
Aerial mycelia. Total peptonization
on the 20th day. Alkane linearization 6.4 to 8.0.
No pigment formation and none
Coagulation Developing in the form of a brown
Ring, without aerial mycelium. Total pepto
nization between the 15th and 20th day.
Brown soluble pigment. Easy
Alkalinization. There is no coagulation Strong hydrolysis as early as the 10th day Strong hydrolysis but slow
(weak 10th day, strong 20th day)

Table III + carbon
source Sucrose Lactose Raffinose +
+ Ramnose Xylose Salicin Mannitol Calcium malate ... growth
Mutant UV-9 | S. lavendulae 7 Cl J

55

6o

It is known that the original strain Streptomyces lavendulae 7 Kl a first as Eurymycin called antibiotic, but it turned out to be identical to neomycin. That from

Strain S. lavendulae 7 Kl, mutant UV-9, formed antibiotic ussamycin is biological in terms of its and physicochemical properties of neomycin and its components are different. In acid hydrolysis, for example, it supplies amino acids, so it is presumably a polypeptide. Im ascending Paper chromatogram in the system n-butanol — pyridine — acetic acid — water (15:10: 3:12) it shows an Rf value of 0.74 while using neomycin stains Has Rf values> 0.1 and another with Rf = 0.13 to 0.14. With descending chromatography in the system water-saturated n-butanol with the addition of 2% toluenesulfonic acid is found for ussamycin an Rf value of 0.75 and for neomycin from 0.25 to 0.27.

Other antibiotics obtainable by cultivating Streptomyces lavendulae are known, such as Streptothricin, streptin, lavendulin, antibiotic 136,

5 6

Streptolin, streptothricin VI and valacidin. This decomposition occurs. In acidic hydrolysis antibiotics differ from usamycin in (6-n-hydrochloric acid, 20 hours, 100 ° C) a crude product in the following way: Streptothricin gives off acidic hydrolysis of usamycin, a mixture of amino hydrosis 3 ninhydrin-positive substances that do not contain acids , some of which are on paper chroinato- \ amino acids (cf. Korzybski and Ku- 5 graphischem way provisionally identified as follows rylowicz, "Antibiotica", 1961, p 408, lines 9: glycine, alanine, leucine, serine , Asparabis 12), namely / i-lysine, an amino sugar and a gic acid, glutamic acid, arginine, lysine.
2-Amino-imidazolidine derivative, while ussamycin is used to produce the antibiotic ussamycin

the acid hydrolysis ^ amino acids, as in the strain S. lavendulae 7 Kl, mutant UV-9, is carried out in the exemplary embodiment. Streptin was produced in aqueous form, a carbon and nitrogen source and only as a concentrate, the substance itself was grown with nutrient solution containing inorganic salts, was not isolated and chemically characterized in more detail until it had a significant antibacterial effect (cf.Merck Index, 7th edition, 1960, P. 980). Contains streptin, and the ussamycin is then isolated,
is effective against mycobacteria (cf.W aksma η Carbon and nitrogen sources are e.g.

and Lechvalier, »Actinomycetes and their anti-15 carbohydrates, such as glucose, sucrose, lactose, biotics ", Baltimore 1953, p. 227; Woodruff et al., Starch, alcohols such as mannitol, glycerin, amino acids, J. Bacteriol., 52, p. 502 [1946]) and therefore certainly not e.g. glycine, peptides, proteins and their breakdown identical to ussamycin, which is anti-mycobacterial products such as peptone or tryptone, meat extracts, is not active, as shown in Table 1 below, water-soluble fractions of cereal grains, such as maize can be seen. In contrast to ao or wheat, lavendulin gives distillation residues of the alcohol usamycin a negative Sakaguchi reaction (cf. manufacture, Cornsteep liquor, yeast, seeds, especially Korzybski and K u r y 1 0 w i c z, a. a. Cit., P. 419, those of the rapeseed and soy plants, the cotton plant, last line), so does not have any guanidino groups on ammonium salts, nitrates, in question. Inorganic like ussamycin, which contains the amino acid arginine. The nutrient solution contains salts, for example chlorides, Antibiotic 136 was not isolated in pure form 25 carbonates, sulfates, nitrates, phosphates of alkali (cf. B o h o η o s et al., Arch. Biochem., 15 [1947], and alkaline earth metals, from Magnesium, Zinc, Man-S. 217, line 2), but should, in its chemical property, be iron.

be similar to streptothricin (cf. loc. cit., cultivation takes place aerobically, for example in

