DE1230525B - Process for the production and recovery of (+) - 3-cyclohexen-1-glyoxylic acid and / or (+) - 3-cyclohexene-glycine and / or (+) - 3-cyclohexen-1-hydroxyacetic acid and their salts and esters - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. Cl .:

Number:
File number:
Registration date:
Display day:

C12d

German class: 30h-6

P33743IV a / 30h

March 4th 1964

December 15, 1966

The invention relates to a method for producing and obtaining at least one of the antibiotic compounds (+) - 3-cyclohexene-1-glyoxylic acid, (+) - 3-Cyclohexene-glycine and (+) - 3 - cyclohexene - 1 - hydroxyacetic acid by cultivating Streptomyces antibioticus ATCC 11 891 or ATCC 14 890 in an aqueous, carbohydrate, source of organic nitrogen as well as inorganic Nutrient medium containing salts under submerged aerobic conditions to the medium essential antibiotic efficacy is imparted, extraction of the antibiotics from the medium and if necessary, conversion of the antibiotics into salts or esters in a manner known per se.

The process according to the invention consists in particular in that after fermentation has ended the culture filtrate at a pH value below about 5 with a cation exchange resin present in its H + form brings into contact and the (+) - 3-cyclohexene-glycine adsorbed by the cation exchange resin eluted, the liquid material passing through the cation exchanger at pH 1.5 with methyl isobutyl ketone extracted, the methyl isobutyl ketone solution extracted with an aqueous phosphate buffer, the buffer solution acidified over methylene chloride with a mineral acid to pH 4 and from the Methylene chloride layer the (+) - 3-cyclohexene-1-hydroxyacetic acid and from the aqueous layer the (-t-) -3-Cyclohexen-1-glyoxylic acid wins.

The Streptomyces antibioticus ATCC 11891 was already used for the production of the basic antibiotic Oleandomycin is used (see U.S. Patent 2,757,123). But no one knew that this microorganism other antibiotic substances, namely (+) - 3 - cyclohexene -1 - glyoxylic acid, (+) - 3-Cyclohexene-1-glycine and (+) - 3-Cyclohexene-1-hydroxyacetic acid generated. These antibiotics that get rid of the basic oleandomycin in the Liquid were previously discarded.

Process for the production and extraction of (+) - 3-Cyclohexene-1-glyoxylic acid and / or (+) - 3-Cyclohexenglycine and / or (+) - 3-Cyclohexene-1-hydroxyacetic acid and its salts and esters

Applicant:

Chas. Pfizer & Co., Inc.,

New York, N.Y. (V. St.A.)

Representative:

Dr. W. Beil, A. Hoeppener

and Dr. H. J. Wplff, lawyers, Frankfurt / M.-Höchst, Adelonstr. 58

Named as inventor:

Walter Daniel Celmer,

New London, Conn. (V. St. A.)

Claimed priority:

V. St. v. America of March 5, 1963 (262 829), July 5, 1963 (292 848)

Streptomyces antiobioticus ATCC 14890, which has not yet been described, only forms the method according to the method antibiotics to be produced and obtained and no oleandomycin. The cultural characteristics this microorganism are summarized below. (In Table I, (R) refers to to the "Color Standards and Nomenclature" by R i d g w a y.)

Table I.
Cultural characteristics of Streptomyces antibioticus ATCC 14890

medium Growth amount Aerial mycelium and spores Soluble pigment Remarks 609 747/299 Glucose Aspa-
ragin agar Well well, about »easy
mouse gray «(R) light brown Reverse pale yellow on the
Edges, turning brown towards the center More synthetic
Agar very weak very thin, pale
cinnamon no Colorless back

Continuation university;

medium Growth amount Aerial mycelium and spores Soluble pigment Remarks Inorganic Well good »easy mouse gray-brown Backside brown, .spore chains Medium # 1 gray «to straight to curved and in a loop after Gauze "Mouse gray" (R) fen, scattered along the hyphae; broad elliptical spores; Calcium weak, weak, about no Reverse light yellow, calcium malate Malät agar flat "Mouse gray" (R) weakly resolved Cellulose Well plentiful, about no "Mouse gray" (R) Skimmed milk moderate, in no dark brown im no coagulation, partial hydro tan upper hydrolysis lysis, change in the pH value Wrestling ized part, in the from 6.5 to 6.9 lower part about "Sayal Brown" after R id gway Kartoifel pieces Well well, gray gray to black vegetative mycelium yellow Nutrient agar weak to
moderate pale gray-white pale yellowish brown Back whitish Glucose agar Well good, off-white dark brown Gray-red-brown reverse side Emerson agar Well good, light gray white dark brown Reverse gray-red-brown, vegetative Mycelium yellow Yeast extract the end well, about dark brown Yellow back Agar after drawn "Mouse gray" (R) P r i d h a m Agar after Well well, about dark brown Back dark brown Hickey & "Mouse gray" (R) T r e s η e r gelatin moderate, flat whitish, moderate dark brown Reverse light yellow, good Liquefaction after only 7 days Starch agar weak, moderate, about no Reverse pale yellow, no hydrolysis flat »Olive gray« to of strength; Spore chains straight "Mouse gray" (R) to curved, short, densely heaped Organic no nitrite even after 3 weeks Nitrate broth formation, nitrates still present Inorganic no nitrite even after 3 weeks Nitrate broth formation, nitrates still present Peptone iron after 16 hours of incubation Agar Hydrogen sulfide "formed

Occasionally it has been found that some media change the color imparted to them somewhat. Color changes were found especially after several transfers. The reasons for this are currently unknown. The change in appearance has no noticeable effect on the ability of the organism to produce antibiotic substances.

The Streptomyces antibioticus ATCC 14890 grows on many media used for the cultivation of Streptomycetes. A satisfactory medium is. Pridham's Yeast Extract Agar. It consists of a mixture of 4.0 g yeast extract, 10.0 g malt extract and 4.0 g dextrose in 1000 ecm of water, which is adjusted to a pH of 7.3 with sodium hydroxide. Then 20.0 g of agar are added and the mixture is steamed for 15 to 20 minutes. It is then ^{sterilized at 121 °} C. for 15 minutes. The culture should grow at 28 ^° C.

Another suitable medium is one containing 10 g / l cerelose (dextrose hydrate), 5 g / l Curbay-BG (Molasses residue), 10 g / l corn starch, 10 g / l soy flour and contains 5 g / l sodium chloride. It is brought to a pH of 7.0 with 1N potassium hydroxide then add 10 g / l calcium carbonate

1 230 5.2%

given, and then ^{sterilized at 121 °} C. for 30 minutes. Inoculation is done with 2 to 7% of a 40 to 60 hour old culture grown in the same medium. The fermentation may be carried out at about 26 to 3O ⁰ C in 4-1 glass containers which are mechanically ventilated with stirring, about 65 to about 130 hours. The progress of the fermentation can be followed by stirring the effectiveness of the fermentation solution against Aerobacter aerogenes.

