DE1012032B - Manufacture of Angolamycin - Google Patents

Description

GERMAN

The invention relates to the production of a new crystalline antibiotic, hereinafter referred to as "Angolamycin" · is called, its extraction, purification and the production of its salts.

Angolamycin is a base and, when crystallized from a benzene-ether mixture, forms colorless crystals that melt at 133 to 136 °. Angolamycin crystallizes from certain solvents, for example from diisopropyl ether or dioxane, in a second, needle-shaped crystal modification, m.p. = 165 to 168 °. The values of the elemental analysis indicate the gross formula C _{50 ± 1} H _{89 ± 2} O ₁₈ N. Calculated using this formula, angolamycin contains three methoxy groups, two N-methyl groups, at least eight C-methyl groups, determined using the Kuhn-Roth method, and five active hydrogen atoms (Zerewitinoff). Angolamycin is optically active, [a] ² J = - 64 ° in chloroform, and has a strong band in the ultraviolet at 240 ταμ (10g £ = 4.16 in ethanol). In the infrared spectrum, recorded in Nujol, bands appear at 2.93 μ, 3.53 μ, 5.84 μ, 5.92 μ, 6.15 μ, 6.81 μ, 7.06 μ, 7, among others , 25μ, 7.59μ , 7.87μ, 8.16μ, 8.56μ , 9.16μ, 9.34μ, 10.02μ , 10.29μ , 11.00μ , 11th , 91 µ, 12.66 µ and 13.20 µ. When dissolved in methyl cellosolve water (80:20), angolamycin has a pK value of 6.74. In the case of hydrogenation in ethanol with a palladium-calcium carbonate catalyst, 1 mole of hydrogen is taken up, while in glacial acetic acid with a platinum oxide catalyst, 3 moles of hydrogen are consumed.

Angolamycin bears some similarity to carbomycin and in terms of physical constants with erythromycin on. From carbomycin it can be easily identified by means of the color reactions of Fischbach and Le vine (Antibiotics and Chemotherapy, 3, p. 1158 [1953]). Angolamycin initially gives one dirty yellow, later a brown color, while with carbomycin under the same conditions a purple color until a purple color is obtained.

It differs from erythromycin, except in terms of UV absorption, in its paper chromatography Behavior. In the solvent system methanol (19 parts by volume) - acetone (6 parts by volume) - water (75 parts by volume) the following Rf values were found: Angolamycim 0.78, Erythromycin 0.86. Another difference between angolamycin and erythromycin can be seen in the Hydrolysis of these antibiotics with dilute mineral acids by the resulting, reducing cleavage products behave differently according to paper chromatography.

The salts of angolamycin are derived from the known ones inorganic and organic acids, for example hydrochloric acid, sulfuric acids, the production of acetic, propionic, valeric, palmitic or oleic acid, succinic acid, citric acid, mandelic acid, gluta of angolamycin

Applicant:
CIBA Aktiengesellschaft, Basel (Switzerland)

Representative: DipL-Ing. E, Splanemann, patent attorney, Hamburg 36, Neuer Wall 10

Claimed priority:
Switzerland from June 10, 1955

Dr. Ernst Gäumann and Dr. Vladimir Prelog,

Zurich, Switzerland),
have been named as inventors

minic acid or pantothenic acid. They represent neutral or acidic salts. They are produced through action the corresponding acids to the free base or by double conversion of salts, for example of angolamycin sulfate with sodium pantothenate.

Angolamycin has antibiotic activity against various test organisms. Used one as test method in v'tro for bacteria dilution series of the substance (powers of ten) in glucose broth, for amoeba dilution series in Bacto-Entamoeba medium, carried out for 24 hours at 37 ° are incubated, the following inhibitory concentrations result:

Test organisms Inhibiting concentrations
tration / ig / cm ³ Bacillus megatherium 0.1 to 1 Streptococcus pyogenes 10 Micrococcus pyogenes var. Aureus .. 10 to 100 Micrococcus pyogenes var. Aureus, Penicillin resistant 10 to 100 Corynebacterium diphtheriae 100 Entamoeba histolytica 10 to 100

Angolamycin is not effective in vitro at a concentration of 100 / ig / cm ³ against the following microorganisms: Streptococcus mitis, Streptococcus faecalis, Escherichia coli, Salmonella typhosa, Salmonella schottmuelleri, Shigella sonnei, Pseudomonas aeruginosa,

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Klebsiella pneumoniae (type A), Pasteurella pestis, Vibrio comma (El Tor), Mycobacterium tuberculosis (H 37 Rv), Candida albicans and Candida tropicalis.

