CH632011A5 - Process for the preparation of 7-methoxycephalosporin derivatives - Google Patents

Abstract

The invention relates to a process for the preparation of 7-methoxycephalosporin derivatives having the formula (A) <IMAGE> in which R<2> represents a heterocyclic group containing nitrogen as the ring hetero atom and M denotes a hydrogen atom, alkali metal, alkaline earth metal, heavy metal or bases which form a quaternary salt or amine salt, characterised in that Streptomyces oganonensis ATCC 31167 is cultured in a culture medium which contains nutrients with the addition of a heterocyclic thiol of the formula R<2>SH in which R<2> has the abovementioned meaning, or of a salt of the heterocyclic thiol or a compound of the formula R<2>-S-S-R<2> in which R<2> has the same meaning already mentioned. The products prepared according to the invention have antibiotic properties. They can also be used as intermediates for the preparation of useful antibiotics.

Description

       

  
 

** WARNING ** beginning of DESC field could overlap end of CLMS **.

 



   PATENT CLAIMS
1. Process for the preparation of 7-methoxycephalosporin derivatives having the formula (A)
EMI1.1
 wherein R2 represents a heterocyclic group containing nitrogen as ring heteroatom and M represents a hydrogen atom, alkali metal, alkaline earth metal, heavy metal or bases which form quaternary salt or amine salt, characterized in that Streptomyces oganonensis ATCC 31167 is grown in a culture medium which contains nutrients with the addition of a heterocyclic thiols with the formula
R2SH, wherein R2 has the meaning given above, or a salt of the heterocyclic thiol or a compound having the formula
R2-S-S-R2, in which R2 has the same meaning already mentioned.



   2. The method according to claim 1, characterized in that R2 is a 1-methyl-1 H-tetrazol-5-yl group and M represents a hydrogen atom.



   3. The method according to claim 1, characterized in that R2 represents a 5-carboxymethylthio-1, 3,4thiadiazol-2-yl group and M represents a hydrogen atom.



   4. The method according to claim 1, characterized in that R2 represents a 5-methyl-1,3,4-thiadiazol-2-yl group and M represents a hydrogen atom.



   5. The method according to claim 1, characterized in that R2 is a 1, 3,4-thiadiazol-2-yl group and M is a hydrogen atom.



   The present invention relates to a process for the preparation of 7-methoxycephalosporin derivatives having the formula (A)
EMI1.2
 wherein R2 represents a heterocycle-containing heterocycle containing nitrogen and M represents a hydrogen atom, alkali metal, alkaline earth metal, heavy metal or bases which form quaternary salt or amine salt, characterized in that Streptomyces oganonensis ATCC 31167 is grown in a culture medium which is nutrient with the addition of a heterocyclic thiol with the formula
R2SH, in which R2 has the meaning given above, or a salt of the heterocyclic thiol or a compound having the formula R2-S-S-R2, in which R2 has the same meaning already mentioned.



   As a nitrogen-containing heterocyclic group, which is indicated by R2 in the general formula above, the following are mentioned for clarification: a carboxymethylthio-1,3thiadiazol-2-yl group, a 1-methyl-1 H-tetrazol-5-yl group, a 5-methyl-1,3,4-thiadiazol-2-yl group, a 1, 3,4-thiadiazol-2-yl group, etc.



   The cationic group represented by M which forms the salt of cephalosporin means an inorganic group or an organic group. As the inorganic group, an alkali metal such as sodium, potassium, etc .; a calcareous metal such as calcium, magnesium, barium, etc .; as well as a heavy metal such as iron, copper, zinc, etc.; bases which form quaternary salts or amine salts, for example triethylamine, diethanolamine, piperidine, morpholine, etc., serve as the organic radical.

  Practical examples of compounds of this invention are 745-amino-5-carboxyvaleramidof345-carboxymethylthio-1, 3,4thiadiazol-2-yl> thiomethylj-methoxy-A 3-cephem4-carboxylic acid, 75-amino-5-carboxyvaleramido> 3- (1 -methy1-1 H-tetrazol-5-yl) thiomethyl-7-methoxy-h 3-cephem4-carboxylic acid, 745-amino-5-carboxyvaleramidof7-methoxy: 345-methyl-1,3,4-thiadiazol-2-yl ) thiomethyl-A 3-cephem4-carboxylic acid, 745-amino-5-carboxyvaleramidof7-methoxy-341, 3,4-thiadiazol-2yl) thiomethyl-A3-cephem4-carboxylic acid, and the salts of these compounds.



   Some 7-methoxy-3-heterocyclothiomethylcephalosporins are described in British Patent 1,321,412 (1970) as compounds obtainable by chemical synthesis, but no practical physical and chemical properties of these compounds are given in this British patent.



   The object of this invention is to create new 7-methoxycephalo



  to provide sporine derivatives and a process for the preparation of these derivatives by fermentation.



   Cephalophorins have excellent antimicrobial activities against gram-positive and gram-negative bacteria, and among these compounds the cephalosporin derivatives with a methoxy group in the 7-position and a heterocyclic thiomethyl group in the 3-position have very excellent effects in the treatment of serious diseases caused by infection with bacteria such as Pseudomonas and Proteus against which ordinary antibiotics are ineffective, or caused by infection with bacteria which are not sensitive to ordinary cephalosporins which have no methoxy group in the 7-position.



