DE2660659C2 - Process for the preparation of 7-methoxycephalosporin derivatives - Google Patents

Description

wherein R ^{2 is} an imidazolyl, pyrazolyl, triazolyl, tetrazolyl or thiadiazolyl group which can be substituted by an alkyl or carboxyalkylthio group, and M denotes a hydrogen atom or a cationic salt-forming radical, characterized in that Streptomyces oganonensis ATCC 31167 in one Culture medium is grown, which nutrients with the addition of a heterocyclic thiol with the formula

R ² SH,

wherein R ^{2 has} the meaning given above, or its salt or a compound with the formula

R ² —S — S — R ² ,

wherein R ^{2 has} the same meaning already mentioned.

30 Proteus against which ordinary antibiotics are not effective, or which are caused by infection with the bacteria which are not sensitive to ordinary cephalosporins which do not have a methoxy group in the 7-position.

These compounds are usually made by first making the appropriate compounds that have a Have acetoxymethyl group or a carbamoyloxymethyl group in the 3-position, are produced, namely by a fermentation process, and then these compounds with a heterocyclic Thiol compound implemented.

The invention has the advantage that the compound with a heterocyclic thiomethyl group in the 3-position can be obtained directly by a single fermentation stage. The compound so obtained is the compound with the general formula A.

The compounds of Formula A have excellent antimicrobial activities of their own, and continue to do so can have the antimicrobial properties and the antimicrobial Spectra of the compounds can be increased or changed by adding the acyl group side chain in the 7 position represented by

The invention relates to the method characterized by the claim.

As a typical group, which is indicated by R ² in the above general formula, the following are mentioned for clarification: a carboxymethylthio-U ^ -thiadiazol-2-yl group, an i-methyl-1H-tetrazol-5-yl group, a 5-methyl-1,3,4-thiadiazol-2-yl group or a 1,3,4-thiadiazol-2-yl group.

The cationic residue represented by M which constitutes the salt of cephalosporin means an inorganic one Residue or an organic residue. Examples of the inorganic residue are an alkali metal such as sodium, Potassium; an alkaline earth metal such as calcium. Magnesium, Barium; and a heavy metal such as iron, copper, zinc; Examples of the organic radical are base-forming quaternary salts, amine salts, e.g. B. triethylamine. Diethanolamine, piperidine, morpholine.

Some 7-metlloxy-3-heterocyclotlliomethylcephalo- so sporins are described in GB-PS 1321412 (1970) as compounds obtainable by chemical synthesis, however, there are no practical physical and chemical properties in this British patent of these compounds. DE-OS 2304226 also discloses the production of such compounds.

The object of this invention is to provide a process for the production of these derivatives by fermentation to deliver. tio cephalosporins have excellent antimicrobial properties Have activities against gram-positive and gram-negative bacteria, and among these compounds, the Cephalosporin derivative having a methoxy group in the 7-position and a heterocyclic thiomethyl group in the 3 position quite excellent effect in the treatment of serious diseases caused by infection with bacteria such as Pseudomonas and H, N

HOOC

CH- (CH ₂ ) ₃ -CO-

with an i'nd:: n acyl group. such as B. α-aminophenylacetyl group, ot-carboxyphenylacetyl group. oc-sulfophenylacetyl group, a-hydroxyphenylacetyl group. Pyridylthioacetyl group, thiadiazolylthioacetyl group, triazolylacetyl group, cyanomethylthioacetyl group, trifluoromethylthioacetyl group. Thus, the compounds with the formula A can also be used as intermediates in order to prepare these derivatives with these acyl groups. For example: 7ß-Cyanomethylthioacetamido-7oc-methoxy-3- (1 methyltetrazol-S-ylthiomethyO-A -'-cephem ^ -carboxylic acid and 7a-methoxy-3- (1-methyltetrazol) -5-ythiomethyl) -7ß- ( trifluoromethylthioacetamido) -A ³ -cephem-4-carboxylic acid.

The compounds of the formula A have the properties of an amphoteric material, because the compounds have one amino group and two carboxy groups in the molecule, and this makes the isolation and Cleaning of the connections made troublesome.

In this invention a new stream is used, namely Streptomyces oganonensis Y-G19Z. the one previously by the inventors from the ground near the city Ogano, Chichibugun, Saitama District, Japan. This strain is in the Microbial Institute Industry. Industrial Science and Technology Branch. Ministry of International Trade and Industry, Japan, under an accession number FERM-P 2725 and also in the American Type Culture Collection, 12301 Parklawn Drive, Rockville. marryland 20852 (USA) under ATCC No. 31167 deposited been. The mycological properties of the strain are as follows:

I. Morphological characteristics of Streptomyces oganonensis Y-G19Z strain:

The trunk grows on both natural and synthetic media to form a well-branched one Substrate mycels. while the formation of atmospheric mycelium is insufficient. because the spore formation / u is low. The spore chains are straight, belong to the R "(rectus) or RF (Rectiflexiblcs) type and wear 10 to 50 spurs in each chain. The spurs are

elliptical, spherical or cylindrical in shape and have a size of 0.45 to 0.60x0.55 to 0.90 μ. the Spore surface is smooth. Neither flagellate spores nor sporangium are observed.

II. Culture characteristics of the S. oganonensis Y-G19Z strain:

Medium Growth Atmospheric Soluble

ric mycelium pigment

Czapek's

Glucose Czapek's

Glucose Aspa ^ agin-

Glycerin asparagine

Inorganic Salt Starch

Tyrosine agar

Iron and Hete Extract Tyrosine Agar

Culture medium agar

Bennett's agar

Calcium malate agar potato pieces

Blood agar

Loeffer's Serum Medium

very little sparse

know white

well tolerable

cream yellow yellowish gray

good low

know white

good low

white to white to yellowish-white gelbüch-white good low

yellowish-gray yellowish-gray to slightly yellowish-brown good low

pale yellow yellowish gray

Well

weak yellow to yellowish brown good

pale yellowish brown

Well

pale pale brown

moderately creamy color good

pale yellowish brown

Well

olive-gray to dark olive-gray good

Well

brownish white to yellowish gray

good, powdery slightly orange to slightly brown good

brownish-gray, pale orange to pale pink none

Well

yellowish gray to slightly brownish gray none

no

no

very low

no

no

no

weak weak yellowish-gray very little light brownish-gray

pale yellow-brown

very weak

no

no

brownish gray to dark yellowish brown

yellowish-gray to dark reddish-brown

no

Cellulose degradation negative

Hemolysis positive solubilization of calcium

malate positive

Utilization of carbon compounds by S. oganonensis Y-G19Z:

Carbon source exploitation

Glucose +

Arabinose +

Sucrose -

Xylose +

Inositol -

Mannitol +

Fructose +

Rhamnose -

Raffinose -

Characteristics of Streptomycc; oganonensis Y-Gl 9Z strain:

1. It belongs to the non-chromogenic Strepioirvces Tribe;

2. Its atmospheric mycelium is straight without branching (R or RF type);

3. Spores are spherical or elliptical;

4. Spore surface is smooth;

5. There are pale yellowish-gray to pale yellowish-brown growths on various media;

6. The color of the spherical mycelium is brownish-white, yellowish-white and yellowish-gray;

7. The produced antibiotic substance Y-G19Z-D3 belongs to the 7-methoxycephalosporin group.

When researching known strains exhibiting the above properties, am next upcoming streptomycetes the following are considered:

Streptomyces globisporus, described in S. A. Waksman: "The Actinomycetes" 2, 218 (1961) and International Journal of Systematic Bacteriology, 18, (4) 324-325 (1968).