Angolamycin is also active in vivo. When administered to those infected with Streptococcus pyogenes Mice are given -10 to 50 mg / kg s.c. on the 6th day 100% survivors and 67% survivors on day 10.

The toxicity of angolamycin is low: 500 mg / kg

Pigment light brown; Starch hydrolysis 0.5 to 0.8 cm after 4 days.

9. Nutrient agar: pustular growth, brownish-yellow; Aerial mycelium velvety, ash gray; Pigment reddish brown. Of the

Strain Ä 7489 does not form aerial mycelium.

10. Potato: growth like lichen, light brown; Aerial mycelium velvety, milk white, then ash gray; Pigment | brownish to pitch black.

11. Carrot: pustular growth, brownish-yellow; Air-

injected subcutaneously once, become velvety in mice without i ° mycelium, with strain A 6677 chalk white to gray, Endure harm. in the strains A 6905 and A 7489 white-gray to pink;

The antibiotic angolamycin is produced in the pigment pitch black. '

Culture of a new actinomycete species of genus 12. Litmus milk (Difco No. B 107): growth ring-

Streptomyces, with none of the in Bergeys »Manual · shaped, brown, later surface growth; Luftfflytsel of Determinative Bacteriology ", 1st edition, or in 15 ash gray; Pigment dark brown; Peptonization ajit "Actinomycetes and their Antibiotics ". Von Waksman coagulation; Litmus slightly reddish. ., ',. ■' ■ '■'

and Lechevalier, 1953, is identical and hereinafter referred to as Streptomyces eurythermus is described. It has been isolated three times so far, from soil samples collected in Cuanza (Angola), in Kisantu (Congo) and Regensberg (Switzerland). All of these three tribes produce this new antibiotic and agree morphologically and physiologically with only minor differences

match. You will be in our laboratories and in the 25 Lechevalier by the color of the Luftmycels and the Federal Institute of Technology, Institute for special pigment formation; ^{: iiliil1}

Botany, Zurich, kept under the designations A 6677, A 6905 and A 7489.

Streptomyces eurythermus forms a substrate mycelium from long hyphae and a 30th hyphae on almost all nutrient media
dark brown, diffusing pigment. The aerial mycelium
is dense and usually gray, with individual, not in clusters
united chains of conidia. The latter are a
typical feature of the genus Streptomyces. The individual conidia are ovate to spherical and smooth, 35 Woodruff) closest to Waksman and Henrici, dotjh. _s their size is 0.8 to ίμχ 0.6 to 0.7μ. The wax is the growth on potatoes, carrots and MSM ·: g ,,

The temperature is relatively little dependent on the temperature, it is very different. ■;>' IM

The fungus Streptomyces eurythermus develops at both 18 ° and 58 °, according to the method / 1 |

good, but the optimum is between 25 and 32 °. by T. G. Pridham and D. Gottlieb, J. Bacteriology,

For further characterization, the 40 56, p. 107 (1948) investigated in the following, when using the growth of Streptomyces eurythermus on different carbon sources as follows: · 'Different nutrient media are described. The culture media
1 to 8 and 12 were based on W. Lindenbein, Arch.
Mikrobiol., 17, p. 361 (1952), hergesteEt.

1. Synthetic agar: pustular growth, light brown; Aerial mycelium sparse, initially white-gray, later ash-gray; Pigment brown. The A 7489 strain forms a more abundant, dense, velvety, ash-gray aerial mycelium.

2. Synthetic solution: Submerged growth, ^ "" - ^ ι,., »» ^^ ν. ». , ^ ^ Flakes and sediment, light brown; Pigment brown. 50

3. Glucose broth: growth sparse, sediment puste- raffinose + sodium succinate + * lig, brownish yellow.

4. Glucose agar: growth pincushion to wrinkled, light brown; Aerial mycelium velvety, white-gray to greenish-gray; Pigment auburn.

5. Glucose asparagine agar: growth thin and veil-like, white-yellow; Aerial mycelium velvety, white-gray to ash-gray; Pigment auburn. The aerial mycelium from Strain A 7489 is slightly pink on the surface.