After a satisfactory level of antibiotic activity has been achieved, the active substances can be isolated by methods known per se. A particularly suitable method is as follows: The fermentation solution is filtered, brought to a pH value of 2.5 ± 1 and ^{extracted with 1} ^ to ¹ % of its volume of methyl isobutyl ketone or ethyl acetate. The organic layer is extracted by shaking with an aqueous sodium phosphate solution, the aqueous layer is separated off and extracted with an equal volume of methylene chloride. The methylene chloride layer is separated and treated with an equal volume of cyclohexene or heptane; the concentration of the resulting solution to about a quarter of its volume results in a precipitate of crystalline (+) - 3-cyclohexene-1-glyoxylic acid.

The crystalline antibiotic can be recrystallized its potassium salt is further purified from a mixture of methanol and isopropanol will. The less pure acid becomes 1.3 to 1.4 molar in about 10 parts by weight of methanol Solution solved. Then 4 n-methanolic potassium hydroxide is added in an amount that corresponds to the corresponds to the amount of acid originally used, and the precipitated solid is filtered off. That The filtrate is concentrated to about half its volume and, while stirring, with 1.5 to 2.0 parts by volume Isopropanol added. The potassium salt which separates out is filtered off and dried second batch is obtained by concentrating the mother liquor. As mentioned above, the Antibiotics to be produced and obtained according to the invention also from the known Streptomyces antibioticus strain ATCC 11891 produced, which forms oleandomycin in addition to these antibiotics.

The new Streptomyces antibioticus strain ATCC 14890 does not have the ability to produce oleandomycin and differs in other respects from the oleandomycin-producing Streptomyces antibioticus ATCC 11891. The differences are summarized in Table II.

Table II

Differences between ATCC 11891
and ATCC 14890

medium

Potato pieces

Emerson's agar

ATCC 11891

vegetative
Mycelium
yellowish green

Colonies with
gray spurs

vegetative
Mycelium yellow

Colonies with
greyish white spores

medium 5 Calcium ATCC 11891 ATCC 14890 strength Malate agar concentric no wax 10 inorganic Growth rings Medium # 1 rings after Gauze no resolution chess resolution ♦ soluble pig soluble pig ment light pink ment dark brown, back brown, back side dark side brown brownish red

55

ATCC 14890 To the valuable new antibiotics from fermentation solutions obtained by fermentation of the ATCC 11891 strain will be the Treated liquids previously discarded in the following way: By adding 85% phosphoric acid is brought to a pH of about 1 to about 5. Then it becomes about a quarter volume Methyl isobutyl ketone was added and the mixture was stirred for about 15 minutes. Then the aqueous layer separated and discarded. The organic layer is made six times, each with a quarter of its volume extracted on 0.35% disodium phosphate solution. The organic layer is discarded. the The aqueous layer is extracted with about a tenth of its volume in methylene chloride, the organic Layer separated and the aqueous layer brought to a pH of 2 with phosphoric acid. The aqueous layer is then extracted with about one tenth its volume of methylene chloride and the methylene chloride is distilled off in vacuo. The residue becomes about 50 parts by weight Dissolved methanol and the solution with 2 N-methanolic potassium hydroxide to a pH of 6.7 ± 0.1 brought. The solvents are then distilled off in vacuo. The crystalline residue is triturated with isopropanol, the crystals are filtered off and dried.

In general, for the production and production of the new antibiotics according to the Invention a) Streptomyces antibioticus ATCC 11891 or Streptomyees antibioticus ATCC 14890 in an aqueous nutrient medium, "the carbohydrate, a source of organic nitrogen and inorganic Containing salts, grow under submerged aerobic conditions until one opposed to the medium Aerobacter aerogenes is given substantial antimicrobial activity; Then it will be

b) the culture medium to a pH value below 5 acidified,

c) the culture medium in contact with a comminuted ion exchange resin in acidic form brought up the S-cyclohexene-l-glycine is essentially completely adsorbed,

d) the 3-cyclohexene-1-glycine adsorbate is separated off and

e) the cyclohexene-glycine obtained from the adsorbate.

A particularly suitable method for obtaining the pure compounds from their mixtures is shown in the following scheme:

Total fermentation solution,
acidified

Filtrate
pH = 2.5 ± 1

Cation exchange resin H ± form

outflowing

material

pH = 1.5

MIK

Eluate

(+) - 3-Cyclohexene-1-glycine

consumed

acidic material

pH = 9.5

MIK

- J.

more alkaline
MIC extract

Oleandomycin

consumed alic material acid extract

MIK
3.5% Na ₂ HPO ₄

in H ₂ O or

4.3% K2HPO4

inH ₂ O

consumed
MIK

weak acid

CH ₂ Cl ₂
(+) - 3-cyclohexene-1-hy-

hydroxyacetic acid

strongly acidic

CH ₂ Cl ₂

extract

(-) - 3-cyclo-

hexen-1-gly-

oxylic acid aqueous extract

pH = 4.0

CH ₂ Cl ₂

aqueous phase

pH = 1.5

CH ₂ Cl ₂

consumed aqueous
material

MIK = methyl isobutyl ketone and CH ₂ Cl ₂ = methylene chloride.

The above separation scheme shows that when using the Streptomyces antibioticus strain ATCC 14890, which does not produce oleandomycin, the steps indicated within the dashed lines are not used Need to become.

According to the separation scheme, the entire fermentation solution is first filtered and brought to a pH value of 2.5 ± 1. It is then treated with so much cation exchange resin in its H ⁺ form that all of the glycine present is removed. Usually 7% resin, based on the volume of the fermentation solution, is sufficient; however, the required amount can be determined analytically. The type of resin used is not critical. However, it has been found that a sulfonated polystyrene resin, the particles of which ^{pass a sieve of 56 to 400 mesh / cm 2} , e.g. B. Amberlite 1R-120, which has been converted from the sodium form to the H ⁺ form with a mineral acid and washed to pH 4 with deionized water, is particularly useful. The treatment with the ion exchanger is preferably carried out continuously in a tower. After the fermentation solution has been introduced into the tower, its lower seal is opened and the material flowing out is collected until its volume corresponds to the volume of the fermentation solution. It was found that the pH of the effluent decreased to 1.5 to 1.7. The outflowing material, which when Streptomyces antibioticus ATCC 11891 is used, contains oleandomycin and cyclohexene-glyoxylic acid and cyclohexenoic hydroxyacetic acid, is saved for recovery of these compounds. The tower is then washed with deionized water until the effluent

Material has reached a pH of about 4.0 to 4.3. The wash water is discarded.