However, if you consider the well-known S. globisporus. which is described in the above literature, with the When comparing strain Y-G19Z, it differs in the following properties, which are given in the table:

Tabel

Features: Y-G19Z

S. globisporus

III. Physiological properties of S. oganonensis Y-G19Z strain:

negative

positive

positive, weak

positive, weak

positive

positive, weak

Size of the 0.45-0.60x0.55-0.90 1.2-1.4x8-2.0 Spore (µ) ■ or 0.9-1.4

spherical no

Tyrosine formation Nitrate reduction Skimmed milk coagulation Skimmed milk peptonization Hydrolysis of starch liquefaction of gelatin

Liquid

Pigment or

Glycerine

Asparagine

medium

Rhamnose negative

Recovery

Strong-strong

hydrolysis

Skimmed milk positive

Coagulation

Skimmed-weak

Peptonization

Production positive

by Cephalo-

sporin anti

biotica

yellowish to greenish-yellow

positive weak negative strong

negative

From the differences given in the table above, it can be seen that the was used in this invention Strain is a new strain different from the above known strains.

Since the Y-G19Z strain has been confirmed as a new strain from the above observation results is, it has been referred to as "Streptomyces oganonensis".

As already stated in the comments on the Streptomyces oganonensis Y-19Z strain, is a 7-methoxycephalosporin antibiotic producing strain. As similar tribes that belong to the Streptomycetes, the following are known to be producers of 7-methoxycephalosporin antibiotics:

Streptomyces griseus, Streptomyces viridochromogenes, Streptomyces fimbriatus, Streptomyces halstedii, Streptomyces rochei, Streptomyces cinnamonensis, Streptomyces chartreusis and Streptomyces lactamdurans (cf. Japanese Patent Laid-Open No. 3286/71 and Beigische Patent 7,64160) and Streptomyces lipmanii (U.S. Patent 3,719,563); Streptomyces clavuligerus (Japanese Patent 45,594/74). Streptomyces wadayamensis (Japanese Patent Application Laid-Open No. 26488/74). Streptomyces jumonjinensis (Japanese Patent Application Laid-Open 42 893/74). Streptomyces heteromorphus and Streptomyces panayensis (Japanese laid-open specification 53 594 / 75). and Streptomyces chartreusis SF-1623 (Japanese Patent Laid-Open Nos. 82291/75 and 121488/75).

The preparation of the discussed compound A is carried out by cultivating the 7-methoxycephalosporin antibiotic-producing strain mentioned in a conventional culture medium to which an addition of heterocyclic thiol compound corresponding to the heterocyclic thio group R ² —S— has been added. to be inserted in the 3 position.

Examples of the heterocyclic thiols added to the culture medium in this invention are: imidazolthiol. Pyrazolthiol. Triazol thiol, tetrazol thiol. Methyltetrazolthiol.thiadiazolthiol.

These heterocyclic rings can carry an alkyl or carboxyalkylthio group.

These heterocyclic thiols can be used as their salts. These salts are inorganic salts, such as alkali metal salts. Alkaline earth metal salts. Ammonium salts and the salts with organic bases, such as triethylamine. Triethanolamine. Dicyclohexylamine, lysine. Arginine. Histidine, basic water-soluble antibiotics, e.g. B. Kanamycin. Alkaloids, basic proteins. The salts having high water solubility can be used selectively. if necessary, and further if the heterocyclic thiols have strong toxicity to the antibiotics having producing strains, the sparingly water-soluble salts can be used selectively.

The disulfides R ² —S — S — R ² can also be used.

The cultivation is carried out by the usual conventional methods for growing microorganisms performed, but submerged culture in a liquid culture medium is preferred. It can synthe- μ table culture media, semi-synthetic culture media and natural culture media are used. For the Composition of the culture medium can be glucose. Sucrose, mannitol, glycerin, dextrin, starch and vegetable oils are used as carbon sources, and meat extract, peptones, gluten meal, cottonseed meal, Soybean meal, peanut meal, fish meal. Grain pulp. Dry yeast. Yeast extract. Ammonium sulfate, Ammonium nitrate, urea and other organic and inorganic nitrogen sources can be used as nitrogen donors Find use. Likewise, if necessary. Metal salts such as sulfates, nitrates, chlorides. Carbonates. Phosphates of sodium, potassium. Magnesium, calcium, zinc and iron added to the culture medium will. Furthermore, if necessary. Materials that favor the formation of antibiotics, or Defoaming agents such as methionine. Cysteine, Methyloieat, Lardöi, Siiiconöi, surface-active agents, in appropriate Way to be added to the culture medium.

The above-mentioned heterocyclic thiol, its salt or its derivative is usually added in a concentration of 0.1-5 mg / ml, preferably 0.5-2 mg / ml as the heterocyclic thiol. It can be added to the culture medium as a fall-like addition before the cultivation or else in different subdivided portions in the initial stages of the cultivation. It is generally favorable to carry out the cultivation under aerobic conditions. The cultivation temperature is usually between about 18 ° C to about 35 ⁰ C., preferably 30 ⁼ C. Good results are obtained when the pH of the culture medium at about 5-10. preferably about 6-8 is held. The cultivation time depends on the composition and the temperature of the culture medium used. but generally it takes about 3 days to about 10 days. The produced material is selectively enriched in the medium after the cultivation is finished.

The antibiotic produced can be isolated and obtained from the culture broth by the usual methods that are common for the isolation of antibiotics from the cultivated mycelium medium. The manufactured Antibiotic is mainly present in the culture broth. After the mycelium has been separated from the Culture medium solution by centrifugation or filtration becomes the produced effective material from the filtrate extracted; that is, the produced material is separated, pulled out again and purified from the filtrate. Devices and methods are used here, which are used in general for the production of antibiotics, as are methods that highlight the differences the solubility in a suitable solvent take advantage of the differences in adsorptive affinity

uit »ouch

Differences in the separation methods are based in two liquid phases.

These methods can be used in a suitable combination or repeated. Some practical examples of the invention 7-MethoxycephaIosporin compounds produced are named below:

I. 7- (5-Amino-5-carboxyvaleramido) -3- (5-carboxy-methylthio-1,3,4-thiadiazol-2-yI) thiomethyl-7-methoxy-A ³ -cephem-4-carboxylic acid;

OCH,

HOOC-CH-CH ₂ CH ₂ Ch ₂ CONH

NH,

N-N

CH ₂ -SI JS-CH ₇ COOH

The physical and chemical properties of the Compound I produced are the following:

1. white powder;

2. Beginning of melting at 156-160 C. Brown discoloration occurs here a. and the decomposition begins at about 170 C;

3. Slightly soluble in water, sparingly soluble in methanol, and very sparingly soluble in other organic ones Solvents;

4. Umphoteric material with a positive ninhydrin reaction:

5. It has the ultraviolet absorption spectrum shown in FIG. 1 of the accompanying drawings when it is in a 1/100 M phosphate buffer solution with a pH vuii 6.4 is measured, the absorption maxi-! 5 mum is 287 ιτιμ;

6. It has the infrared absorption spectrum according to FIG. 2. when it is measured in the potassium bromide tablet. This results in absorptions at 3413cm ^"1, 2920cm" ^1, 1763cm ^"1, 1620cm ~ ', 20 13i5 cm" ¹ and 1380 cm ^"1;

7. When the nuclear magnetic resonance spectrum is determined using TMS as an external standard in heavy water, the following signals are obtained: fi value (ppm): 2.35 (4H, multiples). 2.96 (2H. Multiplet), 4.00 (3H. Singlet), 3.73-4.33 (2H, quartet, J = 18 Hz). 4.25 (IH. Multiples), 4.44 (2H. Singlet), 4.42-4.91 (2H. Quartet. J = 14 Hz), 5.63 (IH. Singlet);

8. The product produced in the purest state at the moment has the following elementary-analytical properties Values: C: 35.95%. H: 3.87%. N: 10.85%. S: 18.33%:

9. It gives ot-aminoadipic acid on hydrolysis with 6 η hydrochloric acid:

10. After N-chloroacetylation of the compound and conversion of the product into the methyl ester, the mass spectrum of this compound gives the following fragment with m / e 392. That is, (1,3,4-thiadiazol-2-yl) thiomethyl-A ³ -cephem- 4-carboxylic acid.

The following are the results of various chromatographic analyzes and paper electrophoretic ones Investigations of the compounds I, II, III and IV prepared are indicated.