   These compounds are usually prepared by first preparing the corresponding compounds having an acetoxymethyl group or a carbamoyloxymethyl group in the 3-position by a fermentation process, and then reacting these compounds with a heterocyclic thiol compound.



   On the one hand, the present invention also has such an advantage that the compound having a heterocyclic thiomethyl group in the 3-position can be obtained directly by a single fermentation step. The compound thus obtained is the compound having the general formula (A).



   The compounds of formula A themselves have excellent antimicrobial activities, and further the antimicrobial properties and the antimicrobial spectra of the compounds can be increased or changed by the acyl group side chain in the 7-position represented by
EMI2.1
 with another acyl group, such as. B. a-aminophenylacetyl group, a-carboxyphenylacetyl group, a-sulfophenylacetyl group, a-hydroxyphenylacetyl group, pyridylthioacetyl group, thiadiazolylthioacetyl group, triazolylacetyl group, cyanomethylthioacetyl group, trifluoromethyl group, etc. are exchanged. 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: 713-cyanomethylthioacetamidoJamethoxy-341-methyltetrazol-5-ylthiomethyl) t 3-cephem4-carboxylic acid and 7a-methoxy-341-methyltetrazolS5-ylthiomethylS 7ss4trifluormethylthioacetamidofA 3-cephem4-carboxylic acid.



   The compounds of the formula A have the properties like an amphoteric material because the compounds have one amino group and two carboxy groups in the molecule, and thus the isolation and purification of the compounds produced is troublesome.



   As a microorganism which is used for the production of 7-methoxycephalosporin antibiotics and which belongs to the Streptomycetes, a new strain is used in this invention, namely Streptomyces oganonensis Y-G19Z, which was previously invented by the inventors from the soil near the city of Ogano, Chichibugun, Saitama District (Japan). This strain is in the Institute of Microbial Industry, Branch for Industrial Science and Technology, Ministry of International Trade and Industry, Japan, under a deposit number FERM-P 2725 and also in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 (USA) on August 21, 1975 under ATCC No. 31167 has been deposited.



   The mycological properties of the strain are as follows: 1. Morphological characteristics of Streptomyces oganonensis Y-G19Z strain
The strain grows on both natural and synthetic media to form a well-branched substrate mycelium, while the formation of atmospheric mycelium is insufficient because the formation of spores is too low.



  The spore chains are straight, belong to the R- (rectus) or RF- (Rectiflexibles) type and carry 10 to 50 spores in each chain. The spores are elliptical, spherical or cylindrical in shape and have a size of 0.45 to 0.60 x 0.55 to 0.90 u. The spore surface is smooth. Neither flagellate spores nor sporangium are observed.



  II. Culture characteristics of the strain S. oganonensis Y-G19Z: Medium growth Atmospheric Soluble
Mycelium pigment Czapek's agar very low sparse no white white glucose good tolerable no Czapek's agar cream yellow yellowish gray very low glucose good low no asparagine white white agar glycerin good low no asparagine white to white to agar yellowish white yellowish white inorganic good low no Salt starch - yellowish gray yellowish gray agar to weak yellowish brown tyrosine agar good low weak weak yellowish yellowish gray weak yellowish gray iron and good good very low yeast extract - weak yellow brownish white light brownish tyrosine agar to yellowish to yellowish gray brown gray Culture medium - good good,

   powdery weak agar weak weak yellowish brown yellowish brown orange to weak brown Bennett's agar good good very weak weak brownish gray, yellowish brown weak orange to weak pink calcium malate- moderate no no agar cream-colored
Potato good, well brownish gray pieces slightly yellowish gray to dark yellowish brown to slightly yellowish brown brownish gray blood agar good no yellowish gray olive gray to dark dark olive reddish brown gray Loeffer's good no no serum medium
III.

  Physiological properties of S. oganonensis Y-G19Z strain: tyrosine formation negative, nitrate reduction positive
Skim milk coagulation positive, weak Skim milk peptonization positive, weak Hydrolysis of starch positive Liquefaction of gelatin positive, weak cellulose breakdown negative Hemolysis positive Solubilization of calcium malate positive
Exploitation of carbon compounds through. oganonensis Y-G19Z carbon source exploitation glucose + arabinose + sucrose xylose + inositol mannitol + fructose + rhamnose raffinose Characteristic features of Streptomyces oganonensis Y-G19Z strain can be summarized as follows:
1. It belongs to the non-chromogenic Streptomyces strain;
2. Its atmospheric mycelium is straight without branching (R or RF type);
3. Spores are spherical or elliptical;
4th

  Spore surface is smooth;
5. There are pale yellowish gray to pale yellowish brown plants on various media;
6. The color of the spherical mycelium is brownish white, yellowish white and yellowish gray;
7. The generated antibiotic substance Y-G19Z-D3 belongs to the 7-methoxycephalosporin group.



   When researching known strains exhibiting the above properties, the closest streptomycetes can be considered to be the following: Streptomyces globisporus, described in SA Waksman: The Actinomycetes 2,218 (1961) and International Journal ol Systematic Bacteriology, 18, (4) 324 -325 (1968).



