DE1617924A1 - Erizomycin and process for its preparation - Google Patents

Description

The invention relates to the new antibiotic erizomycin, which is used to inhibit the growth of various microorganisms, e.g. Eseherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Staphylococcus aureus, or Streptococcus faecalis, can be used, as well as a method for its production. The antibiotic according to the invention is produced by an erizomycxn-producing microorganism, preferably Streptomyces griseus var. erizensis in an aqueous Nutrient medium, preferably containing a source of assimilable carbohydrates and assimilable nitrogen, cultured under aerobic conditions until the medium has acquired significant antimicrobial activity and by isolating erizomycin from the medium.

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Erizomycin is a basic substance,

which is capable of the growth of certain organisms, in particular the growth of bacteria, e.g. Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Salmonella typhimurium, Staphylococcus aureus, Streptococcus faecalis, Streptococcus hemolyticus or Streptococcus viridans and inhibit them can be used alone or in combination with other antibacterial agents to prevent or reduce growth to serve the number of such organisms in various environments. For example, erizomycin can be used as an oil preservative can be used, for example, as a bacteriostatic agent to inhibit the growth of Proteus vulgaris, which is used for this it is known to cause spoilage in cutting oils. It is also used in washing liquids for sanitary purposes useful, for example for washing hands and cleaning equipment, flooring or furniture in contaminated rooms or laboratories; it is also used as an industrial preservative useful, e.g. as a bacteriostatic detergent for cleaned clothes and for the impregnation of papers and tissues, it is also useful for suppressing the growth of sensitive organisms in plate procedures and other microbiological media. It can also be used as a feed additive to promote the growth of animals, e.g. mammals, Birds, fish and reptiles.

The description includes two drawings, of which FIG. 1 shows the infrared absorption spectrum of erizomycin

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and Figure 2 depicts the paper chromatography of erizomycin.

Chemical and physical properties of erizomycin.

Elemental Analysis: Calculated for C ₂₇ H ₅₂ N ₄ O ₈ : C 59.99; H, 5.96;

N 10.37; 0 23.68.

Found: C, 59.97; H, 6.05;

N 10.46; 0 22.88. Molecular weight: 540 (mass spectrometer).

Ultraviolet spectrum:

Erizomycin has the following UV absorption spectra:

In ethanol solution: Λ Max. , (in τη / ^υ 1 a in ethanol •
• 221 (sh) 42.01 255 (sh) 9.45 262 8.56 269 . 7.52 303 16.38 350 0.32 Optical rotation: [^ : 3d ^{5 β} ^ ^{82 0} (c = 1.35, Solubility: At room temperature

The approximate solubilities (in mg / ml) are as follows:

Water: <1
95 Io ethanol:> 10 ethyl acetate:> 10 acetone:> 10

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Methylene chloride: > 100 Benzene: Λχ. ^ 2 Skellysolve: B: c ι n-Eutanol: Z "" ** ¹ * · s 5 214-217 ⁰ C

Melting point; Infrared spectrum;

The infrared absorption spectrum

the erizomycin in mineral oil is shown in Figure 1 of the drawings

shown; Erizomycin shows absorption beams at the following wavelengths (dimensions in cm):

3445 (M) 1572 (M) 1325 (M)

3460 (S). 1540 (M). 1314 (S)

3440 (S) 1522 (S) 1300 (S)

3200 (W) 1505 (M) 1293 (S)

3060 (W) 1472 (M) '1278 (M)

2950 (S) (OeI) 1465 (M) 1272 (W)

2920 (S) (OeI) 1458 (M) (OeI) 1247 (S)

2850 (S) (OeI) 1447 (S) 1225 (S)

2730 (W) 1420 (M) 1195 (M)

2620 (W) I ₃₈₈ (μ) n ₉ i (κ)

! 725 (S) 1384 (M) 1152 (S)

¹⁶ 55 (S) 1376 (M) (OeI) 1145 (S)

ⁱ⁶ 45 (S) 1360 (M) 1130 (M)

¹⁶²⁰ (W) 1355 (M) 1097 (M)

¹⁶⁰⁰ (M). 1338 (S) 1087 (S)

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1069 (S) 952 (W) 808 (W)

1062 (S) 952 (W) 802 (M)

1047 (W) 911 (M) 772 (M)

1035 (W) '888 (W) 720 (M)

1024 (W) 885 (W) '706 (M) -

1012 (W) 367 (W) 698 (M)