The obtained Rf values of these compounds in thin layer chromatography using microcrystalline Cellulose (Avicel "SF) are shown in the following table:

- <^ ^N V. S-CH ₂ COOCH,

40

H "N V-CH ₂ -S-

COOCH,

With all of the above results in mind, it can be seen that this compound is a 7-methoxycephalosporin compound, since the compound gives an absorption at 1763 cm " ¹ (cyclic lactam) in the infrared absorption spectrum and the presence of the signals at 4.00 ppm (3H, singlet. 7-OCH ₃ ), 5.63 ppm (1H.singlet.6-CH), 3.73-4.33 (2H, quartet, J = 18Hz, 2-CH ₂ ) and 4.42-4.91 (211, quartet. J = 14 Hz, 3-side chain CH ₂ ) in the nuclear magnetic resonance spectrum. The compound gives a-aminoadipic acid on acid hydrolysis, furthermore the compound gives absorptions at 4.44 ppm (2H, singlet, CH ₂ of - S — CH ₂ —COOH) in the nuclear magnetic resonance spectrum and also the fragment of m / e 392 in the mass spectrum of the derivative, for these reasons it has been decided that the compound has the above structure into which the heterocyclic thiol has been introduced.

I1. 7- (5-Amino-5-carboxyvaleramido) -3- (1-methyl-1H-tetrazol-5-yl) -thiomethyl-7-methoxy-A ³ -cephem-4-carboxylic acid;

III. 7- (5-Amino-5-carboxyvaleramido) -7-methoxy-3- (5-methyl-1,3,4-thiadiazol-2-yl) methyl-A ³ -cephem-4-carboxylic acid.

IV.7- (5-Amino-5-carboxyvaleramidoV7-methoxy-3-

Compound I 0.39 0.37

Compound II 0.39 0.34

Compound III 0.43 0.43

Compound IV 0.39 0.38

Cephalosporin C 0.37 0.36

7-methoxycephalosporin C 0.41 0.36

Cephamycin C 0.37 0.36

0.32 0.31 0.40 0.33 0.31 0.32 0.31

Developing solvent system used (volume ... ratio):

1. I sopropanol: n-butanol: acetic acid: water (21: 3: 7: 9).

2. n-butanol: acetic acid: water (4: 1: 2).

3. n-butanol acetic acid: water (6: 1.5: 2.5).

The control sample. Cephamycin C is 7- (5-amino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-A ³ -cephem-4-carboxylic acid.

The Rf values obtained from paper partition chromatography using Wattman "No. 1 filter paper and a developing solvent system of n-butanol: acetic acid: water (4: 1: 2 by volume) are determined as follows:

Rf value

Connection 1 0.40 Connection 11 0.39 Cephalosporin C 0.36 7-methoxy-cephalosporin C 0.40 Cephamycin C. 0.35

The compounds were then analyzed using a Hitachi® 635 high speed liquid chromatography apparatus analyzed and thereby achieved the following results:

Column: 3 χ 500 mm column made of stainless steel Resin: Hitachi «2610 (cationic exchange resin)

Developing solvent system: 0.2 M citrate buffer solution (pH 3.6);
Flow rate: 0.6 ml / min; Media speed: 1.0 cm / min.

Retention time

Compound I 5 min 33 see

Connection II 6 min 00 see

Compound III 9 min 35 sec

Cephalosporin C 5 min 18 sec

7-methoxy-cephalosporin C 5 min 09 see Cephamycine 5 min 18 sec

The compounds A of this invention have excellent antimicrobial activities; these are for used in the prophylaxis and treatment of diseases in humans and animals. They can also be used in suitable as intermediates for the preparation of other effective 7-methoxycephalosporin derivatives

Find a twist. In such cases it is preferred that the To isolate the compound of the formula A as a pure product or as a highly concentrated crude product. Particularly when the substituent group

H ₁ N

HOOC

CH- (CH ₂ ) J-CONH-

in the 7-position of the compound of the formula A is replaced by another acylamide group, the amphoteric properties disappear and the acidic product thus modified becomes well suited for isolation and it becomes soluble in organic solvents. The subsequent reaction may be carried without difficulty _{leads]. 5} Hence the modification of the connection of the form! A, as already explained above, is preferred for use in industrial practice.

example 1

20th

Preparation of 7- (5-amino-5-carboxyvaleramido) -3- (S-carboxymethylthio-O.'t-thiadiazol-1-yOthiomethyl-V-methoxy-Ä ³ -cephem-4-carboxylic acid 1:

A culture medium containing 1% starch. 1% glucose, 1.5% soybean meal, 0.5% yeast extract, 0.1% dipotassium hydrogen phosphate. Containing 0.05% magnesium sulfate and 0.3% sodium chloride, was filled into a 500 ml Sakaguchi bottle with 100 ml each and ^{sterilized at 120 °} C. for 20 minutes. Each bottle was inoculated with Streptomycesoganonensis Y-G19Z slamm and cultured for 48 hours at 30 ° C.

In further batches, the same culture medium was filled into 2000 ml Sakaguchi bottles with 400 ml each and ^{sterilized at 120 °} C. for 20 minutes. It was inoculated with the same strain at a concentration of 2-3% and ^{cultivated at 30 °} C. for 24 hours in order to obtain a culture.

In addition, 60 liters of a culture medium containing 7% strength. 2% gluten flour, 2% soybean flour, 0.8% glycerin, 0.1% casamino acid, 0.01% ferric sulfate and 55 g sodium hydroxide, each filled into two 100 liter fermenters together with 10 ml defoaming agent. It was ^{sterilized at 120 °} C. for 30 minutes. Each medium was inoculated with 800 ml of the culture was inoculated culture, and then 24 hours at 3O ¹ C "5 was cultured. In an aqueous sodium hydroxide solution, 5-mercapto-1,3,4-thiadiazole-2-thioacetic acid was dissolved, and the resulting solution was sterilized under high pressure and added to each fermentor up to 0.05% of the inoculum and then cultured for 90 hours.

After the cultivation was complete, the culture broth was adjusted to pH 2.0 and then with filter aid mixed. The mixture was filtered using a filter press. The filtrates became with each other combined, and 100 liters of the filtrate mixture were obtained. The filtrates were adjusted to pH 3.0 with aqueous sodium hydroxide solution set and passed through a 12 liter Amberlite * XAD-2 column. The column was with Washed 30 liters of water and eluted with 30 liters of aqueous acetone. The eluate was collected and concentrated to 5.5 liters. After removing the impurities that had formed, water was added to the residue, to get 10 liters of solution. The solution thus obtained was adjusted to pH 3.5 with dilute aqueous Adjusted hydrochloric acid solution and then passed through a 3 liter Amberlite® IRA-68 (Cl-type) column. After washing the column with 6 liters of water, an aqueous solution (pH 7.2) containing 1 M sodium nitrate and 0.1 M sodium acetate, eluted. About 5 liters of solution containing antimicrobial active material were obtained obtain. The solution was adjusted to pH 3.0, passed through a one liter Amberlite "XAD-2 column, and after washing the column with water, the column was washed with aqueous 50% acetone. It about 400 ml of aqueous solution containing antimicrobial active material was obtained. By lyophilization the solution was about 18 g of the crude product produced Connection I received.

Then 18 g of the crude powder was subjected to column chromatography using about 800 ml DEAE-Sephadex * A-25 (acetic acid type) subjected. The column was filled with a small amount of 0.5 M ammonium bromide-acetic acid buffer solution filled. It has been fractionated into the effective components. The antimicrobially active fractions obtained were collected, passed through a 500 ml Amberlite® XAD-2 column, and after washing the column with water, the Column eluted with an aqueous 25% acetone solution. The eluates obtained were evaporated to dryness.