The ion exchange resin containing adsorbed glycine is used in an amount of 15 to 20 percent by volume the original fermentation solution with 1.5 N ammonium hydroxide solution treated. After the pH of the eluate has risen above about 5, the eluate is collected until about 10 to 15% des Volume of the original fermentation solution are preserved. The eluate is in a vacuum at about 30 to 40 narrowed to about two thirds or half of its volume. During this stage, the excess ammonia removed. Then the pH is brought to 4.5 with mineral acid and further concentrated until the concentrated solution contains a total of about 10 percent by weight solids, which are filtered off and washed with a little water. The filter cake is with 95% Washed ethyl alcohol and air dried. The product obtained (+ J-S-cyclohexene-1-glycine can be made by suspending in about 100 parts by weight of water, adjusting the pH of the suspension with mineral acid to 1.75, treatment with about two thirds of the glycine weight of decolorizing charcoal, filtering and adjusting the pH value with a basic solution (e.g. potassium hydroxide) can be cleaned to 4.5. The crystalline suspension formed is filtered after standing overnight. The crystals obtained are to constant weight dried.

To isolate the (+) -3-cyclohexene-l-hydroxyacetic acid, the stored eluate is, if necessary, brought to a pH of 1.5 with a mineral acid and with about a fifth of its Volume of methyl isobutyl ketone (MIK) extracted. If according to the above scheme of Streptomyces antibioticus ATCC 11891 was used, the basic oleandomycin formed remains after the removal of the methyl isobutyl ketone extract in the spent acidic material. For this it can Oleandomycin e.g. B. by adjusting the pH to 9.5 with an alkaline agent, extracting with methyl isobutyl ketone, adjusting the pH to 1.5 with a dilute mineral acid, washing of the acidic extract with chloroform and then with heptane.

The methyl isobutyl ketone fraction containing the cyclohexene-1-hydroxyacetic acid contains, is with a buffer solution of aqueous 3.5% disodium hydrogen phosphate or aqueous 4.3% dipotassium extracted from hydrogen phosphate. Of this about a fifth of the volume of the organic solution is sufficient. The spent methyl isobutyl ketone layer is discarded and the aqueous buffer with a mineral acid over methylene chloride on one brought pH of 4.0. The methylene chloride layer is separated and the solvent is evaporated. The residue obtained is (+) -3-cyclohexene-1-hydroxyacetic acid. The (+) - 3-cyclohexene-1-glyoxylic acid can pass through from the aqueous phase Adjusting the pH with a mineral acid over methylene chloride to 1.5 can be obtained. the The methylene chloride phase is separated off, the same volume of isopropanol and the methylene chloride are added distilled off. The isopropanol solution is adjusted to pH 5.5 with 3 N methanolic potassium hydroxide brought to 6.0 and the solution concentrated. The precipitated crystals are filtered off and with Washed isopropanol. In this way, the (-r) -3-cyclohexene-1-glyoxylic acid is obtained in the form of it pure crystalline potassium salt.

The new compounds with antibiotic effect obtainable according to the method can be in an can be converted into salts and esters in a known manner. The salts are obtained z. B. by mixing the stoichiometrically equivalent amounts of acid with alkali metal or alkaline earth metal hydroxides in

ίο aqueous solution and evaporation of the water. Suitable hydroxides are above all sodium hydroxide, potassium hydroxide and calcium hydroxide.

The lowest water solubility of (i -) - 3-cyclohexene-glycine. that represents an amino acid is in the so-called isoelectric range for one pH from about 4 to about 8. The water solubility is determined by the preparation of the salts by reaction with stoichiometrically equivalent amounts of alkali metal or alkaline earth metal hydroxides,

z. B. sodium hydroxide, potassium hydroxide and calcium hydroxide improved. It can through implementation with mineral acid or organic acids, e.g. B. phosphoric acid, sulfuric acid, hydrochloric acid and Trichloroacetic acid can also be produced with addition salts to the amino group.

3-Cyclohexene-1-glyoxylic acid which can be prepared according to the invention and their derivatives have the general formula

in which R is hydrogen, an alkali metal, alkaline earth metal or an alkyl group having 1 to 3 carbon atoms means.

The ring carbon atom on which the glyoxyl group is attached is asymmetrical. 3-Cyclohexene-1-glyoxylic acid and their derivatives therefore appear in optically active forms and their racemic mixtures. The optically active stereoisomer, which rotates the plane of polarized light to the right and is hereinafter referred to as the (+) shape is antibiotic. The antibiotic effectiveness is particularly striking opposite gram-negative organisms, with no acute toxic side effects. Also against gram-positive microorganisms there is a certain abundance present. However, it is generally somewhat weaker.

The antibiotic effectiveness of (+) -3-cyclohexene-1-glyoxylic acid (R = H) towards a large number of organisms is particularly pronounced. It is therefore used in remedies for human and Used in veterinary medicine and agriculture, as well as in technical fermentations Scale,. To contamination by microorganisms to prevent. It is also useful for medical diagnostic procedures and for Separation and classification of organisms.

The in vitro effectiveness of (-j -) - 3-cyclohexenglyoxylic acid against a group of microorganisms that cause various diseases, was detected in Escherichia coli, Proteus vulgaris, Aerobaster aerogenes and Salmonella typhosa. In vivo, (+) -3-cyclohexene-glyoxylic acid has the ability to protect mice against infection

609 747 299

ΐί

to protect death caused by Escherichia coli. For example, (+) -3-cyclohexene-1-glyoxylic acid protected 50% (five out of ten, PDso) of the mice that had been infected intraperitoneally with Escherichia coli, strain 462, when the animals were given multiple oral doses ( ¹ IZ, 4, 24, 48 hours after infection) of 800 mg / kg. When administered subcutaneously, the corresponding PD50 was 500 mg / kg. The potassium salt of (+) -3-cyclohexene-1-glyoxylic acid showed only low acute toxicity in rats. No deaths were observed when administered orally at 2000 mg / kg or when administered intravenously at 300 mg / kg. The intravenous LD50 was 400 mg / kg.