992 (W) 850 (W) 670 (W)

973 (W) 835 (W) 662 (M)

960 (W) 822 (W)

In KBr, the erizomycin shows absorption bands at the following wavelengths (dimensions in cm):

1248 (S)

1224 (S)

1190 (M)

1150 (S)

1132 (S)

1085 (S)

1068 (S)

1050 (M)

1025 (M)

1015 (W)

995 (W)

965 (W)

930 (W)

907 (W)

3485 (W) 1595 (M) 3420 (W) 1575 (M) 3,370 (M) 1540 (M) 3210 (W) 1538 (S) 3035 (M) 1515, (S) 2980 (M) 1477 (M) 2940 (M) 1465 (M) 2880 (M) 1458 (S) 2760 (W) 1448 (S) 2630 (W) 1424 (M) 1725 (S) • 1380 (M) 1679 (S) 1358 (M) 1655 (S) 1320 (S) 1645 (S) 1290 (S) - 5 _ 00981 1/1649

890 (W) 876 (W) 835 (W) 810 (M) 780 (M) 755 (M) 717 (M) 660 (M)

The band intensities are with

"S", "M", and "W" respectively denote and are indicated approximately in relation to the background absorption in the vicinity of the bands. An "S" band is a band of the same order of magnitude as Intensity as the strongest band in the spectrum; Have "M" bands an intensity between one third and two thirds of the strongest band and "W" bands have an intensity of. less than a third of the strongest ties. These estimates are based on a percentage permeability scale made.

rapierchromatogramtn

Erizomycin shows a characteristic

Paper chromatogram shown in Figure 2 of the drawings using the following solvent systems:

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I. 1-butanol, water (84:16), 16 hours.

II.L-butanol, water (84:16) plus 0.25 i »p-toluenesulfonic acid, 16 h. -

III. l-butanol, acetic acid, water (2: 1: 1), 16 h.

IV. 2 # piperidine (v / v) in 1-butanol, water (84:16), 16 h.

V. l-butanol, water (4:96), 5 h.

VI. l-butanol, water (4:96) plus 0.25 # p-toluenesulfonic acid, 5 h.

Antitumor activity of erizomycin

'Erizomycin is effective against

KB cells (human epidermal cancer cells) in tissue cultures. The ID ₅₀ is 4.4 / Ug / ml of erizomycin.

Antibacterial efficacy of erizomycin tube dilution test

Test microorganism

Minimum inhibition concentration in / Ug / ml .

Escherichia coli Klebsieila pneumoniae Proteus vulgaris Salmonella paratyphi Salmonella pullorum Salmonella typhimurium

125 125, 250 250

62.5 125

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Streptococcus faecalis 125

Streptococcus hemolyticus 250

Streptococcus viridans 250

Staphylococcus aureus 250

The tube dilution test method

was carried out on BHI medium (Brain Heart Infusion Broth, Difco, Detroit, Michigan, USA). The test tubes (13 mm 100 ram) were produced in the usual way, as described by EB Snell, Vitamin Methods, Vol. I, Academic Press, Inc., New York 1950, p. 327. Were incubated test organisms, the 18 hours at 37 ⁰ C, to inoculate the test medium were used. The determinations were made after 17 hours.

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. . ■ 3

The microorganism

The actinomycete strain used according to the invention for the production of erizomycin is Streptomyces griseus var. erizensis var. nova. One of its tribal characteristics is the production of erizomycin. An offshoot culture of the living organism can be obtained from the collection of the North utilization and Research Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois, U.S.A., can be obtained. Its depository number is NRRL 3242.

Streptomyces griseus var. Erizensis was with

Compared

Streptomyces griseus Waksman / 4, ATCC 1013 "Z ^ Both microorganism cultures showed cream-colored to cream-olive-colored air wax; convoluted sporophores; smooth-walled Spores and grew best at 24 to 28 ° C. Streptomyces griseus var. Erizensis stands out over Streptomyces griseus due to the production of the antibiotic erizomycin and due to the color differences shown in the tables below for Ektachrome, Culture and Parb characteristics are given

Streptomyces griseus var. Erizensis and Streptomyces griseus Waksman # 4, ATCC 10137 are used in the following Tables compared:

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Appearance on Ektachrom microscopic features KuItürme r kmale

Growth on Carbon Compounds in Synthetic Medium Table V - Color Characteristics

Table I Table II Table ItI Table IV

Table I.