   However, if one compares the known S. globisporus, which is described in the above literature, with the strain Y-Gl9Z, this differs in the following properties, which are given in the table:
Table properties Y-Gl9Z S.

   globisporus size of the spore (lot) 0.45-0.60x0.55-1.2-1.4x8-2.0
0.90 or 0.9-1.4 spherical liquid pigment or no yellowish to glycerin-asparagine medium greenish yellow rhamnose utilization negative positive starch hydrolysis strong weak skim milk coagulation positive negative skim milk peptonization weak strong production of positive negative cephalosporin -Antibiotica
From the differences given in the table above, it appears that the strain used in this invention is a new strain which differs from the known strains mentioned above.



   Since the Y-Gl 9Z strain has been confirmed as a new strain by the above observation results, it has been called Streptomyces oganonensis.



   As has already been stated in the case of the Streptomyces oganonensis Y-1 9Z strain, it is a 7-methoxycephalosporin antibiotic-producing strain. As similar strains belonging to the Streptomycetes, the following are known as producers of 7-methoxycephalosporin antibiotics: Streptomyces griseus, Streptomyces viridochromogenes, Streptomyces fimbriatus, Streptomyces halstedii, Streptomyces rochei, Streptomyces cinnamondomyces (Streptomyces undisptamcesdirectiscesiscesiscesiscesiscesamisurisdemptomsces (Streptomyces cinnamonensomyces and Streptomyces cisamonisis chartis, Streptomyces undisamptomsces, Streptomyces cisamonisces, Streptomyces undisamptomsces (Streptomyces cinnamon chart, Streptomyces cinnamon chart and Streptomyces cinnamon chart.

  Japanese Patent Laid-Open No. 3286P7 1 and Belgian Patent No. 764 160) and Streptomyces lipmanii (US Pat. No. 3,719,563), Streptomyces clavuligerus (Japanese Patent 45,594 / '74), Streptomyces wadayamensis (Japanese Patent Laid-Open No. 26488p74), Streptomyces Japanese Patent Application No. 8948774 (Japanese Patent Application Laid-Open Publication No. 26488p74) ), Streptomyces heteromorphus and Streptomyces panayensis (Japanese Patent Application 53594P75), and Streptomyces chartreusis SF-1623 (Japanese Patent Applications 82291r75 and 121 488 / '75).



   The preparation of the discussed compound is carried out by cultivating the above-mentioned 7-methoxycephalosporin antibiotic-producing strain in a common culture medium to which an addition of heterocyclic thiol compound has been added corresponding to the heterocyclic thio group to be introduced in the 3-position.



   Examples of the heterocyclic thiols added to the culture medium in this invention are: pyrrole thiol, imidazole thiol, dihydroimidazole thiol, pyrazole thiol, triazole thiol, tetrazole thiol, methyl tetrazole thiol, pyridinthiol, diazine thiol thiol, thiazole thiol, dihydrothiaziazole thiol, thiazole thiol, thiol, diol thiol thiol , Benzimidazolthiol, Benzoxazolthiol, Benzothiazolthiol, Triazolpyridinthiol, Purinthiol etc. These heterocyclic rings can have one or more substituents such as a halogen atom, an amino; Nitro, alkyl, hydroxy, alkoxy, aryl, aralkyl; Furyl; Thienyl, oxazolyl, carboxy, carboxymethyl, carboxyalkylthio; Carboxyalkyloxy group etc.



   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 etc., 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, etc.



   The salts with high water solubility can be used selectively, if necessary, and further, if the heterocyclic thiols are highly toxic to the antibiotic-producing strains, the sparingly water-soluble salts can be used selectively.



   Also two molecules of the same heterocyclic thiol compound connected by the S-S bond can be used, such as. B. R2S-SR2.



   The novel antibiotics obtainable according to this invention are prepared by cultivating the strain of the Streptomyces species mentioned in claim 1 in a culture medium with addition of the heterocyclic thiol compound already described above or the salt or its derivative R2-S-S-R2. The cultivation is carried out according to the usual conventional methods for growing microorganisms, but submerged culture in a liquid culture medium is preferred. Any culture medium containing nutrients for the Streptomyces strains producing 7-methoxycephalosporin antibiotics can be used. Synthetic culture media, semi-synthetic culture media and natural culture media containing the above-mentioned nutrients can be used in this invention.

  For the compilation of the culture medium, glucose, sucrose, mannitol, glycerin, dextrin, starch, vegetable oils etc. can be used as carbon sources, and meat extract, peptones, glue meal, cottonseed meal, soybean meal, peanut meal, fish meal, grain stillage, dry yeast, ammonium sulphate extract, yeast sulfate extract , Ammonium nitrate, urea and other organic and inorganic nitrogen sources can be used as nitrogen donors. Also, if necessary, metal salts such as sulfates, nitrates, chlorides, carbonates, phosphates, etc. of sodium, potassium, magnesium, calcium, zinc and iron can be added to the culture medium.

  Furthermore, if necessary, materials which favor the formation of antibiotics or defoaming agents such as methionine, cysteine, cystine, methyl oleate, lard oil, silicone oil, surfactants, etc. can be appropriately added to the culture medium.