The products of the process can be used in pure, raw form to treat infections will. This includes filtered fermentation solutions, such as those with the Streptomyces antibioticus ATCC 14890 obtained, or solid or liquid concentrates from these fermentation solutions. For administration to A non-toxic carrier is of course used for humans and animals, with toxicity here being a disadvantageous one Effect is understood in comparison to the dose administered without a carrier. It can be liquid or solid pharmaceutical carriers can be used, e.g. B. water, aqueous ethanol, isotonic salt solutions or glucose. Starch, lactose, calcium phosphate, animal feed or mixtures of different materials as found in a filtered fermentation solution. Both the oral and parenteral administration gives satisfactory results, albeit parenteral administration may be preferable until a suitable diet is put together for the patient in question is. For this purpose, solutions or suspensions in water, oils, such as Peanut oil, or other pharmaceutically acceptable solvents or carriers can be used. Furthermore, solid preparations for extemporaneous dilution can be prepared which various Buffering agents, local anesthetics and other drugs, e.g. B. antibiotics, hypnotics, analgesics as well contain inorganic salts to give the preparation the desired pharmaceutical properties to rent.

The levorotatory or (-) forms of 3-cyclohexene-1-glyoxylic acid and their derivatives, which are biologically inactive, have for the production of the antibiotic dextrorotatory forms Value. The left-turning form can be, for. B. by physical or chemical processes to one Mixture of (+) - and (-) - forms are racemized, expediently by treatment with a basic reagent such as potassium hydroxide. Then the active (-f -) - form according to conventional Procedure, e.g. B. by fractional crystallization, can be obtained from the mixture.

Although the stereoisomers are very stable at neutral and acidic pH values, they are racemized with increasing alkalinity of their solutions. For example, at the following pH values, the stated percentage of original rotation is retained after 19 hours: pH 7 = 99%. pH 8.9 - 86%, pH 9.5 - = 78%, pH 10.4 - 45% and pH 11.5 = 0%. At a pH of 12, all optical activity is lost in about 30 minutes. All measurements were carried out with the potassium salt in buffered 5% phosphate solutions at 25 C, with the exception of the measurement that was not carried out in buffered form at a pH of 12.
To obtain the (-) - form from the (-) - form, it is particularly useful to racemize the potassium salt in sodium hydroxide solution and the mixture consisting of the (- t -) - form and (-) - form in itself known way to separate by fractional crystallization of the salts with optically active basic alkaloids. For this purpose, the potassium salt of the optically active form can be dissolved in about 50 parts of water and the solution can be made basic up to a pH of 12.2 by adding 10 η aqueous sodium hydroxide. The progress of the racemization can easily be followed in a polarimeter. After about 1.5 hours at 25 ° C., the solution has lost its optical rotation. After a further half hour, the racemate can be extracted with the same volume by adjusting the solution with 50% phosphoric acid to a pH value of about 2 Methylene chloride, separation of the organic layer, drying of the organic layer and evaporation of the solvent. The racemate remains as a crystalline residue, which is separated into the pure (+) - and (-) - form that the salts with optically active basic alkaloids fractionally crystallized, the mother liquor removed for further crystallization and then the optically active acids regenerated.

The glycine compounds which can be prepared by the process according to the invention have the general characteristics formula

(«) CH - NHR ₁
C-OR

Il ο

in which a) R is hydrogen, an alkali metal or alkaline earth metal, an alkyl group with up to 10 carbon atoms, the phenyl group, alkyl-substituted phenyl groups with up to 10 carbon atoms or the benzyl group and Ri hydrogen, alkanoyl with up to 10 carbon atoms, the benzoyl radical, alkylierle benzoyl radicals with up to 10 carbon atoms or the benzyloxycarbonyl group. They also include acid addition salts of these compounds.

The compound in which R is hydrogen melts in its pure crystalline form with decomposition at above about 300 C; [«]" - 110 (± 10 ") (c - 1 in 2% aqueous HiPOi). In potassium bromide, characteristic absorption in the infrared range at 2.90. 3.40 (crystal accumulation). 3.65. 3.80, 4.70, 6.30. 6.60, 7.05, 7.15, 7.25, 7.60, 7.95, 8.25, 8.65, 9.20, 9.55, 9.80, 10.45, 10.65, 10, 90, 11.30, 11.60, 12.00, 12.40, 12.95, 13.70 and 14.10 μ (see diagram).

The ring carbon atom marked "1" on which the aminoacetic acid residue is located, and the Amino group-bearing, with "" marked carbon

The side chain's material atom is asymmetrical. Because of these two asymmetric carbon atoms 3-cyclohexene-1-glycine occurs in four optically active forms. The present invention relates in particular to that of the possible optically active stereoisomers of the general Formula II, which rotate the plane of polarized light to the right and hereinafter referred to as (+) shapes will. These forms have the same antibiotic properties as (+) - 3-cyclohexene-1-glyoxylic acid and their derivatives and are used in the same manner as these compounds.

(+) - 3-Cyclohexene-l-glycine of the above formula is particularly valuable as an antibiotic, in which R is hydrogen.

An optically inactive compound of the above formula II, in der.R hydrogen, is already known, as well as their antibiotic effect (cf. E d e 1 s ο η et al. in Journal of the American Chemical Society, Vol. 80, p. 2698 [1958]). The (+) -3-cyclohexene-1-glycine obtainable according to the invention differs from the "known optically inactive 3-cyclohexene-1-glycine" in several respects. The (+) - cyclohexene-l-glycine has a much stronger antibacterial when used in the same amount Efficacy against gram-negative organisms than the compound according to E d e 1 s ο n.

Thus, the (+) -3-cyclohexene-1-glycine according to the invention is essential to Escherichia coli more effective than the synthetic compound according to E d e 1 s ο n.

Table III shows the smallest inhibitory concentrations in y / cc of the optically inactive 3-cyclohexene-1-glycine in comparison to the corresponding values of (+) -3-cyclohexene-l-glycine according to the Invention specified. The measurement was carried out using standard dilution methods.

Table III

Smallest inhibiting concentration opposite
various bacteria, y / ccm

() -3-cyclohexene 3-cyclohexene bacteria 1-glyein (according to the l-glycine present (after Ed el son) Invention) Aerobacter aerogenic .... 1.95 31.2 Escherichia coli 0.95 1.95 Proteus vulgaris 7.8 250 Pseudomonas .. > 250 250 Salmonella .... 0.95 31.2 Streptococcus 125 125 Staphylococcus 250 250 Pasteurella .... > 250 > 250

In Table IV are the zones of inhibition against Aerobacter aerogenes for the (+) - 3-cyclohexene-1-glycine according to the invention and the 3-cyclohexenl-glycine according to E d e 1 s ο η indicated. The tested The larger the diameter of the zones of inhibition, the more effective the connection. The measurement took place according to the agar diffusion method.