Appearance of S. griseus var. Erizensis and S. griseus ATCC 10137 on Ektachrome *

Agar medium

S. griseus var »erizensis

S. griseus ATCC 10137

Bennett · s surface back

reddish white yellow

Czapek ¹ s sucrose surface reddish-white

Colorless back

Malto se-tryptone Surface back

Peptone-iron surface back side

0.1 # tyrosine surface back side

Casein starch surface back

reddish white brown

somewhat white yellow-brown

somewhat white pale yellow

reddish-white reddish-brown cream-reddish yellow-brown

reddish-white colorless

reddish

reddish brown

no air growth yellow-brown

somewhat white pale yellow

reddish-white reddish-brown

* Dietz, A., "Ektachrome Transparencies as Aids in Actxnomycete Classification, "Annals of the New York Academy of Sciences, 60: 152-154, 1954.

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Table II

Microscopic features of S. griseus var. Erizensis and S. griseus, ATCC 10137

S. griseus var. Erizensis

S. griseus ATCC 10137

Light microscope

Electron microscope
Direct

Carbon footprint

Sporophores long, tortuous

Spurs smooth rectangular

Spores furrowed with large surface markings

Some spores with a dark, central spot

Sporophores straight to convoluted

Spores smooth, sausage-like

Spores furrowed with large surface markings and a dark middle spot

Table II

Cultural characteristics of S. griseus var. Erizensis
and S. griseus, ATCC 10137

medium

S. griseus var. Eri ^ ensis

S. griseus ATCC 10137

Peptone iron agar

Surface white

Yellow back

Otherwise melanin

something white yellow
Melanin -

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Table III (port setting)

Calcium Malate Agar Surface of the back Other

Gluco se asparagine

Surface of the back Other

Skimmed milk agar surface back side miscellaneous

Tyrosine agar surface back pigment

Xanthine agar surface back side other

Yeast extract-malt extract-agar surface Reverse side otherwise

Casein starch agar surface back side other

somewhat white, creamy reddish pale reddish. pigment Malate dissolved

quite reddish-white

cream

sth. yellow pigment

very weakly white yellow-brown yellow-brown casein dissolved

cream yellow-brown yellow-brown tyrosine dissolved

cream

yellow

pale yellow pigment

Xanthine dissolved

cream

olive brown

pale yellow pigment cream

White

no pigment malate dissolved

cream

yellow

yellow pigment

no air growth yellow-brown ■ yellow-brown casein dissolved

White
yellow-brown yellow-brown tyrosine dissolved

something white yellow

Pale yellow pigment xanthine dissolved

cream

yellow-brown pale yellow pigment

olive-creme

reddish reddish brown

sth. reddish pigment sth. reddish pigment

Hydrolyzed starch hydrolyzed starch ■

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fd

Table III (port setting)

Bennett's
18 ⁰ C surface
back
pigment cream olive
yellow
no pigment creamy white
olive
a little olive 24 ⁰ C surface
back
pigment cream olive
olive brown
no pigment cream brown
yellow-brown
something yellow 28 ⁰ C surface
back
pigment cream olive
olive
a little olive cream brown
yellow-brown
something yellow 37 ⁰ C surface
back
pigment no air growth
slightly colorless
no pigment very weak cream
blurred
yellow-brown
something yellow 55 ⁰ C no growth no growth Czapek's sucrose
Agar
18 ⁰ C surface
back some cream olive
cream some cream olive
cream 24 ⁰ C surface
back a little cream.
swim
White some cream ver
swam
cream 28 ° C surface
back cream
cream cream olive
cream olive 37 ⁰ C surface
back no air growth
colorless no air growth
colorless 55 ° C no growth no growth Maltose tryptone
Agar
18 ⁰ C surface
back
pigment cream
olive
no cream olive
olive
a little olive

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Table III (port setting)

Maltose tryptone
Agar
24 ⁰ C surface
Back.
pigment cream
olive brown
no cream
yellow-brown
something yellow 28 ⁰ C surface
back
pigment cream olive
olive
a little olive cream
yellow-brown
something yellow 37 ⁰ C surface
back
pigment no air growth
colorless s
no no air growth
yellow-brown
yellow-brown 55 ⁰ C no growth no growth Simple gelatin complete ver
liquid complete ver
liquid Nutrient gelatin complete ver
liquid complete ver
liquid Synthetic nitrate
broth somewhat colorless
vegetative wax
at the bottom colorless upper
flats and
flaky growth
at the bottom