P. 215, line 9), which, as stated above, is very dormant surface culture, or preferably subdivided from ussamycin. Antibiotic 136 undergoes shaking or stirring with air or separates itself through its antibacterial spectrum, oxygen in shake flasks or the known ones in particular through its effectiveness against ferments. As the temperature, such an acid-resistant microorganisms (see ibid., P 219) is between 18 and 4O ⁰ C, preferably 27 ⁰ C. Vorzugsvon Ussamycin. Streptolin supplies in the acidic way one cultivates in several stages, ie one hydrolysis among other things / 3-lysine and an amino 35 first a culture in a liquid nutrient medium sugar (cf. Korzybski and Kurylowicz, then in the ratio of about 1:20 in the loc. Cit., P. 414, lines 15 and 16). These substances actual production medium is inoculated. The first liquid culture obtained in the acid hydrolysis of ussamycin is obtained, for example, by not obtaining. Streptothricin VI, also called S VI, is inoculated into a culture grown on potato nutrient medium (cf. Hutchison, Swart and W aksma η, 40 from 8 to 10 days of age in the liquid nutrient medium cited in Korzybski and Korylowicz, allowed to grow for 48 hours and loc. cit., p. 411), has been found to be identical to Strepto- then to the production medium,
thricin proved (S wart, J. Am. Chem. Soc, 71, The isolation of the antibiotic from the culture

P. 2943 [1949]). Valacidin is not a peptide antibiotic filtrate is carried out according to methods known per se. like ussamycin because it has a negative ninhydrin reaction. 45 Adsorbents can be used, e.g. B. Active gives (See German Auslegeschrift 1 112 608, column 3, carbon, such as Norit, activated earth, such as Fuller earth Line 37); it also differs by or floridin, or resin adsorber, such as asmit. the its reddish-brown color (loc. cit., column 1, line 50) elution of the adsorbates is expediently carried out with of the colorless ussamycin. Mixtures of organic miscible with water

Ussamycin is an organic base and forms 50 solvents with water or aqueous acids, according to salts. In the form of the hydrochloride it is e.g. B. with water — methanol, dilute hydrochloric acid— very soluble in water. It is also soluble in methanol. Methanol (pH 2.0), dilute acetic acid — methanol, In acetone and other organic solvents, water — methanol — glacial acetic acid — butanol. One can like ether, benzene, chloroform, petroleum ether, it is the antibiotic also on ion exchange resins practically insoluble. It can adsorb with sulfonic acid groups, especially with those containing acid groups Azo dyes such as helianthin like resins such as Amberlite IRC-50. The elution will. It also forms a Reineckat. The antibiotic takes place, for example, with dilute acids or kum Ussamycin has the same purity as it has previously been possible with mixtures of methanol and dilute acids, could be obtained a light yellow color, which further can also take the antibiotic directly from it

remains unchanged between pH 3 to 8. The following 60 are precipitated from the culture filtrate, e.g. B. in the form of the color test are negative: Maltol, Glucosamine, Molisch, Reinneckates. The transfer of the poorly soluble Millon. The ninhydrin and sakaguchi tests are salts in easily soluble salts of the antibiotic becomes positive. The antibiotic is relatively stable: a solution that is strong either with mineral acids or with ion exchange acid can resin for 10 minutes on the water bath, e.g. B. on Amberlite IRA-400, carried out, heated, and alkaline solutions remain at 65. This method can also be used to enrich the room temperature for 30 minutes. Will antibiotic be used
on the other hand neutral solutions or solutions in 5% iger An enrichment of the antibiotic is also

Sodium hydroxide solution heated on a water bath for 30 minutes, achieved by using aqueous or alcoholic

aqueous solutions of the salts with an excess of organic, water-miscible solvents, such as acetone, dioxane, added, whereby inorganic salts and other accompanying substances are precipitated and eliminated will.

Another enrichment and purification method is chromatography, e.g. B. Adsorption chromatography on various materials, such as Norit, aluminum oxide, magnesium silicates, as well as partition chromatography with cellulose, starch, Silica gel as a carrier or chromatography on ion exchange resins, e.g. B. at Dowex-50, Amberlite IRC-50.

Furthermore, the antibiotic can be mixed between two immiscible ones by countercurrent distribution according to Craig Solvent phases are enriched.

The salts of the antibiotic and its components are derived from inorganic or organic Acids, for example from hydrochloric acid, sulfuric acids, phosphoric acids, nitric acid, acetic acid, Propionic acid, valeric, palmitic or oleic acid, succinic acid, citric acid or mandelic acid. Her Production takes place by the action of the corresponding acids on the free base or by doubling them Implementation of salts.

The antibiotic ussamycin and its salts have a high antibiotic effectiveness against it gram-positive microorganisms, a smaller one compared to gram-negative microorganisms and none Effect against acid-resistant microorganisms in a concentration of up to 100 μg / ml (see Table 1).

Table 1
Antimicrobial spectrum of the antibiotic UV-9

Microorganisms activity
(y / ml) B. subtilis 9
B. subtilis 27
B. anthracis
B. mycoides
B. cereus
S. aureus W
S. aureus ATCC
M. citreus
Sarcina lutea
St. haemolyticus
K. pneumoniae
S. typhosa
E. coli N
Sh. paradysenteriae
A. aerogenes
N. catarrhalis
Pr. Morganii
Pr. Mirabilis
Pr. Vulgaris
Br. Suis 0.1 to 0.2
5.0 to 10.0
> 100.0
> 100.0
> 100.0
0.2 to 0.4
0.2 to 0.4
2.0 to 4.0
0.2 to 0.4
6.0 to 8.0
20.0 to 40.0
15.0 to 20.0
20.0 to 40.0
15.0 to 20.0
60.0 to 80.0
8.0 to 10.0
60.0 to 80.0
80.0 to 100.0
60.0 to 80.0
4.0 to 6.0
4.0 to 6.0
4.0 to 6.0
> 100.0
> 100.0
> 100.0
40.0 to 60.0
> 100.0
40.0 to 60.0 Br. Melitensis
Br. Abortus
Mycobacterium 607
Mycobacterium phlei
Mycobacterium smegmatis
N. asteroides
C. albicans IBB-50
Cr. neoformans ENCB