Then, a mixed solvent of isopropanal: water (7: 3 volume ratio) was used to make the obtained residue of the product of column chromatography using microcrystalline cellulose (AviceF). prepared with a mixed solvent of the same composition given above. The antimicrobially active fraction obtained was applied dropwise to a thin-layer plate made of Avicel® SF, and developed with a mixture of isopropanol: n-Buianol: acetic acid-water (21: 3: 7: 9 volume ratio). Pyridine solution of 0.25% ninhydrin was then used The fractions with an Rf value of 0.39 were then collected and the fraction was evaporated to dryness in vacuo at about 45-50 ° C. The residue obtained was subjected to column chromatography with microcrystalline cellulose (Avicel *) The filling was prepared with a solvent mixture of isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9 volume ratio) The antimicrobially active fraction thus obtained was then thin-layer chromatography with AviceF SF using the above Subjected to a solvent mixture and further processed with the same aftertreatment. The fractions with the Rf value 0.39 were sat ammelt and evaporated to dryness in vacuo. The product residue was further purified by microcrystalline cellulose column chromatography using a solvent mixture of butanol: acetic acid: water (6: 1.5: 2.5 in the molar ratio). The purified active fraction was evaporated to dryness and then taken up in a small amount of distilled water. The solution was developed in a Sephadex * G-10 column using water. The fractions with antimicrobial activity were collected and subjected to thin layer chromatography, as already explained above, using a mixed solvent of n-butanol: acetic acid: water (6: 1.5: 2.5 by volume). The fractions with Rf values 0.32 were collected, concentrated and then subjected to lyophilization. About 80 mg of white 7- (5-amino-5-carboxyvaleramido ") -3- (5-carboxymethylthio-1,3,4-thiadiazol-2-yl) thiorethyl-7-methoxy-A ³ -cephem-4-carboxylic acid were obtained obtain.

Example 2

7- (5-Amino-5-earboxyvaleramido) -3- (1 -methyl-1H-tetrazol-5-yl) thiomethyl-7-methoxy-A'-cephem-4-carboxylic acid Il:

A culture medium containing 1% starch. 1% glucose. 1.5% soybean meal. 0.5% yeast excl. 0.1% dipotassium hydrogen phosphate. Containing 0.05% magnesium sulfate and 0.3% sodium chloride, was placed in 500 ml Sakagu.'hi bottles filled with 100 ml each and sterilized at 120 ° C. for 20 minutes. Then each medium was using Streptomyces oganonensis Y-G19Z strain inoculated. Afterward was cultivated at 30 ° C. for 48 hours. Then the aforementioned culture medium was divided into two liters Sakaguchi bottles filled with 400 ml each. After slicing for 20 minutes at 120 ° C, each was Medium inoculated with 2-3% of the culture broth obtained above and then added for a further 24 hours Cultivated at 30 "C to obtain an inoculation stock.

60 liters of a culture medium containing 7% strength. 2% Embers?! Imehl. 2% sqja oil. 0.8% glycerin. 0.1% Casamino acid. 0.01% ferric sulfate and 55 g sodium hydroxide was added to two liter fermenters 10 ml of defoaming agent each filled. After sterilizing for 30 minutes at 120'C, each medium was with 8 (X) ml of the inoculation mixture prepared above added, followed by cultivation at 30 ° C. for 24 hours. Then i-methyl-5-mercapto-1H-tetrazole dissolved in aqueous sodium hydroxide solution. The solution was sterilized at high pressure and closed added to the culture broth to adjust its concentration to 0.05%. The mixture was 90 hours further cultivated.

After the cultivation was completed, the thus formed The culture broth is adjusted to pH 2.0 and then a filter aid is added while stirring, the mixture filtered using a filter press. The resulting filtrates were combined and approximately 100 liters of filtrate mixture obtained.

The filtrate was adjusted to pH 3.0 with an aqueous sodium hydroxide solution, passed through a 12 liter Amberlite 'XAD-2 column, and after washing the column with 30 liters of water, the column was eluted with 30 liters of an aqueous 50% acetone solution. The eluate was concentrated to 5.5 liters, and the concentrate was adjusted to pH 3.5 with dilute aqueous hydrochloric acid solution and passed through a three-liter Amberlite 'IRA-68 (CI type) column. The column was washed with 6 liters of water and with an aqueous solution (pH 7.2). containing 1 M sodium nitrate and 0.1 M sodium acetate, fractionated. About 5 lines of a solution containing an antimicrobial active material were obtained. The solution was adjusted to pH 3.0, passed through a one liter Amberlite "XAD-2 column, washed with water, and eluted with an aqueous solution of 50% acetone. About 400 ml of aqueous solution containing the antimicrobial active material was obtained By lyophilizing the solution, about 54 g of the crude powder of Prepared Compound II was obtained. The crude powder was subjected to column chromatography with about 800 ml of DEAE Sephade? ·. * 'A-25 (acetic acid type) filled with a Small amount of 0.5 M ammonium bromide-acetic acid buffer solution, subjected to fractionate the active components. The antimicrobial active fractions were collected, passed through a 500 ml Amberlite ⁸ XAD-2 column. The column was washed with water and with an aqueous solution of 25 % acetone

eluted. The antimicrobially active fractions were collected and then evaporated to dryness in vacuo.

The residue formed was column chromatographed using microcrystalline cellulose (Avicel "), filled with a solvent mixture from isopropanol: water (7: 3 volume ratio) using the solvent mixture with the composition given above. The antimicrobially active fraction obtained was collected, applied dropwise to a thin-layer plate of Avicel "SF, with a mixed solvent from n-butanol: acetic acid: water (6: 1.5: 2.5 volume ratio) developed and a solution of 0.25% ninhydrin in pyridine was sprayed on, and then the staining was developed by heating. Then the fractions with the Rf value 0.31 were collected. The fractions were concentrated under reduced pressure and dried. The formed retreat was a column chromatography with,, microcrystallinecr Cellulose (Avicel "). The company .. .ng was with a solvent mixture of isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9 volume ratio) prepared. The antimicrobially active fractions obtained were subjected to thin layer chromatography subjected to Avicel * SF with the solvent mixture with the same composition mentioned above, using the same procedure as given above, was applied. The fractions with the Rf value 0.39 were collected and taken to dryness in vacuo evaporated.

The residue thus formed was subjected to microcrystalline cellulose column chromatography using a mixed solvent of n-butanol: acetic acid: water (6: 1.5: 2.5 volume ratio) to purify the effective component. The active fraction thus purified was evaporated to dryness in vacuo, dissolved in a small amount of distilled water and developed in a Sephedex * G 10 column using distilled water. The fractions with antimicrobial activity were collected and subjected to thin layer chromatography using a mixed solvent of n-butanol: acetic acid: water (6: 1.5: 2.5 volume ratio) as indicated above. Then the fractions with the Rf value 0.31 were collected, concentrated and then lyophilized. About 60 mg of white 7- (5-amino-5-carboxyvaleramido) -3- (1-methyl-1H-tetrazol-5-yl) thiomethyl-7-methoxy-A ³ -cephem-4-carboxylic acid were obtained .

Example 3

Preparation of 7- (5-amino-5-carboxyvaleramidö) -7-methoxy-3- (5-methyl-1.3.4-thiadiazol-2-yl) thiomethyl-A ³ -cephem-4-carboxylic acid III:

A culture medium containing 1% starch. 1% glucose, 1.5% soybean meal. 0.5% yeast extract. 0.1% dipotassium hydrogen phosphate. 0.05% magnesium sulfate and 0.3% sodium chloride was filled in 500 ml Sakaguchiflaschen ml each and sterilized for 20 minutes at 100 ^c C TlO. Each medium was then inoculated with Streptomyces oganonensis Y-G19Z strain, and cultured for 48 hours at 30 ⁰ C. Another culture medium as described above was filled in Zvveiliter ml Sakaguchiflaschen with 400 and sterilized for 20 minutes at 12O ⁰ C. Each medium was treated with the above cultured broth at a 2-3% concentration.

inoculated and then cultivated for 24 hours at 30 ⁼ C, thereby obtaining a Wurd Animpfkukur ::.