   The above-mentioned heterocyclic thiol, its salt or its derivative R2-S-S-R2 is usually added in a concentration of 0.1-5 mg / ml, preferably 0.5-2 mg / ml, as a heterocyclic thiol. It can be added to the culture medium as a drop-like addition before cultivation or in various divided portions in the entry stages of the cultivation. It is generally convenient to carry out cultivation under aerobic conditions. The cultivation temperature is usually between about 18 "C to about 35" C, preferably around 30 "C. Good results are obtained if the pH of the culture medium is kept at about 5-10, preferably about 6-8.

  The cultivation timing depends on the composition and the temperature of the culture medium used, but generally about 3 days to about 10 days are required. The material produced is selectively enriched in the medium after the cultivation is complete.



   The material made in accordance with this invention can be isolated and recovered from the culture broth by the usual methods common for isolating antibiotics from the cultured mycelium medium. The antibiotic produced according to the invention is mainly present in the culture broth. After separating the mycelium from the culture medium solution by centrifuging or filtering, the effective material produced is extracted from the filtrate; that is, the material produced is separated, extracted and cleaned of the filtrate.

  Here devices and methods are used which are generally used for the production of antibiotics, such as methods which take advantage of the differences in solubility in a suitable solvent, which take advantage of the differences in adsorptive affinity for different adsorbents, or which differ in the separation methods in two liquid phases.



   If necessary, these methods can be used in a suitable combination or repeatedly.



   Some practical examples of the new 7-methoxycephalosporin compounds of this invention are given below.



      1,745-amino-5-carboxyvaleramidof345-carboxymethyl-thio-1,3,4-thiadiazol-2-yl) thiomethyl-7-methoxy-h 3-cephem4carboxylic acid;
EMI4.1
 Addition compound: 5-mercapto-l, 3,4-thiadiazole-2-thioacetic acid;
EMI4.2

The physical and chemical properties of the compound I with the general formula A
EMI4.3
 are the following:
1. white powder;
2nd

  Melting begins at 156-160 "C. This turns brown and the decomposition begins at about 170" C;
3. Easily soluble in water, sparingly soluble in methanol, and very sparingly soluble in other organic solvents;
4. amphoteric material with positive ninhydrin reaction;
5. it has the ultraviolet absorption spectrum according to FIG. 1 of the accompanying drawing when it is measured in a 1/100 M phosphate buffer solution with a pH of 6.4, the absorption maximum being 287 ml.
6. it has the infrared absorption spectrum according to FIG.



  2 if it is measured in the potassium bromide tablet. There are absorptions at 3413cm-1, 2920cm1, 1763cm- ', 1620cm1, 1515cm-1 and 1380 cm';
7. When determining the nuclear magnetic resonance spectrum using TMS as the external standard in heavy water, the following signals are obtained: 6value (ppm): 2.35 (4H, multiplet), 2.96 (2H, multiplet), 4.00 ( 3H, singlet), 3.73-4.33 (2H, quartet, J = 18 Hz), 4.25 (1 H, multiplet), 4.44 (2H, singlet), 4.42 - 4.91 ( 2H, quartet, J = 14 Hz), 5.63 (1 H, singlet);
8th.

  The product, which is currently in its purest state, has the following elementary analytical values: C: 35.95%, H: 3.87%, N: 10.85%, S: 18.33%; 9. it gives a-aminoadipic acid on hydrolysis with 6N hydrochloric acid; 10. After an 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 mle 392, ie
EMI5.1

Taking all the above results into account, it can be seen that this compound is a 7-methoxycephalosporin compound because 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-OCH3), 5.63 ppm (1 H, singlet,

   6-CH), 3.73 - 4.33 (2H, quartet, J = 18 Hz, 2-CH2) and 4.42 - 4.91 (2H, quartet, J = 14 Hz, 3-side chain CH2) in the nuclear magnetic resonance spectrum. The compound gives a-aminoadipic acid in acid hydrolysis, the compound also gives absorptions at 4.44 p pm (2H, Singlett, CH2 of -S-CH2 COOH) in the nuclear magnetic resonance spectrum and also the fragment of nile 392 in the mass spectrum of the derivative; for these reasons, it has been decided that the compound has the above structure in which the heterocyclic thiol has been introduced.



  II. I.745-Amino-5-carboxyvaleramidof3A 1 -methyl- 1 H-tetrazol-5 ylstiomethyl-7-methoxy-h 3-cephem4-carboxylic acid:
EMI5.2
 Addition compound: 5-mercapto-l-methyl-1 H-tetrazole;
EMI5.3

The physical and chemical properties of compound II of formula A produced
EMI5.4


 <tb> <SEP> R2 <SEP> = <SEP> N <SEP>, <SEP> M <SEP> = <SEP> H <SEP>)
 <tb> <SEP>, <SEP> 7N
 <tb> <SEP> CII
 <tb> are <SEP> the <SEP> following:

   <SEP> 3
 <tb>
I. White dust;
2nd  gives brown discoloration and decomposition at 160-170 "C;
3rd  easily soluble in water, slightly soluble in methanol and sparingly soluble in other organic solvents;
4th  amphoteric material with positive ninhydrin reaction;
5.  it will be shown in Fig.  3 shown ultraviolet absorption spectrum obtained when measured in 1/100 M phosphate buffer solution with a pH of 6.4.  The absorption maximum is 273 mm;
6.  it will be shown in Fig.  4 infrared absorption spectrum obtained when measuring the potassium bromide tablet. 