Table IV

Comparison of the zones of inhibition
Aerobacter aerogenes

40

45

50

55

60

These data clearly show that the clockwise connection obtainable according to the invention is opposite most gram-negative bacteria at significantly lower concentrations is more effective than the known optically inactive compound.

link
per slice
(15 mm). (-) -3-cyclohexene
1-glycine (according to
the present
Invention). 3-cyclohexene
l-glycine (after
E de 1 s ο n). V mm mm 2000 50 39 1000 45 34 500 44 32 250 40 29 125 39 '25 62.5 36 19th 31.25 34 16 15.6 30th NZ *) 7.8 26th NZ 3.9 24 NZ 2.0 21 NZ 1.0 17th NZ 0.5 16 NZ

*) NZ inhibition zone could not be observed.

Differences in their ability to target mice were also found between the two compounds to protect against Escherichia coli infections. If there is a lot of Escherichia coli that killed all mice of a control group of 10 mice, it was found that all mice of the Group of ten were killed when using the optically inactive synthetic 3-cyclohexene-l-glycine after E d e 1 s ο η were treated, while four mice of the group of ten survived when they have been treated with the right-handed mold according to the invention. The dose of the two compounds was 800 mg / kg s.c.

There are also clear differences in the infrared spectra. Is special in this context to point out the absorptions of the compound of the invention at 7.15, 7.25, 12.95 and 14.2 microns, which for the compound after E d e 1 s ο η are absent, as well as an absorption at 7.35 microns by the compound according to Edelson, which is absent from the compound of the invention.

The reasons for the differences in the infrared spectra and in the antibiotic activity are not yet fully understood. It is assumed, however, that the antibiotic according to E de 1 s ο η is D, L -allo- 3-cyclohexene-1-glycine.

Since infrared measurements do not differentiate between enantiomorphic forms, but do differentiate geometric isomers, it is obvious that the glycine according to E de 1 s ο η represents the (-t-) -allo- 3-cyclohexene form and not an (i) racemate , that from the

(+) - 3-Cyclohexene can be obtained according to the present invention.

The novelty of the glycine which can be prepared according to the invention is also based on other properties of the (+) - 3-Cyclohexene-l-glycine and related compounds. The catalytic hydrogenation of the glycine according to the invention results in the absorption of one molar equivalent of hydrogen and yields the dextrorotatory cyclohexane glycine of the following formula:

H-C-NH ₂
CO ₂ H

[a] " = + 20 ° (c in 5N HCl).

This L-amino acid is known (see R u d man et al., Journal of the American Chemical Society, Vol. 74, p. 551 [1952]). The reduction proves the absolute configuration of the a-amino substituent of the compounds obtainable according to the invention. In order to avoid ambiguity, the term öS, which is used by C a h η et al. in Experientia, Vol. 12, p. 81 [1956] and is known to those skilled in the art, used to develop the Configuration of the asymmetric center, which is denoted by «in the above formula II, define.

In the system according to C a h η et al. becomes any of the four possible stereoisomeric 3-cyclohexene-1-glycine designated as follows:

■ Enantimorph forms ■

H - C - NH ₂

CO ₂ H

A.
(«S: 1 R)

H''

H ₂ N-C-H

CO ₂ H
C.
(α R: 1 R)

H - C - NH ₂

HO ₂ C

(α S: 1 S)

The compound obtainable according to the invention has the stereochemical configuration A (denoted f / S: 1 row). This results in the connection after E d e 1 s ο η, if you think of them as (±) -allo-3-cyclo-

denotes hexene-1-glycine, a 1: 1 mixture of the Glydenes with the configurations C and D («R: 1 R and S: 1 S). These configurations were as follows certainly.

After the a-substituent of the (+) -3-cyclohexene-1-glycine obtainable according to the invention has the S configuration, the configuration of the asymmetric position denoted by "1" in formula II is determined by reducing the position " 1 «with hydrogen with a significant decrease in the right-hand rotation ([«] "= 110 ± 10 ^J -> [u] a - —20) brought about symmetry. The asymmetrical position "1" must therefore contribute in a positive direction to the observed rotation of the (+) - 3-cyclohexene-1-glycine. As is well known, the absolute configuration of a clockwise asymmetrical position, which is denoted by "1" in formula II, can be recognized by testing analogous connections, the direction of rotation and configurations of which are known. Two such compounds are (R) -limonene and (R) -a-terpineol:

CH ₃

H C-OH

H ₃ C

CH ₃

(R) limes

(R) -a-terpineol
[a] _B = + 95.9 °

The R configuration of these compounds is fixed (see Can η et al., Loc. Cit., P. 85; Richter and Wolff, Reports, Vol. 63, p. 1724 [1930] and Stephan, J. pr. Chem., Vol. 62, p. 63 [1900]). It is therefore clear that the compound obtainable according to the invention in the system according to C a h η has the configuration mS: has 1 R.

The 3-cyclohexene-1-hydroxyacetic acid which can be prepared according to the invention and their derivatives have the general formula

(III)

CHORi
C-OR

Il ο

in which a) R is hydrogen, an alkali or alkaline earth metal, an alkyl group with up to 10 carbon atoms, the phenyl group, alkyl-substituted phenyl groups with up to 10 carbon atoms or the Benzyl group, and b) Ri is hydrogen, an alkanoyl group with up to 10 carbon atoms, the benzoyl group, alkyl-substituted benzoyl groups with up to 10 carbon atoms or an alkali metal or Alkaline earth metal means.

These compounds are also of value as antibiotics and accordingly have the same Areas of application such as the 3-cyclohexene-l-glyoxyl

acid and the S-cyclohexene-l-glycine as well as theirs Derivatives. They can also be used to produce the glycines described above. If you replace z. B. in the (+) -3-cyclohexene-l-hydroxyacetic acid through the hydroxyl group one after the other Halogen and then through the amino group, the end product retains the desired dextrorotatory Configuration. There is no loss of antimicrobial effectiveness during this conversion on.

10

example 1

20th

a) Streptomyces antibioticus ATCC 14890 are grown on an Emerson agar slant, to obtain spores for inoculation of a nutrient medium of the following composition:

Cerelose (dextrose hydrate) 10 g

Curbay-Bg (molasses residue) 5 g

Corn starch 10 g

Soy flour 10 g

Sodium chloride 5 g

This nutrient medium is diluted to 11 with water, then adjusted to pH 7 with potassium hydroxide and ^{sterilized at 121 °} C. for 30 minutes. It is then cooled and then inoculated under aseptic conditions with spores of Streptomyces antibioticus ATCC 14890. Cultivation takes place for 2 days at 28 ^° C. in Fernbach flasks on a shaking device. The fermentation solution-mycelium mixture obtained in this way is added to 20 parts by volume of a sterile fermentation medium of the same composition and the pH is adjusted to 7. The mixture is then ^{stirred at 28 °} C. for 108 hours and aerated.