60 liters of the culture medium containing 7% starch were separated from each other. 2% Glutenr.shl. 2% soybean meal. 0.8% glycerin. 0.1% casamic acid. Containing 0.01% ferric sulfate and 55 g sodium hydroxide, filled into two 100 liter fermenters together with 10 ml defoaming agent. It was sterilized for 30 minutes at 120 ⁰ C and treated with 800 ml of Animpfungskultur inoculated. Cultivation was carried out at 30 ^{° C. for 24 hours.} Then, 2-mercapto-5-methyl- in 1.3.4-thiadiazole was dissolved in an aqueous sodium hydroxide solution and sterilized under high pressure and added to the culture broth so that the concentration thereof in the broth became 0.05%, followed by further cultivation for 90 hours. ! 5

After the end of cultivation, the culture broth was adjusted to pH 2.0 and added with filter aid while stirring mixed. The mixture was filtered using a filter press and the filtrates were combined and give about 100 liters of filtrate mixture.

The filtrate was adjusted to pH 3.0 by addition of aqueous sodium hydroxide solution and then passed through a 12 liter Amberlite "XAD-2 column. The Column was washed with 30 liters of water. It was eluted with 30 liters of 50% aqueous acetone. That Eluate was concentrated to 5.5 liters and the concentrate was adjusted to pH 3.5 with dilute aqueous hydrochloric acid solution and by a 3 liter Amberlite 'IRA-68 (Cl-type) column given. The column was washed with 6 liters of water. It was made with a aqueous solution (pH 7.2). containing 1 M sodium nitrate and 0.1 M sodium nitrate, eluted. It was about Receive 5 liters of an antimicrobial active material. The solution was adjusted to pH 3.0 and by a Given one liter Amberlite XAD-2 column. After washing the column with water, the column was washed with a aqueous solution of 50% acetone eluted. There was about 400 ml of aqueous solution containing the antimicrobial contained active material. The product was lyophilized.

Using a solvent mixture of n-butanol: acetic acid: water (4: 1: 2 volume ratio) the residue formed was subjected to column chromatography using microcrystalline Cellulose (Avicel *). filled with the solvent mixture of the same composition given above. Then the obtained ones became antimicrobial active fractions fractionated and applied drop-shaped to a thin-layer plate from Avicel * SF and with a solvent mixture isopropanol: acetic acid: water (21: 3: 7: 9 in volume ratio) developed. A pyridine solution of 0.25% ninhydrin was sprayed over and the color developed with heating. The fractions with the Rf value 0.43 were collected and brought to dryness at 45-50 ° C. in vacuo. The residue obtained became the Subjected column chromatography with microcrystalline cellulose (AviceP). The column was prepared with a mixed solvent of acetonitrile: water (7: 3 volume ratio). The obtained antimicrobial active Fraction was subjected to thin layer chromatography with Avicel * SF. as subjected to the above procedure. Then the fractions with the Rf value 0.43 were collected and evaporated to dryness. It became 0.78 g get raw powder.

The powder was dissolved in a small amount of distilled water and placed in a column of Sephadex "G 10 developed using distilled water.

The antimicrobially active fractions were fractionated and subjected to thin layer chromatography using a mixed solvent of n-butanol: acetic acid: water (4: 1: 2 volume ratio) as indicated above. The fractions with the Rf value 0.43 were collected, concentrated and lyophilized. 82 mg of white 7- (5-amino-5-carboxyvaleramido) -7-methoxy-3- (5-methyl-1,3,4-thiadiazol-2-yl) -thiomethyl-A ³ -cephem-4-carboxylic acid were obtained .

Example 4

Using the same procedure as given in Example 1, but using a solution of bis (5-carboxymethylthio-1,3,4-thiadiazol-2-yl) disulfide, prepared by dissolving the disulfide in hydrous methanol and sterilizing by filtration through a Millipore filter in this example, instead of the solution of 5-mercapto-1,3.4-thiadiazole-2-thioacetic acid prepared in water using an aqueous sodium hydroxide solution and sterilized under high pressure, 23 g of crude powder of the prepared compound 1 was obtained. By purifying the product, as indicated in Example 1, about 45 mg of 7- (5-amino-5-carboxyvalerarmdoJ-S-fS-carboxymethylthio-l ^^ - thiadiazol-2-yl) thiomethyl-7-methoxy-A ³ -cephem-4-carboxylic acid (compound 1).

Example 5

Using the same procedure outlined in Example 2 but using a Solution of bis (1-methyl-1H-tetrazo! -5-yl) disulfide. made by dissolving the disulfide in water containing Methanol and sterilization by filtration using a Millipore filter in this example instead of a solution of 1-methyl-S-mcrcapto-IH-'ctrazole. manufactured by Dissolve the tetrazole in water using an aqueous sodium hydroxide solution and sterilize high pressure, about 26 g of the crude powder of compound II was obtained. By cleaning the product, as indicated in Example 2, about 37 mg of 7- (5-amino-5-carboxyvaleramido) -3- (l-methyl-lH-tetrazole-5 - yl) thiomethyl - 7 - methoxy-A'-ccphem -4 -carboxylic acid (Compound II) obtained.

Example 6

Using the same procedure as outlined in Example 3, by employing a solution of bis (5-methyl-1,3.4-thiadiazol-2-yl) disulfide. prepared by dissolving the disulfide in hydrous methanol, and sterilizing by filtration medium; Millipore filter in this example instead of the solution before 2-Me ^ aPtO-S-InCIhVl-1.3.4-thiadiazole. prepared in water using an aqueous sodium hydroxide solution and sterilization at high pressure, about 19 j of crude powder of compound III is obtained. By cleaning the product, as indicated in Example 3, about 50 mg of 7- (5-amino-5-carboxyvaleramido) -7-methoxy - 3 - (5 - methyl -1,3.4 - thiadiazol - 2 - yl) thiomethyl-Δ ' ¹ cephem-4-carboxylic acid (compound III).

Example 7

Preparation of 7- (5-Amino-5-auboxyvalenimido) - "? Melhoxy-3- (1.3.4-thiadiazol-2-yl) thiomethyl-A ^J -cephem 4-carboxylic acid IV.

A culture medium, which 1% starch, 1% glucose, 1.5% soybean meal, 0.5% yeast extract, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate and 0.3% sodium chloride, was in 500 ml Sakaguchi bottles with each 100 ml filled, sterilized for 20 minutes at 120 ° C and inoculated with the Streptomyces oganonensis Y-G19Z-Siamm. Cultivation was then carried out at 30 ° C. for 48 hours. Another prominent culture medium was filled ml in two-liter Sakaguchiflaschen with 400 and sterilized for 20 minutes at 12O ⁰ C. Then 2-3% of the culture broth prepared according to the above procedure was inoculated. Cultivation was carried out at 30 ° C. for 24 hours to obtain an inoculum.

Separately, 60 liters of a culture medium containing 7%! 5 strength. 2% gluten flour. 2% soybean meal, 0.8% glycerin. 0.1% casamino acid, 0.01% ferric sulfate and 55 g sodium hydroxide in two 100 liter fermenters, filled with 10 ml defoaming agent and ^{sterilized at 120 °} C. for 30 minutes. Then 800 ml of the inoculation culture were inoculated. Cultivation was carried out at 30 ° C. for 24 hours. Then a solution of 2-mercapto-1.3.4-thiadiazole. prepared by dissolving the thiadiazole in water using an aqueous sodium hydroxide solution and sterilizing under high pressure, added to the culture broth so that the concentration of the thiadiazole became 0.05%. The batch was then further cultivated for 90 hours.

After the cultivation was complete, the culture broth was adjusted to pH 2.0 and with stirring using filter aid offset. The mixture was filtered using a filter press and the filtrates were combined to give about 100 liters of the filtrate mixture.