  Absorbances at 3413 cm-1, 2920cm- ', 1765 cm-',
1620 cm- ', 1515 cm1 and 1390cm-1 found;
7.  the nuclear magnetic resonance spectrum, measured using TMS as an external standard in heavy water, gave the following signals: 6value (ppm): 2.36 (4H, multiplet), 2.96 (2H, multiplet), 3.98 (3H, Singlet), 4.38 H, H, multiplet), 4.50 (3H, singlet), 5.59 H, H, singlet);
8th.  the material obtained in the purest state so far gave the following elementary analytical values: C: 37.48%, H:. 4.25%, N: 16.74% and S: 10.90%;
9.  a-aminoadipic acid is obtained when hydrolyzed with 6N hydrochloric acid. 

  5-Mercapto-1-methyl-1 H-tetrazole is obtained by hydrolysis in methanol by Dowex 50 W (H type);
Taking all of the above results into account, the compound is a 7-methoxycephalosporin compound because the compound gives signals at 3.98 ppm (3H, singlet, 7-OCH3) and 5.59 ppm (1H, singlet) , 6-CH) in the nuclear magnetic resonance spectra and absorption at 1765 cm- '(cyclic lactam) in the infrared absorption spectrum.  Furthermore, a-aminoadipic acid is obtained by acid hydrolysis.  

  Furthermore, the fact of absorption at 450 ppm (3H, singlet, tetrazole-N-methyl) in the nuclear magnetic resonance spectrum and also the fact that 5-mercapto-l-methyl-1H-tetrazole is obtained by mild hydrolysis means that the compound has been assigned the structure set forth above into which the heterocyclic thiol has been introduced. 



      111. 745-Amino-5-carboxyvaleramidof7-methoxy-3- (5-methyl-1,3,4-thiadiazol-2-yl) thiomethyl-h 3-cephem4-carboxylic acid. 
EMI5. 5
  



  Addition compound: 2-mercapto-5-methyl-1, 3,4-thiadiazole:
EMI6. 1

The physical and chemical properties of compound III with formula A
EMI6. 2nd
 are the following:
1.  light yellow white powder;
2nd  shows no definite melting point, gives brown discoloration and decomposition at about 170 "C;
3rd  easily soluble in water, slightly soluble in methanol, but insoluble in other organic solvents;
4th  amphoteric material with positive ninhydrin reaction;
5.  this results in Fig.  5 shows the ultraviolet absorption spectrum when measured in a 11100 M phosphate buffer solution with a pH of 6.4, the absorption maximum being 272 mu;

  ;
6.  it will be shown in Fig.  6 infrared absorption spectrum shown when measured as a potassium bromide tablet obtained with absorptions at 3420 cm- ', 2930 cm', 1765cm- ', 1610 cm-', 1515cm-1 and 1385 cm- ';
7.  the nuclear magnetic resonance spectrum, measured using TMS as an external standard in heavy water, gives the following signals: b-value (ppm): 2.34 (4H, multiplet), 2.95 (2H, multiplet), 3.19 ( 3H, singlet), 3.72-4.31 (2H, quartet, J = 18 Hz), 3.99 (3H, singlet), 4.26 (1 H, multiplet), 4.40-4.95 ( 2H, quartet, J = 14 Hz), 5.63 (1H, singlet);
8th.  the purest compound currently produced has the following elementary analytical values: C: 37.82%, H: 4.01%, N: 12.90%, S:

   14.97%
9.  a-aminoadipic acid is obtained when hydrolyzed with 6N hydrochloric acid.  2-Mercapto-5methyl-1,3,4-thiadiazole is also obtained if 50 W (H-type) is hydrolyzed in methanol with Dowex. 



   Taking all of the above results into account, it can be seen that the compound is a 7-methoxycephalosporin compound because the compound has the absorption at 1765 cm-1 (cyclic lactam) in the infrared absorption spectrum.  Furthermore, the signals at 3.99 ppm (3H, singlet, 7-OCH3), 5.63 ppm (1 H, singlet, 6-CH), 3.724.31 ppm (2H, quartet, J = 18 Hz, 2- CH2), 4.40-4.95 (2H, quartet, J = 14 Hz, 3-side chain CH2). 

  A-aminoadipic acid is obtained in the acid hydrolysis; and further due to the fact that the absorption at 3.19 ppm (3H, singlet, thiadiazole C-CH3) is obtained in the nuclear magnetic resonance spectrum and also 2-mercapto-5-methyl1,3,4-thiadiazole is obtained by mild hydrolysis , the compound having the above-mentioned structure into which the heterocyclic thiol has been introduced is recognized. 