The antibiotic effectiveness of the fermentation solution is determined against Aerobacter aesrogenes. The mycelium is filtered off and the filtrate is adjusted to pH 2 with phosphoric acid. The acidic solution is extracted with a quarter of its volume of methyl isobutyl ketone and the separated organic layer is extracted four times with the same volume of a 0.35% disodium phosphate buffer solution. The buffer extracts are combined and washed with one twelfth of their volume of methylene chloride. The methylene chloride layer is separated and discarded. The washed buffer is adjusted to pH 2 with phosphoric acid and extracted with an equal volume of fresh methylene chloride. The methylene chloride is separated off and concentrated to dryness. The crystalline residue is triturated with cyclohexene and the crystalline (+) - 3-cyclohexene-1-glyoxylic acid obtained is filtered off and dried. F = 55 to 56 ⁰ C; [«]" = + 60 ± 5 ° (in water). It was shown to be highly effective against a wide variety of gram-negative microorganisms both in vivo and in vitro.

b) The potassium salt [a] _D = +50 ± 5 ° (in water) is obtained by dissolving the acid in methanol up to a concentration of 1.3 mol. Then methanolic 4N potassium hydroxide is added up to pH 7 . Some solid that has precipitated is filtered off. The filtrate is concentrated to half its volume, 1.5 parts by volume of isopropanol are added and the potassium salt which has precipitated is filtered off and dried. It can be converted into the calcium salt in the following way.

To a solution of 1 g of crystalline potassium - (+) - 3-cyclohexene-1-glyoxylate 2 ecm of saturated aqueous calcium acetate solution are added to 10 ecm of water with stirring given. The precipitated crystalline calcium salt is filtered off and dried; yield : 660 mg. With stoichiometrically equivalent amounts of magnesium acetate and barium acetate the magnesium or barium salt of (+) - 3-cyclohexene-1-glyoxylic acid.

The sodium salt is obtained by adding a solution of 1.0 g of crystalline (+) -3-cyclohexene-1-glyoxylic acid in 20 ecm isopropanol dropwise with 6 ecm of a normal methanolic sodium hydroxide solution offset. The precipitated sodium salt is filtered off and dried. yield 560 mg.

c) The methyl ester is prepared by dissolving 40 g of crystalline (+) - 3-cyclohexene-1-glyoxylic acid in 120 ml of methylene chloride and treating with 40 ml of methanol and 0.8 ml of concentrated sulfuric acid. The solution is refluxed for 2 hours, cooled and filtered. The solvents are distilled off at 25 ⁰ C under a pressure of 15 ± 5 mmHg. The residue is distilled by increasing the bath temperature to about 140.degree. A first fraction (11.4 g, refractive index: 1.4735) and a second fraction (21.3 g, refractive index: 14740) are distilled off at about 100 ⁰ C. The ethyl or n-propyl ester of 3-cyclohexene-1-glyoxylic acid is obtained with the equivalent amount of ethanol or n-propanol.

Example 2

The procedure of Example 1 is repeated using ATCC 11891 strain. After removing the (+) - 3-cyclohexene-1-glyoxylic acid by extraction at acidic pH the aqueous phase is adjusted to a pH of about 9 and extracted with chloroform. Of the Chloroform extract is concentrated to a syrupy consistency, and retained after standing a crystalline oleandomycin chloroform solvate (cf. Celmer et al., Antibiotics Annual, 1957/1958, P. 478). A methanol solution of the chloroform solvate is distilled to azeotropically remove it. That The concentrate is carefully treated with water until the oleandomycin free base crystallizes is initiated. It was filtered off and found to be identical to the product of the USA patent 2,757,123. In contrast to (+) - 3-cyclohexene-1-glyoxylic acid, this antibiotic is highly effective against gram-positive microorganisms

Example 3

I a) Streptomyces antibioticus ATCC 14890 are grown on an Emerson agar slant, to obtain spores for inoculating a nutrient medium of the following composition :

Cerelose (dextrose hydrate) 10 g

Curbay-BG (molasses residue) .... 5 g

Corn starch 10 g

Soy flour 10 g

Sodium chloride 5 g

The nutrient medium is diluted to 11 with water and to a pH value with potassium hydroxide

609 747 299

set of 7 and ^{sterilized at 121 0} C for 30 minutes. It is then cooled and inoculated with the spores under aseptic conditions. Cultivation takes place for 2 days at 28 ^° C. in Fernbach flasks on a shaking device. The mixture of fermentation solution and mycelium obtained in this way is added to 20 parts by volume of a sterile medium of the same composition, the pH of which is adjusted to 7. The mixture is then ^{stirred at 28 °} C. for 108 hours and aerated. The antibiotic effectiveness of the fermentation solution is determined against Aerobacter aerogenes. The mycelium is filtered off and the filtrate is adjusted to a pH of 2.8 with phosphoric acid.

The filtered fermentation solution is sent through a tower which, based on the total volume of the Fermentation solution, containing 7% of an ion exchange resin (Amberlite IR-120), previously mixed with sulfuric acid and then washed with water until the effluent washing water had a pH of had about 4, so it is in the H + form. The outflowing Material is used to obtain (+) - 3-cyclohexene-1-hydroxyacetic acid and (+) - 3-cyclohexene-1-glyoxylic acid kept.

The resin on which the (+) - 3-cyclohexene-1-glycine is adsorbed is so long with deionized Washed water until the pH of the effluent reached about 4.0. The wash water is discarded. The resin is then with a solution of 1 part concentrated ammonium hydroxide treated in 10 parts of water (about 1.5 N). Having discarded the outflowing material for so long until the pH has risen above 5, the eluate is recovered. You get one Eluate amount of about 15% of the original fermentation solution volume. The eluate is in a vacuum 35 ± 5 ° concentrated to half its volume, with excess ammonia being removed. It is then brought to a pH of 4.5 with phosphoric acid. Then it is further concentrated, until the suspension contains 10% solids. The solids are filtered off and with little Water and then washed with 95% ethanol. The solids contain a mixture of ammonium phosphate and (+) - 3-cyclohexene-l-glycine, from which the glycine is obtained by the following process:

A slurry of 1 part of the solids in 3.3 parts of water is filtered and the filter cake washed with 0.45 parts of water and 0.67 parts of ethanol and air dried. A suspension of 1 part of the dried cake in 21 parts of water is adjusted to a pH of 1.65 with 50% phosphoric acid, then treated with 0.15 part of decolorizing charcoal, stirred for 1 hour and filtered. The filtrate is then adjusted to pH 4.5 with 2.5 liters of 5N potassium hydroxide and stirred overnight. The precipitated (+) - 3-cyclohexene-1-glycine crystals are filtered off and dried to a constant weight; F r ~> 300 ° (decomposition); [α] ο = + 100 ° (c = 1 in 2% aqueous H ₃ PO ₄ ).