The filter, adjusted to pH 3.0, was passed through a γ> 12 liter Amberlite® XAD-2 column. The column was washed with 30 liters of water. It was eluted with 30 liters of aqueous 50% strength acetone solution. The eluates were except for 5.5 liters. The concentrate was adjusted to pH 3.5 and passed through a 3 liter Amberlite * IRA-68 (Cl type) column. The column was washed with 6 liters of water. Elution was carried out with an aqueous solution (of pH 7, 2). Which contained 1 M sodium nitrate and 0.1 M sodium acelate. About 5 liters of a solution containing antimicrobially active material were obtained. The solution was adjusted to pH 3.0 by means of a one-liter Amberlite * XAD-2- Column and washed with water. It was washed with an aqueous 50% acetone solution. About 400 ml of the aqueous solution containing the antimicrobial active material was recovered. The solution was lyophilized. Using a mixed solvent of n-butanol: acetic acid : Water (4 : 1: 2 volume ratio), the residue formed was subjected to column chromatography using microcrystalline cellulose (Avicel ") and prepared with the mixed solvent having the same composition as above. The antimicrobially active fractions were fractionated, placed dropwise on a thin-layer plate made of Avicel®, developed using a mixed solvent of isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9) in a volume ratio. Then a pyridine solution of 0.25% ninhydrin was sprayed on and the stain developed by heating. The fractions with the Rf value b5 0.39 were then collected and evaporated to dryness in vacuo at 45-50 C. The product residue was subjected to column chromatography with microcrystalline cellulose (Avicel *; and a Solvent mixture prepared from acetonitrile: water (7: 3 volume ratio). The antimicrobially active fractions were also subjected to thin layer chromatography with Avicel * SF as in the above procedure. The fractions with the Rf value 0.39 were collected, evaporated to dryness in vacuo 0.92 g of crude powder was obtained.

The crude powder was dissolved in a small amount of distilled water and then subjected to column chromatography using Amberlite * CG-50 (H type) with distilled water, and the antimicrobial active fractions were collected, concentrated and lyophilized. The residue was dissolved in a small amount of distilled water and placed in a column of Sephedex ^? G 10 developed using distilled water. The antimicrobial activity of each fraction was checked. The effective fractions were determined by thin-layer chromatography using a mixture of n-iutanol: acid: water (4: 1: 2 volume ratio), as described above. _nd described, subjected. Then the fractions with the Rf value 0.38 were collected, concentrated and lyophilized. White 7- (5-amino-5-carbo-amido) -7-methoxy-3- (1,3,4-thiadiazol-2-yl) thiomethyl-A'-cephem-4-carboxylic acid were obtained .

Example 8

a) A culture medium which 1 ',. Strength. 1% glucose, 1.5% soybean meal. 0.5% yeast extract, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate and containing 0.3% sodium chloride, was in 500 ml Sakaguchi bottles filled in an amount of 100 ml each. Each bottle was sterilized at 120 ° C for 20 minutes. Each Medium was inoculated with Streptomyces oganonensis Y-G 19Z strain and cultivated at 30 ° C. for 48 hours.

The culture medium was also filled into two-liter Sakaguchi bottles in an amount of 400 ml each and ^{sterilized at 120 °} C. for 20 minutes. The mixture was inoculated with 2-3% of the culture broth, which was prepared as above, and cultivated at 30 ° C. for 24 hours. Such an inoculation culture was obtained.

Separately 60 liters of a culture medium containing 7% starch, 2% gluten flour, 2% soybean flour, 0.8% glycerin, 0.1% casamino acid. 0.01% ferric sulfate and 55 g of sodium hydroxide in two 100 liter fermenters mixed with 10 ml of antifoam agent each, sterilized for 30 minutes at 120 ° C. 800 ml of the inoculated culture were added. which had been prepared according to the above procedure, inoculated. Cultivation was carried out at 30 ^{° C. for 24 hours.} Then a solution of 5-mercapto-1-methyl-1 H-tetrazole. prepared from an aqueous sodium hydroxide solution and sterilized under high pressure, added to the culture broth so that the concentration of the tetrazole became 0.05%. The cultivation was carried out for 90 hours.

After the cultivation was completed. the culture broth was adjusted to pH 2.0 and mixed with filter aid while stirring. The mixture was filtered with a filter press and the resulting filtrates were combined. 100 liters of filtrate mixture containing 100 meg ml of 7- (5-amino-5-carhoxyvaleramido) -7-methoxy-3- (1-methyl-1 H-tetrazoloyl) thiomelhyl-A " ¹ -cephem-4 -carboxylic acid obtained.

b) The filter was adjusted to pH 3.0 and passed through a 12 liter Amberlite ^F XAD-2 column. The column was washed with 30 liters of water and 30 liters

Liters of aqueous 50% acetone eluted. The eluate was concentrated to 5.5 liters. The concentrate was on Adjusted to pH 7.5 using an aqueous sodium hydroxide solution, and the produced compound became isolated.

Example 9

a) Preparation of 7- (5-amino-5-carboxyvaleramido) -3- (1-methyl-1 H-tetrazol-5-yl) thiomethyl-7-methoxy-A ³ -cephem- ^ ncarboxylic acid.

A culture medium containing 1% starch, 1% glucose, 1.5% soybean meal, 0.5% yeast extract, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate and 0.3% sodium chloride was added in 5 (X) ml Sakaguchi bottles each filled in an amount of) 00 ml. Each medium was sterilized at 120 ° C for 20 minutes. The steric; Culture medium was inoculated with the Streptomyces oganonensis Y-G19Z strain. Cultivation was carried out at 30 ^{° C. for 48 hours.} The culture medium prepared above was also filled into two liters of Sakaguchi bottles in an amount of 400 ml each. After sterilizing the medium at 120 ° C. for 20 minutes, the culture medium was inoculated with the culture broth obtained in the above method in an amount of 2-3%. It was cultured for 24 hours at 30 ^c 'C, to obtain a seed culture.

Separately, 60 liters of a culture medium containing 7% starch, 2% gluten flour, 2% soybean egg flour, 0.8% glycerin, 0.1% casamino acid, 0.01 % ferric sulfate and 55 g sodium hydroxide were placed in two hundred liter fermenters along with each Filled in 10 ml of defoaming agent. Each culture medium was sterilized at 120 ° C. for 30 minutes. It was inoculated with 800 ml of the inoculation culture prepared according to the above procedure. Then a solution of i-methyl-5-mercapto-1H-tetrazole, prepared by dissolving in aqueous sodium hydroxide solution and sterilizing under high pressure, was added to each fermenter so that the content was 0.05%. Then the culture broth was further cultivated for 90 hours. After the end of cultivation, the culture broth was adjusted to pH 2.0 and mixed with filter aid while stirring. The mixture was filtered through a filter press and the filtrates were combined to make about 100 liters of filtrate mixture.

The mixture was adjusted to pH 3.0 by the addition of aqueous sodium hydroxide solution. Then became passed through a 12 liter Amberlite® XAD-2 column. The column was washed with 30 liters of water. It was eluted with 30 liters of 50% aqueous acetone. The eluate was concentrated to 5.5 liters. The concentrate was adjusted to pH 3.5 with dilute aqueous hydrochloric acid solution set and passed through a 3 liter Amberlite® IRA-68 (Cl-type) column. The pillar was washed with 6 liters of water and with aqueous solution (pH 7.2) containing 1 η sodium nitrate and 0.1 M Sodium acetate, eluted. About 5 liters of solution containing an antimicrobially active material were won. The solution was adjusted to pH 3.0 and it was passed through a 1 liter Amberlite® XAD-2 column bo given. The column was washed with water. It was eluted with aqueous 50% acetone.

There was about 400 ml of aqueous solution containing antimicrobial active material obtained which has been lyophilized. About 54 g of raw powder were obtained. The b5 crude powders were subjected to column chromatography. For this purpose, 800 ml DEAE Sephadex * A-25 (Acetic acid type) and filled with a small amount of 0.5 M ammonium bromide-acetic acid buffer solution. The effective fractions were isolated. The antimicrobially active fractions thus collected were passed through a 500 ml Amberlite® XAD-2 column. The column was washed with water and with aqueous 25% acetone eluted. The eluate was then evaporated to dryness in vacuo. The dried one Product was a column chromatography with a solvent mixture of isopropanol: water (7: 3 im Volume ratio) by means of microcrystalline cellulose (Avicel®). The column was with the mixed solvent filled with the above composition. The fractions with antimicrobial Activity were applied dropwise to a thin-layer plate made of Avicel * SF and mixed with a solvent developed from n-butanol: acetic acid: water (6: 1.5: 2.5 by volume).