Nutrient nitrate broth

Nitrates reduced to nitrites

strong v / icy air growth on the surface

Little growth at the bottom

Nitrates reduced to nitrites

to

Nitrates nichy nitrites. »* reduced

white growth of air on the surface Cuticle

compact vegetative growth at the bottom

Nitrates reduced to nitrites

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Table III (port setting)

Litmus milk . white growth in air somewhat white

on blue surface- air growth ring on blue upper

surface ring

partial peptonization pH 7.9 peptonization

PH 7.9

Table IV

Growth of S. griseus var. Erizensis and S. griseus ATCC 10137 on carbon compounds in synthetic medium (J. Bact. 56: 107-114, 1948)

S. griseus S. griseus var. Erizensis ATCC 10137

Control (-) (-,

1. D-xylose ++

2. L-arabinose + (+

3. Rhamnose (-) (+) (-) * (-)

4. D-fructose ++

5. D-galactose ++

6. D-glucose '++

7. D-mannose ++

8. Maltose + · +

9. Sucrose (-) (-)

10. Lactose ++

11. Cellobiose ++

12. Raffinose "(-) (-) 13 · Dextrin ++

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Table IV (porting)

14. Inulin (-) - (-)

15. soluble starch + +

16. Glycerin ++

17. Dulcit (+) (-) (-) (-)

18. D-mannitol (-) + + + '

19. D-sorbitol + (-) (-) (-)

20. Inositol (-) (-) (+) (-)

21. Salicin (-) - - (-)

22. Phenol (-) - -.

23. Cresol

24. Na formate · (-) - ■

25. Na oxalate (-) - (-)

26. Na tartrate (-) - (-)

27. Na salicylate (-) - -

28. Na acetate + (+) +

29. Na citrate + (+) +

30. Na succinate ++

+ = good growth
(+) = massive growth
(-) = low growth

- ss no growth

Scattering of the results in different experiments,

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Table V Color characteristics of S. griseus var. Erizensis and S. griseus, AgCO 10157 Agar medium Co'lor Harmony Manual 3rd Ed., 1948

.B.S. Circular 553, 1955

S. griseus var. Erizensis

S. griseus ^ AJOC 10137

5. griseus
var. erizensis

S. griseus, AJCC 10137

Bennett's surface

back

Czapek's sucrose surface

back

Maltose tryptone

Surface back

l-l / 2db (g) leather 2db (g) ivory 89 gta pale yellow

2gc (g) bamboo chamois 3ng (g) yellow-ahom 90 gm greyish yellow

2cb (m) ivory white 2cb (m) ivory

White

2 db (g) ivory 2db (g) ivory 89 gm pale yellow, 90 g: greyish-yellow, 121 m pale yellow-green,

77 m medium yellowish brown i

2ba (m & g) permusch- 2cb (m) ivory- 92 gm yellowish- 92m. yellowish white, 93 white - white white yellowish gray

2ie (g) light mustard brown

3 ng (g) yellow maple 91gm dark gray-yellow, 94 g light
olive-brown, 106 g
light olive

77 m g3? Att medium yellowish brown

The new invention compound obtainable according produced when the developing organism is grown under submer _s s aerobic conditions in an aqueous Mhrmedium. It should also be understood that bottle surface cultures can be used to produce limited quantities. The organism is in a nutrient medium that is a carbon source, e.g. an assimilable carbohydrate, and a nitrogen source, e.g. a

assimilable nitrogen compound or albuminous

bred.
Material containing / Preferred carbon sources include glucose, sand sugar, sucrose, glycerine, starch, corn starch, galactose, dextrin, molasses and the like. Preferred nitrogen sources include corn soft water, yeast, autolyzed brewer's yeast with milk solids, soybean meal, cottonseed meal, corn flour, milk solids, pancreatic hydrolyzate of casein, stillage, fish meal, animal protein fluids, meat and bone waste and the like. A combination of these carbon and nitrogen sources can be used to advantage. Trace metals, for example zinc, magnesium, manganese, cobalt, iron and the like, do not need to be added to the fermentation, since tap water and unpurified ingredients are used as constituents of the medium.