6. Calcium malate agar: growth like lichen, light

Streptomyces eurythermus differs from other known thermophilic species of the genus Streptomyces as follows ^:! ■■■ 'V''-, · ■■

by S. thermophilus (Gilbert) Waksman and HeiirSci through the well-developed aerial mycelium, through the strong pigment formation on gelatine and nutrient agar, as well as through the color of the pigment; '' '*'; '

of S. thermodiastaticus (Bergey) Waksman lind-

from S. thermofuscus (Waksman, Umbreit 'tMl Cordon) Waksman and Henrici through the color of the air mycelium and the pigment formation on gelatin; ■; "■ ^il;

by S. casei (Bernstein and Morton) Waksman ⁷ Af ,, - 'Lechevalier by the color of the air mycelium, the pigment formation and the hydrolysis of the starch. ' ^{ι1ίι! ι} s- ,,

StreptcH is one of the mesophilic streptomycetes: myces eurythermus to the S. antibioticus (Waksmarl uiifl: "'■:

£ -Xylose

i-arabinose

i-rhamnose |

D-fructose

z> -galactose

Sucrose

Maltose

Lactose

Raffinose

Inulin

B-mannitol

B-sorbitol

Dulcit

Meso-inositol

Salicin

Sodium acetate

Sodium citrate

Sodium succinate

55

- ^ ■■■ ... 7 I;

₊ :., 0. + ■■■

■ 4i * f

Where: · f,; +) good growth, safe use of the relevant Carbon source,,, *

(+) weak growth, use of the relevant carbon source questionable, ■ '* ■'. '.'.

(-) very weak growth, use of the relevant carbon source unlikely,: ⁱ ; ■ - no growth, no use of the relevant; Brown; Aerial mycelium dusty, ash gray; Pigment dark carbon source. _;

Brown. The aerial mycelium from strain A 6905 is brownish- The present invention is what the production of the

Gray. As far as angolamycin is concerned, not i | esi on the use

7. Gelatin prick at 18 °: growth very sparse; Streptomyces eurythermus or other description :, pigment dark brown. Liquefaction is limited in the case of strain A 6677 65 corresponding strains, but affects ¹ '' weakly after 40 days, in the case of strain A 6905 strong (2.2 cm) also the use of variants of these organisms ,,! after 34 days and strain A 7489 thick (2.0 cm), such as for example, by mutation or selectioning; '' ί ^^ ;;;:... after 31 days otter particular under the action of ultraviolet ¹ =

8. Starch plate: pustular growth, golden yellow; Air x-rays or oil from nitrogen mustard mycelium velvety, initially snow-white, later gray; Turn 70.

5 6

To generate the angolamycin, the own compounds themselves, in particular the angolamycin

Effects of streptomyces eurythermus containing sulfates, the angolamycin hydrochloride, acetate and

Streptomycetes strain, e.g. in aqueous, inorganic pantothenate, further which by means of hydrogenation or oxy-

Salts, nitrogen-containing compounds and given conversion products obtained, as well as the

if nutrient solution containing carbohydrates aerobically 5 breakdown products of angolamycin, as they are, for example, in the

grows until these hydrolysis have a substantial antibacterial effect.

shows, and the angolamycin is then isolated from the culture filtrate Angolamycin, its salts, its abovementioned re-isolated, conversion and fission products or equivalent

The nutrient solution contains inorganic salts, for example, mixtures can be used as remedies, e.g. in the form of pharmaceuticals Chlorides, nitrates, carbonates, sulfates of alkalis, chemical preparations are used. These contain Alkaline earths, magnesium, iron, zinc, manganese. On stick- the compounds mentioned in a mixture with a for Substance-containing compounds and, if appropriate, those suitable for enteral, parenteral or local application Carbohydrates and growth-promoting pharmaceuticals organic or inorganic Substances are mentioned, for example: Amino acids and their carrier material. Such substances come in for the same Mixtures, peptides and proteins as well as their hydrolysates, i5 question, which do not react with the new compounds, such as peptone or tryptone, meat extracts, water-soluble such. B. gelatine, lactose, starch, magnesium components of cereal grains such as corn and wheat, of stearate, talc, vegetable oils, benzyl alcohol, gum, Distillation residues from alcohol production: polyalkylene glycols, petrolatum, cholesterol or others of yeast, beans, especially the soybean plant, of known excipients. The pharmaceutical pre-seeds, for example the cotton plant, also GIu- 2 ° parate can e.g. as tablets, dragees, powder, kose, sucrose, lactose, starch etc. ointments, creams, suppositories or in liquid form as