In vivo, the antibiotic has an anti-tumor effect against the carcino-sarcoma Walker 256 when tested on white rats (Sprague Dawley). The toxicity of the new antibiotic is low. In white rats given 50 mg / kg of the antibiotic intraperitoreally daily for 10 days no abnormality was noticed.

The antibiotic ussamycin and its salts can be used as remedies in veterinary and human medicine, z. B. in the form of pharmaceutical preparations, use. These include the above Compounds mixed with a pharmaceutical suitable for enteral or parenteral administration organic or inorganic carrier material. For the same, such substances come into consideration, who do not respond to the new connections, such as B. gelatin, lactose, starch, magnesium stearate, vegetable oils, benzyl alcohol, or other known excipients. The pharmaceutical Preparations can e.g. B. as tablets, dragees, powders, suppositories or in liquid form as solutions, Suspensions or emulsions are present. If necessary, they are sterilized and / or contain auxiliary materials, such as preservatives, stabilizers, wetting agents or emulsifiers. You can have others too Contain therapeutically valuable substances. The antibiotic can also be used as a disinfectant and preservative as well as be used as feed supplement.

The invention is described in the following example. The temperatures are in degrees Celsius specified.

example

a) Streptomyces lavendulae UV-9 is made according to the method of two stages under aerobic conditions grown in a nutrient solution containing 30 g of glucose per liter of tap water, 12.5 g of Cornsteep liquor (50%)> Contains 5 g of dry brewer's yeast, 5 g of ammonium nitrate and 4 g of calcium carbonate. The sterilized nutrient solution shows a pH of 6.5 to 6.8. The inoculation is carried out with a culture which is grown in potato nutrient medium in 125 ml Erlenmeyer flask is 8 to 10 days old. One eighth of this culture will be Transfer each into Fernbach bottles of 2000 ml content with 300 ml nutrient solution. After 48 hours Incubation with shaking (approx. 200 rpm), the liquid cultures in a ratio of 1:20 are poured into the Production medium inoculated.

b) In the same way as described under a), S. lavendulae UV-9 is grown in a nutrient solution, the per liter of tap water 20 g soy flour, 20 g glucose, 5 g sodium chloride and 2 g calcium carbonate contains. After sterilization, the pH is 6.7 to 6.9.

The culture filtrate obtained according to a) or b) is adsorbed on activated charcoal and eluted with methanol which has been acidified with hydrochloric acid to pH 2.0. The eluate is brought to pH 6, concentrated in vacuo to the syrup thickness, filtered and then diluted three times with methanol. Then acetone is added in excess. The precipitate is filtered off, the filtrate is acidified (pH 2.0) and evaporated to a syrup thickness. An active crude product precipitates out, which is washed with acetone, dissolved in water and precipitated as Reineckat using Reineckesalz. This is filtered and washed several times with water of 60 to 70 ⁰ C, said reineckate goes into solution. The insoluble residue is filtered off and cooled

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Filtrate on OCab. After standing for several hours at 0 ° C, the pure sugar precipitates out as a gel-like precipitate. This is converted into the hydrochloride in a known manner. This is a cream-colored substance that does not undergo any changes in aqueous solution at a pH of 3 to 8. When a neutral solution is heated to 100 ^° C. for 30 minutes, the substance largely loses its activity; It becomes almost completely inactive if it is ^{heated to 100 °} C. for 30 minutes in 5% sodium hydroxide solution.

The maltol, glucosamine, Molisch and Millon reactions are negative, the reaction according to Sakag u c h i and positive with ninhydrin.

In the paper chromatogram in the system n-butanol - pyridine - acetic acid - water (15:10: 3: 12), ascending Development, 20 hours on Whatman no. 1 paper, the Rf value is 0.74. With a downward trend in the system water — saturated n-butanol + 2% toluenesulfonic acid, the Rf value is 0.75.

In the acid hydrolysis (6 N hydrochloric acid, 20 hours at 100 ° C) amino acids are obtained. In two-dimensional System sec-butanol - formic acid - · Water (75: 15: 10) = I and phenol — water (80:20) = II the following amino acids can be detected by staining with ninhydrin: glycine, alanine, Leucine, serine, aspartic acid, glutamic acid, arginine and lysine.

Claims (1)

Claim: Use of the UV-9 mutant from Streptomyces lavendulae 7 Kl for the production of the antibiotic Ussamycin by the usual biological submerged cultivation, extraction of the antibiotic from the culture filtrate and pure preparation. Considered publications:
Korzybski and Kurylowicz, "Antibiotica", 1961, pp. 406 to 411 and 419. 509 777 / 4CS 12.65 © Bundesdruckerei Berlin