Production of the compound obtainable according to the invention can take place at any temperature

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be carried out between about 18 ° and 40 ⁰ O, and preferably between about 25 ° and 3O ⁰ C causes satisfactory growth of the microorganism, for example. Typically, best compound production is achieved in about two to ten days. Usually the medium remains approximately neutral or slightly alkaline during fermentation. The final pH depends partly on the buffer substances that may be present and partly on the initial pH of the culture medium, which is advantageously adjusted to a pH of 6 to 8 before the sterilization.

• If the cultivation takes place in large vessels ·.

and tanks, it is advantageous to use the vegetative form instead of the spore form of the microorganism for the Inoculation use to avoid a pronounced delay in the production of the new compound and the associated to prevent inefficient use of the equipment. Accordingly it is desirable to have one vegetative inoculum in one Prepare nutrient broth culture by inoculating this culture with an aliquot of a soil or slant culture. When a young, active, vegetative inoculum is prepared in this way, it is aseptically placed in large vessels or tanks convicted. The medium in which the vegetative inoculum is produced may be the same as used to produce the new compound, or it may be different be of it as long as it is such that a good one

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Growth of the microorganism is achieved.

The new obtainable according to the invention

Compound is a basic compound with the empirical formula C ₂₇ H ₃₂ N ₄ O ₈ . At room temperature, erizomycin exhibits the following solubilities: approximately less than 1 mg / ml in water; > IO mg / ml in 95 $ ethanol; > 10 mg / ml in ethyl acetate; > 10 mg / ml in acetone; } 100 rag / ml jj.n chloroform; about 2 mg / ml in benzene;

<C 1 mg / ml in Skellysolve B (petroleum ether or η-hexane); and about 5 mg / ml in n-butanol.

A variety of procedures can be used for

Isolation and purification of erizomycin can be used, e.g. solvent extraction, liquid-liquid partition in a Craig apparatus, use of adsorbents, and Chromatography columns. Solvent extraction processes are preferred for commercial production because they are less time consuming and are less expensive and higher yields can be achieved with them.

In a preferred isolation procedure,

are first carried out by conventional methods such as filtration using a filter aid (or by centrifugation) the mycelium and undissolved solids from the fermentation broth severed. The filtered (or centrifuged) broth is with a solvent (preferably »4Φ ethyl acetate) for Erizomycin extracted. The ethyl acetate extract, which contains the erizomycin, is mixed with a tenth volume of water.

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wash _P then the ethyl acetate extract is evaporated under reduced pressure to give a dry crude product of erizomycin. This preparation can be used in environments where higher purity of the antibiotic is not required. This erizomycin preparation can be further purified by dissolving the crude product in a minimal amount of a solvent for erizomycin (preferably ethyl acetate) and adding 10 times the amount of cyclohexane. The precipitate that forms is collected by filtration and dissolved in ethyl acetate. The cyclohexane filtrate is discarded. The ethyl acetate containing the erizomycin is filtered and the filtrate is evaporated to dryness under reduced pressure. The preparation containing dry erizomycin is then dissolved in a solvent for erizomycin (preferably methylene chloride) and the solution is filtered. The filtrate is evaporated to dryness under reduced pressure and gives a relatively pure erizomycin preparation. Crystalline erizomycin can be obtained by subjecting this relatively pure erizomycin preparation to partition chromatography on buffered diatomaceous earth using the solvent system loluene-propylene glycol. Crystalline erizomycin, which was obtained from the chromatography fractions, can be recrystallized from ethanol and results in a highly pure crystalline erizomycin preparation.

Because of the basic ITatm? des erizomycin can do this from the fermentation broth by means of ethyl acetate extraction

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originally obtained crude product can be purified by adding the active substance at a pH of around 2 to 3 in water is extracted by adjusting the pH to 9 to 10 and then back-extraction in ethyl acetate or another solvent in which erizomycin is soluble takes place. After removing the solvent, the residue can by crystallization, Gregenstrom distribution or chromatography, 'as described above, can be further purified.

Wah.lv/eise can the new according to the invention

Compound also obtainable by adsorption on cation exchange resins can be isolated from the filtered broth. Both carboxylic and sulfonic "resins" can be used v / earth. Suitable carboxylic acid resins include the polyacrylic acid resins, which are produced by copolymerization of acrylic acid and divinylbenzene, for example by the process filed on page 87 of Kunin, Ion Exchange Resins, 2nd Edition (1958), John Wiley and Sons, Inc. is obtained will. Carboxylic acid cation exchange resins of this type are used sold under the tradenames Amberlite IRC-50 and Zeokarb 226. Suitable sulfonic acid resins include sulfonated polystyrene resins which are crosslinked with divinylbenzene and which after that described on page 84 of the lanceted work by Kunin Procedures were obtained. Sulfonated cation exchange resins of this type are sold under the trade names Dowex-50, Amberlite IR-120, Haleite HCR, Chempro C-20, Permutit Q and Zeokarb 225 placed on the market.