A pyridine solution of 0.25% ninhydrin was sprayed on and the color was developed by heating. The fractions containing the Rf value 0.31 were collected, under vacuum at 45 - 50 ⁰ C and evaporated to dryness and subjected to column chromatography with microcrystalline cellulose (Avicel *). The column contained a solvent mixture of isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9 by volume). The antimicrobially active fractions were then selected and subjected to thin layer chromatography with Avicel * SF. The same procedure as above was used. The fractions with Rf Wcrt 0.39 were collected and evaporated to dryness in vacuo.

The dried product was placed in a chromatography column with microcrystalline cellulose.

A solvent mixture of n-butanol: acetic acid: water (6: 1.5: 2.5 in volume ratio) was used to achieve a purification of the effective component. The purified active fractions were dried by concentration, dissolved in a small amount of distilled water and placed in a column with Sephadex * G 10 developed using distilled water. The antimicrobial activity of everyone Fraction was checked and the effective fractions were selected and subjected to thin layer chromatography as explained above.

A mixed solvent of n-butanol: acetic acid: water (6: 1.5: 2.5 by volume) was used. The fractions with the Rf value 0.31 were collected, concentrated and lyophilized. About 60 mg of white 7- (5-amino-5-carboxyvaleramido) -3- (1-methyl-1H-tetrazol-5-yl) thiomethyl-7-methoxy-A ³ -cephem-4-carboxylic acid were obtained.

Example 10

a) Preparation of 7- (5-amino-5-carboxyvaleramido) -7-methoxy-3- (5-methyl-1,3,4-thiadiazol-2-yl) thiomethyl-A ³ -cephem-4-carboxylic acid.

A culture medium containing 1% starch, 1% glucose. 1.5% soybean meal, 0.5% yeast extract. Containing 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate and 0.3% sodium chloride was filled into 500 ml sakaguchi vials in an amount of 100 ml each. Each medium was sterilized at 120 ° C for 20 minutes. The culture medium was then inoculated with the Strcptomyces oganonensis Y-G19Z strain. Then 48 hours at 3O ⁰ C was cultivated.

The above culture medium was in 2 liters

Sakaguchiflaschen ml filled in an amount of 400, and sterilized for 20 minutes at 12O ⁰ C.

Then 2-3% culture broth obtained according to the above procedure was inoculated. It has been cultivated for 24 hours at 30 ⁰ C, to obtain a seed culture.

Separately, 60 liters of a culture medium containing 7% starch, 2% gluten flour, 2% soybean flour, 0.8% Glycerine, 0.1% casamino acid, 0.01% ferric sulfate imd Containing 55 g of sodium hydroxide, filled in 200 liter fermenters together with 10 ml of defoaming agent and Sterilized for 30 minutes at 120 ° C.

Then, 800 ml of inoculum culture prepared by the above !: method was inoculated. Cultivation was carried out at 30 ^{° C. for 24 hours.} A solution of 2-mercapto-5-methyl-1,3,4-thiad-azoL prepared with aqueous sodium hydroxide solution and sterilized at high pressure was added to each fermenter so that its content was 0.05% of the culture broth. The cultivation was continued for 90 hours.

After the cultivation was complete, the culture broth was adjusted to pH 2.0. It became a filter aid added with stirring. The mixture was filtered using a filter press. The filtrates were combined and gave about 100 liters of filtrate mixture.

The filtrate was adjusted to pH 3.0 by the addition of aqueous sodium hydroxide solution. The solution was passed through a 12 liter Amberlite * XAD-2 column. The column was washed with 30 liters of water and eluted with 30 liters of aqueous 50% acetone. The eluate is concentrated up to 5.5 liters, to pH 3.5 adjusted with dilute aqueous hydrochloric acid solution and poured into a 3 liter Amberlite * IRA-68 (Cl type) Column filled. The column was washed with 6 liters of water and with an aqueous solution (pH 7.2) which Containing 1 M sodium nitrate and 0.1 M sodium acetate, eluted. There were about 5 liters of an antimicrobial solution contained active material. The solution thus obtained was adjusted to pH 3.0 and in filled a 1 liter Amberlite® XAD-2 column. The column was eluted with 50% aqueous acetone. There were about 400 ml of an aqueous solution containing the antimicrobial material was obtained. The solution was lyophilized. The product was subjected to column chromatography with a mixed solvent of n-butanol: acetic acid: Water (4: 1: 2 in "volume ratio") using microcrystalline cellulose (Avicel *) and a filling with the same solvent mixture as indicated above, subjected. The received antimicrobially active fractions were applied drop-shaped onto a thin-layer plate made of Avicel * SF, and it was with a solvent mixture isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9 in volume ratio) developed. With a pyriciin solution of 0.25% ninhydrin was sprayed to stain by heating. Then the factions were with the Rf value 0.43 and evaporated to dryness under reduced pressure at 45-50 ° C. That The product obtained was column chromatography with microcrystalline cellulose (Avicel *), filled with a mixed solvent of acetonitrile: water (7: 3 in volume ratio). The obtained antimicrobial active fractions were subjected to thin layer chromatography on Avicel * SF as in the above procedure. The factions with an Rf value of 0.43 were collected and evaporated to dryness under reduced pressure. It 0.78 g of a crude powder was obtained.

The product was dissolved in a small amount of distilled water and developed in a column of Sephadex * G 10 using distilled water. The antimicrobial activity of each fraction was checked. The effective fractions were subjected to thin layer chromatography as indicated above using a mixed solvent of n-butanol: acetic acid: water (4: 1: 2 by volume). Then the fractions with the Rf value 0.43 were collected, concentrated and lyophilized. 82 mg of white 7- (5-amino-5-carboxyvaleramido) -7-methoxy-5- (5-methyl-1,3,4-thiadiazol-2-yl) -thiomethyl-A ³ -cephem-4- carboxylic acid obtained.

Example 11

Preparation of 7- (5-amino-5-carboxyvaleramido) -3- (5-carboxymethylthio-1,3,4-thiadiazol-2-yl) -thiomethyl-7-methoxy-A ³ -cephem-4-carboxylic acid:

A culture medium containing 1% starch, 1% glucose, 1.5% soybean meal, 0.5% yeast extract, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate and 0.3% sodium chloride was added to a 500 ml Sakaguchi bottle 100 ml each and ^{sterilized at 120 °} C. for 20 minutes. Each culture medium was then inoculated with the Streptomycesoganonensis Y-G ^ Z strain. It was then cultivated ^{at 30 ° C. for 48 hours.} Another prominent culture medium was in each case filled ml in 2000 ml Sakaguchiflaschen 400, and each culture medium was sterilized for 20 minutes at 12O ⁰ C. Then with 2-3% of the culture broth prepared by the above methods, seeded and then cultured for 24 hours at 3O ⁰ C, to obtain a seed culture.

Separately, 60 liters of a culture medium which contained 7% starch, 2% gluten meal, 2% soybean meal, 0.8% glycerin, 0.1% casamino acid, 0.01% ferric sulfate and 55 g sodium hydroxide, in 200 liter fermenters together with 10 ml of defoamer filled. Each medium was sterilized 30 minutes at 120 ⁰ C and treated with 800 ml of inoculum, prepared by the above Vei drive vaccinated. This was followed by cultivation at 30 ° C. for 24 hours.

Then a solution of bis (5-carboxymethylthio-1,3,4-thiadiazol-2-yl) disulfide, prepared by dissolving the disulfide in hydrous methanol, and sterilizing by filtration using a Milupore filter added in such an amount that the content in the culture broth would be 0.05%. The cultivation was carried out for 90 hours. After the cultivation was finished, the culture broth became up Adjusted to pH 2.0 and added filter aid with stirring. The mixture was made using a Filter press filtered. The filtrates were combined. About 100 liters of a filtrate mixture were obtained.