      IV. 745-Amino-5-carboxyvaleramidof7-methoxy-341,3,4-thia-diazol-2-yl) thiomethyl-h 3-cephem4-carboxylic acid.    
EMI6. 3rd




  Addition compound: 2-mercapto-1, 3,4-thiadiazole;
EMI6. 4th

The physical and chemical properties of compound IV of formula A produced
EMI6. 5
 are the following:
1.  light yellow-white powder;
2nd  it shows no definite melting point, but brown discoloration and decomposition take place at about 175-180 "C;
3rd  easily soluble in water, slightly soluble in methanol, but insoluble in other organic solvents;
4th  amphoteric material with positive ninhydrin reaction;
5.  this results in Fig.  7 shown ultraviolet absorption spectrum when measured in a 1:

  : 100 M phosphate buffer solution with a pH of 6.4, absorption maximum at 274 ml;
6.  it will be shown in Fig.  8 infrared absorption spectrum obtained when measured with the potassium bromide tablet, the absorptions being 3400 cm- ', 2925 cm-1, 1765 cm-1, 1610 cm-', 1515 cm- 'and 1365 cm-' ;
7.  the nuclear magnetic resonance spectrum, measured using TMS as an external standard in heavy water, gives the following signals: 8-value (p p m):

   2.30 (4H, multiplet), 2.93 (2H, multiplet), 3.69-4.29 (2H, quartet, J = 18 3.97 (3H, singlet), 4.26 H, H, Multiplet), 4.45-4.99 (2H, quartet, J = 14 Hz), 5.56 H, H, singlet), 9.85 (1 H, singlet);
8th.  the product currently obtained in the purest state has the following elementary analytical values: C: 37.53%, H: 4.36 :, N: 12.77%, S: 16.42%;
9.  a-Aminoadipic acid is obtained on hydrolysis with 6N hydrochloric acid.  There is also obtained 2-mercapto-1,3,4thiadiazole on hydrolysis in methanol with Dowex 50 W (H type). 



   Taking all the results into account, the result is that the compound is a 7-methoxycephalosporin compound, because the compound gives an absorption at 1765 cm1 in the infrared absorption spectrum, and the signals at 3.97 ppm (3H, Singlett, 7- OCH3), 5.56 ppm (1 H, singlet, 6-CH), 3.69-4.29 ppm (2H, quartet, J = 18 Hz, 2-CH2), 4.45-4.99 (2H , Quartet, J = 14 Hz, 3-side chain CH2) obtained.  a-Aminoadipic acid is obtained by acid hydrolysis. 

  From the facts that the absorption at 9.85 ppm (1 H, singlet, thiazole CH) is present in the nuclear magnetic resonance spectrum and 2-mercapto-1, 3,4-thiadiazole is obtained with mild hydrolysis, the compound is the aforementioned Structure assigned in which the heterocyclic thiol has been introduced. 



   The results of various chromatographic analyzes and paper electrophoretic investigations of the compounds 1, II, III and IV prepared are given below. 



   The Rf values of this compound obtained in thin layer chromatography using microcrystalline cellulose (Avicel SF) are shown in the following table:
1 2 3 Compound 1 0.39 0.37 0.32 Compound II 0.39 0.34 0.31 Compound III 0.43 0.43 0.40 Compound IV 0.39 0.38 0, 33 cephalosporin C 0.37 0.36 0.31 7-methoxycephalosporin C 0.41 0.36 0.32 cephamycin C 0.37 0.36 0.31 Y-Gl 9Z-D3 0.26 0.26 0, 22 YG 1 9Z-D2 0.39 0.32 0.26 Development solvent system used (volume ratio): 1.  Isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9).    



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



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



   The control sample, cephamycin C, is 745-amino-5-carboxyvaleramidof3-carbomoyloxymethyl-7-methoxy-A 3-cephem4-carboxylic acid. 



   The compounds Y-G19Z-D3 and Y-G19Z-D2, which were used in the comparative tests above, are new 7-methoxy-cephalosporin compounds which had previously been prepared from the culture liquid of Streptomyces oganonensis by the same inventors (cf.  Japanese Patent Application No. 



  109 7531'74 and 146 593 / '75) have been isolated. 



   The Rf values obtained in paper distribution chromatography using Wattman No.  1 filter paper and a developing solvent system of n-butanol: acetic acid: water (4: 1: 2 in volume ratio) are as follows:
Rf value Compound 1 produced 0.40 Compound II 0.39 cephalosporin C 0.36 7-methoxy-cephalosporin C 0.40 Cephamycin C 0.35 Y-G 1 9Z-D3 0.24 Y-G 1 9Z-D2 0.25
The compounds were then analyzed using a Hitachi 635 high speed liquid chromatography apparatus and the results were as follows:
Column: 3 x 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. 



   Writing medium speed: 1.0 cm / min. 



   Retention time Compound I prepared 5 min 33s Compound II 6 min 00 s Compound III 9 min 35 s Cephalosporin C 5 min 18 s 7-methoxy-cephalsporin C 5 min 09s Cephamycin C 5 min 18 s Y-G19Z-D3 3min42s Y- G19Z-D2 3min42s
Below are further results of analyzes using the above-described apparatus obtained under the following conditions in the table:
Pillar:> u Bondapa Cis (Manufacturer: Waters Ltd. ) 4 x 300 mm. 