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The antibiotic is treated with an acid,

preferably at a pH lower than the pKa of the cation exchange resin used, eluted from the resin. Satisfactory results are obtained at a pH of about 1 to 6. The eluate is treated with a base, e.g. Sodium hydroxide, or a strongly basic anion exchange resin, Adjusted to a pH of about 7.5 to 8.5, and the antibiotic becomes immiscible with a small spoon Solvent extracted according to the method described above.

en
Anion exchange resins suitable for this purpose are prepared by chloromethylation according to the process specified by Kunin (see above quotation, pages 88 and 89) of polystyrene, if desired with divinic benzene, which is prepared according to the process specified by Kunin (see above quotation, page 84) is crosslinked, and quaternization with trimethylamine or dimethylethanolamine according to the process given by Kunin (cf. the above quotation on page 97) Anion exchange resins of this type are sold under the names Dowex-2, Dowex-20, Amberlite IEA 400, Duolite A-102 and Permutit SI put on the market J '

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The new compound obtainable according to the invention can also be obtained from the broths and other aqueous solutions by adsorption on a surface-active one Adsorbent, for example Florisil (a synthetic silicate of the type described in US Pat. No. 2,393,625) sold from the Florida Society). Decolorizing carbon or decolorizing resins, and eluting the adsorbed material with a solvent. Any of the above Solvent can be used. A suitable decolorization resin is Permu'tit DR (U.S. Patent 2,702,263).

The new compound according to the invention can also purified by successive transfers from the protonized to the non-protonized state and vice versa especially in combination with other types of loading

.Actions, e.g. solvent extractions and washes, chromatography and fractional liquid-liquid extraction. In this way, salts of erizomycin can be used to make the Isolate or purify antibiotic. The antibiotic can, for example, be converted into an insoluble salt, e.g. Picrat, are transferred, which is then subjected to cleaning processes and then to regenerate the free base of the

"Antibiotic is treated with alkali. Or the antibiotic can be converted to a water soluble salt such as the hydrochloride or sulfate, and the aqueous solution of the salt

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can be extracted with various water immiscible solvents before the free base of the antibiotic is regenerated by treating the acidic solution thus extracted with alkali.

Salts of the erizomycin can be used for the same biological purposes such as the free base can be used, or they can be used to purify the antibiotic as described above be used.

Specific acidic salts can be prepared by neutralizing the free base with the appropriate acid to a pH of less than about 7.5, and preferably to a pH of about 2 to 6. Acids suitable for this purpose include salt, sulfur, phosphorus, vinegar, amber, lemon, milk, maleic, fumaric, pamoe, cholesterol, palatinate, mucilage, camphor. , Glutaric, glyfeol, phthalic, wine, lauric, stearic, salidyl, 3-phenylsalicyl, 5-phenylsalicyl, 3-methylglutar, orthosulfobenzoe, cyclohexanesulfame, cyclopentane propion, 1,2- Cyclohexanedicarbon, 4-cyclohexanecarbon, octadecenyl amber, octenylberastein, methanesulfon, benzenesulfon, helianth, Reinecke, dimethyldithiocarbamine, sorbin, monochloroacetic, undecylene, 4 ^I -hydroxyadecylenesulfur, 4-I-hydroxyadecylenesulfur, 4 - , Pifcric, benzoic, cinnamic and similar acids.

- 25 0 0 9811/1649 "~

Erizomycin, the new compound obtainable according to the invention, is effective against Escherichia coli and can reduce, inhibit and eradicate this microorganism due to its antibacterial effectiveness used by slime production in paper mills. It can also be used to represent cultures of life Trichomonas fetus, Trichomonas hominis and Trichomonas vaginalis to extend by removing this from Escherichia coli contamination freed.