The cultivation takes place aerobically, for example in solutions, suspensions or emulsions, resting surface culture or preferably submerged known fermenters. As a 25 or emulsifier. You can also use another temperature, such as between 20 and 50 °. Contain therapeutically valuable substances.
A substantial antibacterial effect is shown by the nutrient solution in the following example generally after I ¹ Z ₂ to 5 days. wrote without thereby restricting the

To isolate the antibiotic from which the mycelium is intended to be the subject of the invention. The tempe-

Separated culture filtrate can, for example, the following values are given in degrees Celsius,

drive serve: The culture filtrate is extracted advantageously.

way at a p _H greater than 7.0 with a non-water Example

Miscible organic solvents such as esters Prepare a nutrient solution with the following composition:

lower fatty acids, for example ethyl acetate or 3 g meat extract, 5 g peptone, 10 g raw glucose, S g

Amyl acetate, chlorinated hydrocarbons, e.g. ethy- 35 sodium chloride, 10 g calcium carbonate and 11 line

lenchlorid, methylene chloride or chloroform, ketones, water, and adjusted to p _H 7.5. This or a lot of

z. B. methyl propyl ketone, methyl amyl ketone or Diisoffaches the same is in 500 cm ³ Erlenmeyern (with each

butyl ketone, alcohols such as butyl alcohol or amyl 100 cm ³ nutrient solution) or in 500-1 fermenters (each with

alcohols, ethers, e.g. B. ethyl ether, diisopropyl ether, 3001 nutrient solution) bottled and 20 to 30 minutes at

Dibutyl ethers or glycol ethers and the like. Sterilized instead of 40 1 atm. Then seeded with up to 10 ° / ₀ of a

Solvent extraction of the culture filtrate, or in partially sporulating, vegetative culture of the strepto-

Combination with such as a further purification myces eurythherm and incubated under good

operation, the antibiotic can also be shaken or stirred and placed in the fermenter

Gain adsorption, for example on activated charcoal or ventilation (with about 1 volume of sterile air per volume

activated earth, such as fuller's earth or floridin, and 45 lumen nutrient solution per minute) at 27 °. After 48 hours

subsequent extraction of the adsorbate, e.g. with a growth, the cultures are filtered with the addition of a

acidic aqueous solution and / or with a filter aid in water, depending on the volume, through a suction filter

at least partially soluble organic solvent, or through a filter press or rotating filter

such as isopropanol, butanol or methyl ethyl ketone. Next and frees the antibiotic-containing aqueous solution

one can use the antibiotic e.g. as a salt of an organ 50 of the mycelium and other solid components,

African sulfonic acid or by precipitation with picric acid 170 1 culture be at _pH 8 with 70 1 ethyl acetate

precipitate directly from the culture filtrate. pulled out in an extractor. This goes the whole

A good cleaning method for the new anti-gram-positive activity in the extract, biotikum represents the distribution between an acidic while the culture filtrate still has an activity against aqueous solution and a fungus that is immiscible with water, especially against Candida tropicalis, The ethyl acetate extract is then used three times with 41 each time Distribution by the countercurrent method in ent- 0.5 η-aqueous acetic acid extracted, with the speaking apparatus. Chromatography is also included in the overall activity against gram-positive germs Cleaning very suitable. The recovery of the pure aqueous phase passes over. The acetic acid extract is made with Antibiotic in crystalline form is taken e.g. 60 concentrated soda solution and made alkaline three times extracted from organic solvents, such as ether, dioxane, with 2.51 ethyl acetate each. Be on it Ether-petroleum ether mixtures, benzene-ether mixtures, the bases again with a total of 3 1 0.5 η-aqueous vinegar or Acetone-petroleum ether mixtures. Extracted to recrystallic acid, with concentrated soda solution in The same solvent or aqueous-free system are used and finally with a total of 11 ethyl organic compounds Solutions such as dilute alcohols are shaken out with acetate. The ethyl acetate solution is with thin acetone, etc., washed with water, dried with sodium sulfate and

The present invention also forms evaporated to dryness in a vacuum at 40 °. It stays

the manufacturing process for the crystalline Angola - 2.39 g of a yellowish amorphous product back,

mycin, its neutral and acidic salts with other antibacterial effects almost like that of the

organic or organic acids also corresponds to the above-mentioned 70 crystalline angolamycins. The so obtained