   Developing solvent system: Acetonitrile: 0.1% acetic acid solution (pH 3.3) (1: 9 in volume ratio). 



     Flow rate: 0.8 ml / min. 



   Writing medium speed: 1.0 cm / min. 



   Retention time Compound I 3 min 14s Compound II 1 min 52s Compound III 2 min 55s Compound IV 1 min 56s
The results obtained by high voltage paper electrophoresis are as follows: Filter paper: Wattman no.  1 developing solvent: 10% acetic acid (pH 2.2). 

 

  Voltage: 42 volts / cm. 



  Running time: 1 hour.  Migration removal Compound I prepared - 3.6 cm compound II - 3.3 cm compound III - 3.6 cm compound IV - 3.9 cm cephalosporin C - 3.5 cm 7-methoxy-cephalosporin C - 3, 4 cm cephamycin C - 3.5 cm Y-G19Z-D3 - 6.1 cm Y-G1 9Z-D2 - 6.1 cm cysteic acid - 7.5 cm glutathione - 1.2 cm
The antibacterial activity of the compounds 1, II, III and IV prepared was then determined.  The results are shown in the following table together with those of cephalosporin C as a comparative substance. 



  Cardiac infusion agar plate method (500 yglml solution used)
The numerical values in the table indicate the diameter (mm) of the inhibition zone.   



     1 II III IV C Sarcina lutea ATCC 9341 0 0 0 0 14.0 Staphylococcus aureus 209 P 0 0 0 0 12.8 Bacillus subtilis ATCC 6633 0 0 0 0 23.1 Escherichia coli NIHJ 19.2 18.7 14.2 13.0 10.4 Klebsiella pneumoniae 21.8 23.5 23.0 23.0 13.0 Salmonella gallinarum 24.0 25.2 23.5 25.1 23.5 Proteus vulgaris OX 19 22.6 20.2 20.0 21.0 17.5 Proteus mirabilis IMF OM-9 22.2 19.8 19.5 23.0 23.0 Note: I: Compound I of this invention II: Compound II of this invention III: Compound III of this invention IV: Compound IV of this invention C: Cephalosporin C (comparison)
Each of the compounds 1, II, III and IV produced according to the invention can be used in various forms, individually or in combination with other medicaments. 



  This means that the compounds of this invention orally, by intramuscular injections, by intravenous injection, etc.  in the form of capsules, tablets, powders, granules, solutions and suspensions.  Various carriers are added to the preparations, for example mannitol, sucrose, glucose, sterilized distilled water, saline and a vegetable oil such as peanut oil, sesame oil.  Furthermore, other ingredients such as stabilizers, binders, antioxidants, preservatives, a lubricant in the manufacture of tablets, suspending agents, viscosity agents, perfume, etc. can be added. 



   As salts of the compounds of formula A, the salts with the cations M defined in claim 1, which are pharmacologically non-toxic or useful, are used.  The dosage of the medication mainly depends on the condition and weight of the host.  It also depends on the mode of administration, e.g.  B.  oral or parenteral administration.  Generally, 50 mg / kg is administered in one dose or in a few divided doses per day. 



   Compounds A 1 of this invention have excellent antimicrobial activities; these are used for the prophylaxis and treatment of diseases in humans and animals.  They can also be used in a suitable manner as intermediates for the preparation of other effective 7-methoxycephalosporin derivatives.  In such cases, it is preferred to isolate the compound of formula Al as a pure product or as a highly concentrated crude product.    



   Especially when the substituent group
EMI8. 1
 in the 7-position of the compound of formula A is replaced by another acylamide group, the amphoteric properties disappear and the acidic product thus modified is well suited for isolation and becomes soluble in organic solvents.  Therefore, the modification of the compound of formula A, as already explained above, is preferred for use in industrial practice. 



   The in vivo effects of compounds II, III are shown below:
106 healthy cells of E. were isolated from 5 healthy male mice of the ddY strain.  coli NIHJ injected intraperitoneally.  After 2 hours, each sample was administered subcutaneously.  The survival percentages after 5 days are given in the table below.  Similar studies were carried out on 105 Proteus mirabilis 1287 cells. 



   The control group consisted of 10 mice each time
E. coli NIHJ Proteus mirabilis 1287 dose 3 1 0.5 0 3 1 0.5 0 (mg / mouse) sample II 100 100 100 0 40 20 0 0 sample III 80 80 40 0 100 20 20 0 The following are examples of this invention clarified in detail. 



  example 1
Preparation of 7- (5-AminoS-carboxyvaleramido> 3- (5-car-boxy-methylthio-1,3,4-thiadiazol-2-yl) thiomethyl-7-methoxy-AD3-cephem4-carboxylic acid I:
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 placed in a 500 ml Sakaguchi bottle filled with 100 ml each and sterilized at 1200 C for 20 minutes.  Each bottle was inoculated with Streptomyces oganonensis Y-G19Z strain and cultured at 30 C for 48 hours. 



   In further batches, the same culture medium was filled into 2000 ml Sakaguchi bottles with 400 ml each and sterilized at 1200 C for 20 minutes.  It was inoculated with the same strain in a concentration of 2-3% and cultured at 30 C for 24 hours to obtain a culture. 