The following examples are preferred embodiments of the inventive method described. All percentages relate to percentages by weight and all solvent ratios relate to volume data, unless otherwise stated.

example 1
A. Fermentation

A soil culture of Streptomyces griseus var. Erizensis, NERL 3242, was used to make 500 ml Erlenmeyer flasks, each containing 100 ml of sterile medium to inoculate; the germ medium contained the following ingredients: Giucose monohydrate 25 g

Pharmamedia * 25 g

Tap water q.s.ad. 1 liter

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* Phannamedia is a technically pure cottonseed flour manufactured by Trader's Oil Mill Company, Fort Worth, Texas, USA.

The pH of the germination medium before sterilization was 7.2. The germinoculum was at for three days Incubated at 28 ° C on a Gump shaker at 250 rpm.

The germ iminoculum prepared as above was used for inoculating 500 ml Erlmeyer flasks, each 100 ml containing sterile fermentation medium was used; the fermentation medium contained the following ingredients

Sucrose 20 g

Soybean flour 20 g

Calcium carbonate 5 g

Tap water q.s.ad. 1 liter.

The fermentation flasks were filled with 5 ml of germ iminoculum inoculated per 100 ml of fermentation medium. The pH of the fermentation medium before sterilization was 7.2. The fermentation flasks were kept for 5 days at a temperature of 28 ° C on a Gump rotary shaker equipped with 250 rpm worked, incubated. The maximum production of the antibiotic in a flask fermentation generally becomes reached after 2 to 3 days, after which the titer of the antibiotic gradually decreases. In a typical shaking

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bottle fermentation was the erizomycin concentration after one day 9 bio units per ml; after 2 days 11 bio units per ml and after 3 days of fermentation time 10 bio units of erizomycin per ml. The determination was made with an agar plate test carried out against the microorganism Bacillus cereus. The determination against Bacillus cereus is carried out on an agar, which was buffered to a pH of 7.4 with phosphate buffer, pH 7.4, carried out. A unit volume (0.08 ml) of the

Solution containing the substance to be determined is placed on a 12.7 mm paper disk, which is then placed on an agar plate which is inoculated with the test organism. The agar plate is then incubated at 32 ° C. for 16 to 18 hours. A bio-unit (BE) is defined as the concentration of the antibiotic covering a 20 mm zone of inhibition under standard test conditions results, defined. If, for example, a fermentation broth has to be diluted in a ratio of 1: 100, by a 20 mm To give a zone of inhibition, the effectiveness of this broth is 100 BU / ml.

B. Extraction

The entire broth (3450 ml) of an erizomycin fermentation, as described above, with 4.4 biounits per ml of erizomycin was filtered with the addition of kieselguhr. The filter cake was washed with one tenth the volume of water. The filtered broth was treated with the washing water

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009811/1649

agree (3410 ml) with 3.8 BU / ml and with an equal volume extracted on ethyl acetate. The ethyl acetate extract was with washed one-tenth the volume of water. The washed ethyl acetate extract was then evaporated under reduced pressure and yielded a dry crude product (350 mg) of erizomycin containing 32.8 µg / mg erizomycin determined according to the B. cereus method.

A raw erizomycin preparation (704 g with a content of 53 pg / mg according to the B. cereus method) obtained after the procedure described above, was dissolved in the smallest possible amount of ethyl acetate and mixed thoroughly added to ten times the volume of cyclohexane. The precipitate that formed was collected by filtration and poured into approximately 2100 ml of ethyl acetate dissolved. The cyclohexane filtrate was discarded. The ethyl acetate solution, which contained the erizomycin, was filtered and the solids were discarded. The filtrate was then taken under reduced pressure at a temperature evaporated to dryness below 40 ° C. The dry residue of the ethyl acetate extract was dissolved in 420 ml of methylene chloride dissolved and the solution was filtered. The solids were discarded and the filtrate was reduced under reduced pressure Evaporated pressure, with a relatively pure, dry Preparation of erizomycin, which weighed 146 g and a content of 200 pg / mg, was obtained according to the B. cereus method

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true, exhibited.

Further purification of the erizomycin preparation was carried out with the aid of a partition chromatography column which was prepared as follows: 40 ml of propylene glycol were added to a "rolling slurry" of 120 g of buffered kieselguhr in "half-saturated" toluene. Stirring was continued for about 10 minutes in order to achieve good mixing. The mixture was poured into a glass column 2.54 cm in diameter and packed at an air pressure of 0.28 kg / cm. The packed column was covered with a layer of 6 mm sea sand. [The "semi-saturated" toluene used above was prepared as follows: 4 ml of propylene glycol were added to 3.79 liters of toluene with good mixing. This resulted in an approximately half-saturated solution which was used as the mobile phase for the partition chromatography column. The buffered kieselguhr used in the partition chromatography column was prepared as follows: 100 ml of a solution of KHpPO were added to 100 g of kieselguhr with thorough mixing. in water (27.2 g / 1) and 100 ml of a solution of Na ₂ HPO. given in water (28.4 g / l). The mixture was then carefully dried at 120 ° C.]