Claims (1)

7 8 2.39 g of crude product are dissolved with gentle warming. Instead of the pure sugar, salts of other substances can also be dissolved in 50 cm ^{3 of} ether. The solution is nitrated and treated with acids, especially sulfuric acids; Inoculated with hydrochloric acid, angolamycin. Prepare acetic acid and pantothenic acid in the course of a few hours,
1.80 g of the antibiotic separate in crystals from the hydrolysis of angolamycin: 18 mg angolamycin F. = 131 to 134 °. For further purification, a ^{solution of the crystals in 2 cm 3 of} 0.5 η-sulfuric acid in the inclusion tube is heated to 100 ° for 5 hours in benzene that is as little hot as possible. The reaction - mixed with ten times the amount of ether. The mixture is then shaken twice with ethyl acetate crystals of the pure antibiotic, whereupon the aqueous phase by adding melts at 133 ° to 136 °. [α]% = - 64 ° (c = 1.30 in Bariumhydroxydlösung is brought to p _H 5 Man chloroform.). 1.0 removes the excreted barium sulphate by centrifugation .... C 60.21%, H 8.78%, N 1.40%, ^ ^{eren and} J ^am P ^ft ®? ^Small supernatant solution in CH ₃ O 8; 80 ° / ₀ , CH ₃ (N) 3.66%, CH ₃ (C) 11.55%, vacuum to dryness. The residue is examined by papjerakt H 0 52 ° / chromatographically, using a mixture of butanol and glacial acetic acid (10: 1) saturated with water by recrystallization from diisopropyl ether or 15. With ammoniacal super nitrate solution dioxane, angolamycin is obtained in the form of two reducing sub-needles on the paper strips, F. = 165 to 168 °. punch with the Rf values 0.28 and 0.65 visible Found ... C 60.49%, H 8.93%, N 1.66%. S ^^ "Under the same conditions, the erythromycin gives two Instead of direct crystallization, the raw? O reducing substances with Rf values of 0.23 and bases can also be purified by chromatography. To 0.32, while from picromycin and from narbomycin only for this purpose 42 mg of raw bases in 2 g of aluminum - a reducing cleavage product (Rf 0.23) is obtained, oxide (activity III according to Brockmann) after the thorough hydrogenation of angolamycin: a) A solution of run method chromatographed, with benzene, 20 mg angolamycin in 5 cm ^{3 of} absolute alcohol Chloroform and chloroform-methanol mixtures eluted for 2 hours with 10 mg palladium catalyst in a, will. With benzene and chloroform only traces of hydrogen atmosphere are shaken. Then it is filtered Impurities detached while the chloroform made the solution and evaporated it in a vacuum. You get Methanol (16: 1) fractions 40 mg of the pure antibiotic dihydroangolamycin as a colorless powder, which included. This is antibiotic active in the above described. Way crystallized. 30 b) A solution of 20 mg of angolamycin in 5 cm ^{3 of} ice-Next can the raw bases by means of countercurrent. vinegar is cleaned for 1 hour with 10 mg of platinum catalyst in one division, with z. B. shaken the following solution hydrogen atmosphere. Then the means systems can be used: solution filtered and evaporated in vacuo. Man a) Mac Ilvaine buffer (p _H 3.15) and chloroform 1: 1 is obtained, the hexahydro-angolamycin as a colorless powder, b) Isopropyl ether (12 parts by volume) - acetone (10 volumes - 3.5 which is antibiotic active.
parts) —Water (10 parts by volume). If the distribution is over 100 levels, you will find the patent access book:
Substance and activity maximum with system a) at level no. 72 and with system b) at level no. 55. The use of Streptomyces eurythermus Reineckat: A solution of 60 mg angolamycin in 40 or one of its properties exhibiting micro- 1 cm ^{3 of} methanol is combined with 15 cm ^{3 of} an ethereal organism of the genus Streptomyces to produce Pure Eckesäure solution (prepared according to Karrer und lung from Angolamycin by usual biological Schmid, HeIv., 29, 1853 [1946]) slowly fell. The breeding, extraction of the antibiotic from the Precipitate is dissolved in methanol and added. Culture filtrate, pure preparation and production of like of ether. Found Cr 3.96%. 45 its salts. © 709 58T / 384 7.57