A recrystallized sample [a] ₀ = +110 (± 10 °) (c = 1 in 2% aqueous H ₃ PO ₄ ) gave the following analytical values:

I b) The sodium salt of (+) - 3-cyclohexene-1-glycine is obtained by treating an aqueous glycine solution with the stoichiometric equivalent Amount of aqueous sodium hydroxide and evaporate the solution to dryness. When using aqueous Solutions of lithium hydroxide, potassium hydroxide, calcium hydroxide and magnesium hydroxide are used get the lithium, potassium, calcium or magnesium salt.

The hydrochloric acid addition salt of (+) - 3-cyclohexene-1-glycine is formed when the stoichiometrically equivalent amount of aqueous hydrochloric acid is added an aqueous glycine solution and evaporating the solution to dryness. The Addition salts produced with phosphoric, sulfuric and trichloroacetic acid.

I c) The methyl ester of (+) - 3-cyclohexene-l-glycine is obtained by using a mixture which which contains glycine in excess over methyl alcohol, in the presence of a catalytic amount Heated hydrogen chloride under reflux and then evaporated the excess solvent. In the same In this way, the ethyl and i-propyl esters of glycine are produced. The t-butyl ester is obtained in that a mixture of the glycine with the stoichiometrically equivalent amount of t-butanol in a non-reactive solvent in the presence of a catalytic amount of sulfuric acid heated under reflux and recovered the ester by distillation.

The following esters were also produced:

CH ₃ (CHa) ₉ -C ₆ H ₅

2-CH ₃ C ₆ H ₄
4-CH ₃ (CHa) ₃ C ₆ H ₄
C ₆ H ₅ CH ₂
4-O2N - C ₆ H ₄

C ₈ Hi ₃ NO ₂ :

Calculated
found

C 61.91, H 8.44, N 9.03;
C 61.84, H 8.31, N 9.08.

I d) The N-acetyl derivative of (+) - 3-cyclohexene-1-glycine is obtained by mixing aqueous solutions of glycine with acetic anhydride at 30 ⁰ C. The mixture is allowed to stand so long, until the reaction is complete. The solvent and the acetic acid formed as a by-product are then distilled off. The product is obtained as a residue.

The N-benzoyl derivative of (+) - 3-cyclohexene-1-glycine is prepared by adding an excess of 20% strength aqueous sodium hydroxide to a suspension of benzoyl chloride in water and the stoichiometrically equivalent amount of the glycine and vigorous shaking. The aqueous solution of the sodium salt is acidified to Congo red with dilute hydrochloric acid and the precipitated solid product is recrystallized from 60% ethanol; F = 174 to 176 ° C; [a] _D = + 77 ° (c = 1 in 0.5 molar aqueous K ₂ HPO ₄ ). In the same way, the N-carbobenzoxy derivative (F = 109 ° C; [a] ₀ = + 60 ° (c = 3.5 in isopropanol), the N-4-i-propylbenzoyl derivative and the Nn-decanoyl derivative of the (+) -3-cyclohexene-l-glycine produced.

The N-acyl derivatives of (+) - 3-cyclohexene-1-glycine can also be esterified. An example

this is the carbobenzoxy derivative of the formula

H 'HC-NH-CO-CH ₂
C = O
O

NO ₂

After recrystallization from isopropyl ether, it melts at 80 to 81 ^° C .; [a] "= + 22.5 ° (c = 2 in dioxane).

Dissolve 3.6 g of the carbobenzoxy derivative and 1.92 ecm of N-ethylpiperidine at -5 ° C. in 50 ecm Acetone and 1.18 ecm of ethyl chloroformate are added to give the mixed anhydride

HC-NH-CO-CH ₂

= O

O - CO ₂ C ₂ H ₅

After one hour at -5 ° C to - 40 ⁰ C cooled. At −3 ° C., a solution of 3.24 g of 6-aminopenicillanic acid in 63 ml of 3% NaHCOe and 35 ml of acetone is added. The reaction mixture is stirred for 15 minutes at -15 ° C. and for 1 hour without external cooling. The solution is brought from 8.4 to pH 7.7 with 50% H ₃ PO _4. Then it is extracted with 100 ecm diethyl ether. The aqueous phase is separated off, adjusted to a pH of 2.5, extracted once with 250 ecm and once with 100 ecm of methyl isobutyl ketone (MIK). The MIK extracts are combined and washed with 50 ecm of water. They are then extracted with 250 ecm of water that has been adjusted to a pH of 7 with 5N KOH solution. The aqueous extracts are dried by freezing. 5.3 g of the potassium salt of the desired penicillin derivative are obtained; [a] _D = + 192 ° (c = 2 in H ₂ O):

Il

HC-NH-CO-CH ₂ -C ₆ H ₅

/ ^s \

C-NH-CH-CH C (CHs) ₂

OCN CH ^ CO ₂ H

A solution of 4 g of this compound in 20 ecm of water is added to 7.5 g of previously reduced 30% palladium on barium carbonate, which is suspended in 70 ecm of water and hydrogenated ^{at 3.52 kg / cm 2 for 0.5 hours.} The catalyst is filtered off, the filtrate is cooled to 6 ° C. and adjusted to a pH of 2.5 with 50% H3PO4 over the same volume of methylene chloride. The phases are separated and the aqueous phase is extracted with an equal volume of methylene chloride. The aqueous phase is adjusted to a pH of 5 with 5N KOH solution and then concentrated in vacuo at 27.degree. The deposited crystals are filtered off, washed with a little water and then with acetone and dried; Yield: 686 mg; [a] ₀ = + 120 ° (c = 1 in water). The microbiological effectiveness of carbobenzoxypenicillin A and its hydrogenated product B are shown in Table V.

Table V

γ of the connection
per slice comparison B. the inhibition zones B. 25th opposite to
B. subtilis, ram 47 40 1000 A. 40 30th 30th 100 42 34 20th 10 36 29 KHZ 35 1 29 22nd KHZ 0.1 22nd 16 against Arobacter
aerogenes, mm A. 19th KHZ *) KHZ KHZ KHZ

*) No inhibition zone.

Both compounds are active antibiotics. The hydrogenated compound shows somewhat greater effectiveness.

II a) To obtain the (+) - 3-cyclohexene-1-hydroxyacetic acid If necessary, the stored material which has flowed out of the ion exchanger is adjusted to pH 1.5 with sulfuric acid. It is then extracted with a fifth of the volume of methyl isopropyl ketone. The inorganic Layer is separated and extracted with a 3.5% disodium phosphate buffer solution. Therefor one fifth of the volume of the organic layer is used. The phosphate buffer solution is from separated from the organic layer and adjusted to pH 4.0 with phosphoric acid. The solution obtained is extracted with methylene chloride (Vs volume). The methylene chloride layer is separated and the solvent is evaporated. The one after The residue remaining on evaporation consists of crude (+) - 3-cyclohexene-1-hydroxyacetic acid.