The filtrate was adjusted to pH 3.0 by adding an aqueous sodium hydroxide solution. It was the solution is poured into a 12 liter Amberlite® XAD-2 column. The column was washed with 30 liters of water and eluted with 30 liters of aqueous 50% acetone. The eluate was concentrated to 5.5 liters. To Removal of the formed insolubles, water was added to the concentrate to make 10 liters Get solution. The solution was adjusted to pH 3.5 with dilute aqueous hydrochloric acid solution, by a 3 liter Amberlite ^ IRA-68 (Cl-T yp) -

Pillar given. The column was washed with 6 liters of water and eluted with an aqueous solution (pH 7.2) containing 1 M sodium nitrate and 0.1 M sodium acetate. About 5 liters of solution containing antimicrobial material was obtained. The solution was adjusted to pH 3.0, filled into a 1 liter Amberlite * XAD-2 column. The column was washed with water and eluted with 50% aqueous acetone. About 400 ml of an aqueous solution containing antimicrobially active material was obtained. By lyophilizing the aqueous solution, about 23 g of crude powder was obtained. Then, 23 g of the crude powder was subjected to column chromatography using 800 ml of DEAE-Sephadex * A-25 (acetic acid type) filled with a small amount of 0.5 M ammonium bromide-acetic acid buffer solution to separate the effective components. The antimicrobially active fractions were collected and applied to a 500 ml Amberlite® XAD-2 column. The column was washed with water. It was eluted with aqueous 25% acetone. The eluate was evaporated to dryness in vacuo. The product was subjected to column chromatography with a mixed solvent of isopropanol: water (7: 3 by volume) using microcrystalline cellulose (Avicel *) using the mixed solvent having the same composition as in the above fractionation of the antimicrobial active fractions. The fractions thus obtained were applied dropwise to a thin-layer plate made of AviceP SF and developed with a solvent mixture of isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9 by volume). Then a pyridine solution of 0.25% ninhydrin was sprayed on and the coloration developed by heating. Then the fractions with the Rf value 0.39 were collected, evaporated to dryness under reduced pressure. The residue was subjected to column chromatography filled with microcrystalline cellulose (Avicel ^"5x), was a mixed solvent of isopropanol. N-butanol: acetic acid: water (21: 3: 7: 9 in volume ratio) was used, the antimicrobially active fractions. were collected and subjected to thin layer chromatography on an Avicel® SF plate, the solvent mixture having the same composition as the above mixture, the same procedure as described above, the fractions with Rf value 0.39 being collected and in vacuo evaporated to dryness.

The concentrate was purified by column chromatography using microcrystalline cellulose and a solvent mixture of n-butanol: acetic acid: water (6: 1.5: 2.5 by volume). The purified active fractions were evaporated to dryness in vacuo. The residue was taken up in a small amount of distilled water and developed with a column of Sephadex® G-10 using distilled water. The antimicrobial activity of each fraction was checked. The effective fractions were subjected to thin layer chromatography as indicated above. A mixed solvent of n-butanol: acetic acid: water (6: 1.5: 2.5 by volume) was used. The fractions with an Rf value 0.32 were pooled, concentrated and lyophilized. About 45 mg of white 7- (5-amino-5-carboxyvaleramido) -3- (5-carboxymethylthio-1.3.4-thiadiazoI-2-yl) thiomethyl-7-methoxy-A ³ -cephem-4-carboxylic acid were obtained .

Example 12

Preparation of 7- (5-amino-5-carboxyvaleramido) methoxy - 3 - (1,3,4 - thiadiazol - 2 - yl) - thiomethyl - A ¹ -c <phem-4-carboxylic acid:

A culture medium containing 1% starch. 1% Glukosi 1.5% soybean meal, 0.5% yeast extract. 0.1% D: kaliumhydrogenphospha'i, 0.05% magnesium sulfate un 0.3% sodium chloride was dissolved in 500 ml Sak guchiflaschen filled with 100 ml each and sterilized for 20 min at 12O ⁰ C. Then each culture medium was inoculated with the Streptomyces oganonensis Y-G19Z strain. Cultivation was then carried out at 30 ° C. for 48 hours. Another prominent culture medium was 2 liters Sakaguchiflaschen filled ml with 400 un-sterilized 20 minutes at 120 "C. Each Kulturmcdiur was inoculated with the culture broth which has been stated above is called. It has been archived for 24 hours at 30 ⁰ C cultivate to a Separately: 60 liters of culture medium, which contained 7% starch, 2% gluten flour, 2% soybean meal, 0.8% glycerin, 0.1% casamic acid, 0.01% ferric sulfate and 55 g sodium hydroxide, were placed in 200 liter fermenters together with 10 ml En filled foaming agent and sterilized 30 minutes at 120. Each culture medium was then inoculated with 800 of the seed culture and then cultured for 24 hours at 30 ⁰ C. then a solution of 2-Me capto-l, 3,4-thiadiazole, prepared with aqueous sodium hydroxide solution and sterilization at high pressure added to each fermenter in such an amount that the content was 0.05% in the culture broth.

Then the cultivation was continued for 90 hours

After the cultivation was complete, the culture broth was adjusted to pH 2.0 and filtered with a filter aid mixed with stirring. The mixture was filtered using a filter press. The filtrate was combined, and about 100 liters of filtrate mixture were obtained. The filtrate was by adding an aq Adjusted sodium hydroxide solution to pH 3.0 and poured into a 12 liter Amberlite * XAD-2 column. Tuesday Column was washed with 30 liters of water and with 31 Eluted with aqueous 50% acetone. The EIu was concentrated to 5.5 liters and diluted to pH 3.5 with adjusted to aqueous hydrochloric acid and then added to a 3 liter Amberlite® IRA-68 (Cl-type) column The column was washed with 6 liters of water. The elution was carried out with an aqueous solution (pH 7.2) containing 1 Sodium nitrate and 0.1 M sodium acetate. E were 5 liters of solution, the antimicrobial active mate rial contains, won. The solution was adjusted to pH 3 (, in a 1 liter Amberlite® XAD-2 column give. The column was washed with water and rn 50% acetone eluted. About 400 ml of an aqueous solution containing antimicrobially active material were obtained which has been lyophilized.

The obtained product was subjected to column chromatography with a mixed solvent of n-butanol Acetic acid: water (4: 1: 2 by volume) using microcrystalline cellulose (Avicel®) al Subjected to filling, the solvent mixture with the same above-specified together was used to select the antimicrobially active: fractions.

The fractions were applied dropwise to a thin-layer plate made of Avicel® SF, and development was carried out with a solvent mixture of isopropanol: η butanol: acetic acid: water (21: 3: 7: 9). A pyridine solution of 0.25 % ninhydrin was sprayed on

and it was stained by heating. Then the fractions with the Rf value 0.39 were collected, below evaporated to dryness at 45 - 50 ° C under reduced pressure. Then the fractions were subjected to thin layer chromatography with Avicel® SF in the manner described above subjected to the fractions with to collect the Rf value 0.39. The fractions were evaporated to dryness to give 0.92 crude powder became. The powder was dissolved in a small amount of distilled water and subjected to column chromatography subjected to distilled water using Amberlite® CG-50 (H type), to select the antimicrobially active fractions. The fractions were then concentrated and lyophilized

sated. The product was dissolved in a small amount of distilled water and poured out on a column Sephadex® G-10 developed using distilled water. The antimicrobial activity of everyone Parliamentary group was examined. The effective fractions were subjected to thin layer chromatography in the above subject to the manner explained. A mixed solvent of n-butanol: acetic acid: water was used (4: 1: 2 by volume) was used to collect the fractions with the Rf value 0.38. The factions were concentrated and lyophilized, giving 75 mg of white 7- (5-amino-5-carboxyvaleramido) -T-methoxyO-O ^ -thiadiazole-Z-yO-thiomethyl-A'-cephem-4-carboxylic acid obtain.

1 sheet of drawings

Claims (1)

Claim: Process for the preparation of 7-methoxycephalosporin derivatives with the formula (A) '■> OCH ₃ (A) HOOC CH- (CH ₂ ), CONH- - ^rS i H ₂ N ^ // 0 COOM ¹⁰