2.2 g of an erizomycin preparation, which after 'the methylene chloride extraction method described above were kept stirring and

- 30.-, 0 0 9 8 11/16 4 9

Heat on the steam bath in "?, 3 ml propylene glycol dissolved. This solution was mixed with 15 g of buffered diatomaceous earth. and rubbed with the smallest possible amount of half-saturated toluene and put on top of the partition chromatography. pillar given. A layer of 6 mm of sea sand was on top of the Given head of the pillar. The column was filled with half-saturated toluene at a flow rate of about 2 ml / min. eluted. 20 ml fractions were collected. Parliamentary groups 26 to 44 were combined, washed with water to remove the glycol and evaporated to dryness under reduced pressure? Yield 698 mg. This material was recrystallized from approx. 10 ml of ethanol ;? Yield 343 mg of crystalline erizomycin with a content of 420 µg / mg by the B. cereus method.

Example 2 Erizomycin hydrochloride

One gram of erizomycin, prepared as described in Example 1, is dissolved in 20 ml of methylene chloride. the Solution is filtered and hydrochloric acid gas is bubbled into the solution. The precipitate of erizomycin hydrochloride that forms is filtered off and dried.

Erizomycin has fungicidal activity as determined by a standard agar dilution plate test. It shows good and low activity against Nocardia asteroides

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Efficacy against Histoplasma capsulatum. Nocardia asteroides, which causes nocardiosis, was raised from the ground and isolated from laboratory air. As a result, erizomycin can be used to clean soils infected with Nocardia asteroides are to treat. Histoplasma capsulatum, which causes histoplasmosis, was made from storage cellars and chicken coops isolated. As a result, erizomycin can be used to treat such environments when associated with histoplasm capsulatum are infected.

0098 11/1 649

Claims (1)

Claims 1. Pharmaceutical preparation, corresponding to at least 9 Bio-units of erizomycin per ecm, a compound, the a) various types of growth inhibition Gram-negative and gram-positive bacteria are effective and essentially pure crystalline shape b) is soluble in ethyl acetate, ethanol, acetone and methylene chloride, c) has the following percentages according to the elemental analysis: C = 59.97 / °, H = 6.05 $, N = 10.46? S, 0 = 22, α) a molecular weight determined by mass spectrometry Tone 540, e) a characteristic infrared absorption spectrum according to FIG. 1 and f) a characteristic paper chromatogram according to FIG attached drawings. 2. Preparation according to claim i in dry form, accordingly at least 32.8 µg / mg on the Bacillus cereus test. i. Erizomycin according to claim 1 in its substantial 009Ö11 / 1649 pure form. 4. Erizomycin according to claim 1, in its substantially pure crystalline form. t). Erizomycin according to claim ι and its acid addition salts. 6. Preparation according to claim 1 in the form of its hydrochloride. 7. Erizomycin ö. A method for the production of the new one defined in claim 1 Antibiotic erizomycin, characterized in that an erizomycin-producing organism under aerobic Conditions grown in an aqueous nutrient medium until the medium has significant antimicrobial activity received, and that the erizomycin from the nutrient medium is isolated. 9. The method according to claim 8, characterized in that a Erizomycinzusammensetzung is prepared containing at least 9 biomoieties ji Erizomycin per ml. 10. The method according to claim 8, characterized in that Streptomyces griseus var. Erizensis is used as the microorganism will. 11. The method according to claim 10, characterized in that the Microorganism is grown in an aqueous nutrient medium that is an assimilable source of carbohydrates and Contains assimilable nitrogen. 12. The method according to claim 11, characterized in that the medium is filtered that the resulting filtrate with 0 0 9 8 11/16 4 9 a water-immiscible solvent ± * for erizomycin is extracted, and that the erizomycin is isolated from the solution mi'ttelextralct. For The Upjohn Company Kalamazoo, Michigan, V.St.A. Lawyer 11/16