An analytically pure product is obtained by recrystallization from benzene; F = 118.5 to 120.5 ° C; [a] o = + 78 ° (c = 1 in H ₂ O).

Calculated
found

C 61.52, H 7.75;
C 61.34, H 7.73.

II b) The (+ ^ - cyclohexene-1-hydroxyacetic acid became in the same way as the (+) - 3-cyclohexene-1-glycine converted into alkali and alkaline earth salts as well as into esters.

Further, the O-acetyl derivative was prepared by the acid was dissolved in a 5% strength aqueous sodium hydroxide solution and a small amount Ice and a slight excess of acetic anhydride were added. The mixture was about 12 hours stirred vigorously, then acidified with 5% hydrochloric acid and the precipitated product filtered off. That O-benzoyl derivative is made by shaking a mixture of about 20 ecm 5% for 20 minutes Sodium hydroxide solution, 0.5 g acid, 5 ecm chloroform and 0.5 g benzoyl chloride and 12 hours long standing made. The chloroform layer is separated and reduced to about 2 to 3 ecm constricted. Then 10 parts by volume of hexene are added. The precipitated product is filtered off and washed with witches. The O-4-i-propylbenzoyl derivative is obtained by the same procedure of 3-cyclohexene-1-hydroxyacetic acid.

III) To obtain the (+) - 3-cyclohexene-1-glyoxylic acid, the aqueous phase is treated with phosphoric acid Brought to a pH of 1.5 via methylene chloride, the methylene chloride layer separated and heated with the same volume of isopropanol until all of the methylene chloride has distilled off. the Isopropanol solution is then with methanolic 3 N potassium hydroxide to a pH of 5.5 set. The crystalline potassium salt of (+) - 3-cyclohexene-1-glyoxylic acid is obtained and is filtered off and is dried.

Example 4

The procedure of Example 3 is carried out using the Streptomyces antibioticus ATCC 11891, which produces oleandomycin in addition to the antibiotics to be produced according to the method, repeated. The isolation the individual compounds from the fermentation solution was carried out in the following way: The filtered fermentation solution is passed through a tower which contains an ion exchange resin in the H + form (Amberlite IR-120, previously made with sulfuric acid and then washed with water until the effluent wash water had a pH of about 4 had achieved). The resin was used in an amount equal to 7% of the total volume of the fermentation solution. The outflowing material is used to obtain oleandomycin, (+) - 3 - cyclohexene -1 - hydroxyacetic acid and (+) - 3-cyclohexene-1-glyoxylic acid stored.

The resin on which the (+) - 3-cyclohexene-1-glycine is adsorbed is washed with deionized water until the pH of the material flowing out has reached about 4.0. The wash water is discarded. The resin is then treated with a solution of 1 part of concentrated ammonium hydroxide in 10 parts of water (about 1.5 N). The effluent is discarded until the pH rises to about 5. Then the eluate is obtained. 15% of the original volume of fermentation solution is recovered as eluate. The eluate is concentrated to half its volume in vacuo at a temperature of 35 ± 5 ° C., excess ammonia being removed. The concentrate is adjusted to a pH of 4.5 with phosphoric acid. It is then further concentrated until the suspension contains a total of 10% solids. The solids are filtered off and washed with a little water and then with 95% ethanol. The solids contain a mixture of ammonium phosphate and (+) - 3-cyclohexene-1-glycine, from which the glycine is obtained in the following manner: A slurry of 1 part solids in 3.3 parts water is filtered. The filter cake is washed with 0.45 parts of water and 0.67 parts of ethanol and air-dried. A suspension of 1 part of the dried cake in 21 parts of water is adjusted to a pH of 1.65 with 50% phosphoric acid, then treated with 0.15 part of decolorizing charcoal, stirred for 1 hour and filtered. The filtrate is adjusted to pH 4.5 with 2.5 1 5 N potassium hydroxide and stirred overnight. The precipitated crystals of (+) - 3-cyclohexene-1-glycine are filtered off and dried until a constant weight is achieved; F, ^ 300 ⁰ C (decomposition); [α] = +100 (c = 1 in 2% aqueous H ₃ PO ₄ ).

The stored material which has flowed out of the ion exchange tower is adjusted to a pH of 1.5 with sulfuric acid to obtain the oleandomycin. It is then ^{extracted with 1} Js volume of methyl isobutyl ketone and the organic layer is stored for obtaining (+) - 3-cyclohexene-1-glyoxylic acid and (+) - 3-cyclohexene-1-hydroxyacetic acid. After extraction with the ketone, the acidic layer contains the oleandomycin as its salt. It is adjusted to pH 9.5 with a sodium hydroxide solution. The oleandomycin released in this way is ^{extracted with 1} Z ₅ volumes of methyl isobutyl ketone. After the solvent has been evaporated off in vacuo, the oleandomycin is obtained as a residue. To obtain the (+) - 3-cyclohexene-1-hydroxyacetic acid and the (+) - 3-cyclohexene-1-glyoxylic acid, the procedure of Example 3 is applied to the stored organic layer.

Claims (2)

Patent claims: 1. A process for the production and extraction of at least one of the antibiotic compounds (+) - 3 - cyclohexene -1 - glyoxylic acid, (+) - 3-Cyclohexen-glycine and (+) - 3-Cyclohexen-1-hydroxyacetic acid, characterized in that that you can get Streptomyces antiboticus ATCC 11891 or ATCC 14890 in an aqueous, carbohydrate, a source of organic Nutrient medium containing nitrogen and inorganic salts under submerged aerobic conditions cultivates the antibiotics until the medium is given substantial antibiotic activity wins from the medium and converts them into salts or Ester transferred. 2. The method according to claim 1, characterized in that after fermentation has ended the culture filtrate at a pH value below about 5 with a cation exchange resin present in its H + form brings into contact and the adsorbed by the cation exchange resin (+) - 3 - Cyclohexenglycine elutes, the liquid material passing through the cation exchanger extracted at pH 1.5 with methyl isobutyl ketone, the methyl isobutyl ketone solution with an aqueous Phosphate buffer extracted, the buffer solution on methylene chloride with a mineral acid pH 4 acidified and from the methylene chloride layer the (+) - 3-cyclohexene-1-hydroxyacetic acid and - (+) - 3-cyclohexene-1-glyoxylic acid from the aqueous layer wins. Documents considered: German Patent No. 946 256; U.S. Patent No. 2,757,123. 1 sheet of drawings