   Furthermore, 60 liters of a culture medium containing 7% starch, 2% gluten flour, 2% soybean flour, 0.8% glycerol, 0.1% casamino acid, 0.01% ferrisulfate and 55 g sodium hydroxide were each combined in two 100 liter fermenters filled with 10 ml of adecanol as a defoamer.  It was sterilized at 1200 C for 30 minutes.  Each medium was inoculated with 800 ml of the culture, and then cultivated at 30 "C for 24 hours. 

  5-Mercapto-1, 3,4-thiadiazole-2-thioacetic acid was dissolved in aqueous sodium hydroxide solution, and the resulting solution was sterilized under high pressure and added to each fermentor up to 0.05% of the inoculum and then cultivated for 90 hours. 



   After complete cultivation, the culture broth was adjusted to pH 2.0 and then mixed with radiolite (as a filter aid).  The mixture was filtered using a filter press.  The filtrates were combined and 100 liters of a filtrate mixture were obtained.  The filtrates were adjusted to pH 3.0 with aqueous sodium hydroxide solution and 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 acetone.  The eluate was collected and concentrated to 5.5 liters.  After the contaminants formed were removed, water was added to the residue to obtain 10 liters of solution.  The solution thus obtained was adjusted to pH 3.5 with dilute aqueous 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, elution was carried out with an aqueous solution (pH 7.2) containing 1 M sodium nitrate and 0.1 M sodium acetate.  About 5 liters of solution with antimicrobial active material were obtained.  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 500% acetone.  

  About 400 ml of aqueous solution containing antimicrobial active material was obtained.  By lyopholizing the solution, approximately
18 g of crude product of compound I obtained.   



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



   Then a mixed solvent of isopropanol: water (7: 3 by volume) was used to subject the residue of the product obtained to column chromatography using microcrystalline cellulose (Avicel) prepared with a mixed solvent of the same composition given above.  The antimicrobially active fraction obtained was applied in drop form to a thin-layer plate made of Avicel SF and developed with a mixture of isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9 volume ratio).  Then pyridine solution of 0.25% ninhydrin was sprayed on and then heated to develop the color.  Then the fractions with an Rf value of 0.39 were collected, 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 mixed solvent of isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9 volume ratio).  The antimicrobially active fraction thus obtained was then subjected to thin-layer chromatography with Avicel SF using the solvent mixture mentioned above and processed further with the same post-treatment.  The fractions with Rf 0.39 were collected and evaporated to dryness in vacuo.  The product residue was further purified by microcrystalline cellulose column chromatography using a solvent mixture of n-butanol: acetic acid: water (6: 1.5: 2.5 in volume 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 solvent mixture of n-butanol: acetic acid: water (6: 1.5: 2.5 in volume ratio).  The fractions with Rf 0.32 were collected, concentrated and then subjected to lyophilization.  About 80 mg of white 7- (5-amino-5-carboxy-valeramido) -3- (5-carboxymethylthio-1,3,4-thiadiazol-2-yl) thiomethyl-7-methoxy-h 3-cephem were obtained -4 carboxylic acid obtained. 



  Example 2
Preparation of 7- (5-amino-5-carboxyvaleramido) -3- (1-methyl-1 H-tetrazol-5-yl) thiomethyl-7-methoxy-h 3-cephem4-car bonic acid II:
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 placed in 500 ml Sakaguchi bottles filled with 100 ml each and sterilized at 120 "C for 20 minutes.  Then each medium was inoculated with Streptomyces oganonensis Y-G197 strain.  The mixture was then cultivated at 30 ° C. for 48 hours. 



  The culture medium mentioned above was then poured into two liters of 400 ml Sakaguchi bottles.  After sterilizing for 20 minutes at 120 ° C., each medium was inoculated with 2-3% of the culture broth obtained above and then cultivated at 30 ° C. for a further 24 hours in order to obtain a vaccination batch. 



   60 liters of a culture medium, which contained 7% starch, 2% gluten flour, 2% soybean oil, 0.8% glycerin, 0.1% casamino acid, 0.01% ferrisulfate and 55 g sodium hydroxide, were in two liters fermenters with 10 ml of adecanol filled as a defoamer.  After sterilizing for 30 minutes at 120 "C, each medium was mixed with 800 ml of the inoculum prepared above, and then cultivated at 30" C for 24 hours.  Then l-methyl-5-mercapto-1H-tetrazole was dissolved in aqueous sodium hydroxide solution. 

  The solution was sterilized at high pressure and added to the culture broth to adjust its concentration to 0.05%.  The mixture was further cultivated for 90 hours. 



   After the cultivation was completed, the culture broth thus formed was adjusted to pH 2.0, and then radiolite as an auxiliary for filtering with stirring was added, and the mixture was filtered using a filter press.  The filtrates obtained were combined and about 100 liters of filtrate mixture were obtained. 



   The filtrate was adjusted to pH 3.0 with 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 (Cl-Type} column.  The column was washed with 6 liters of water and fractionated with an aqueous solution (pH 7.2) containing 1 M sodium nitrate and 0.1 M sodium acetate.  About 5 liters of a solution containing an antimicrobial active material was 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.  Lyophilization of the solution gave approximately 54 g of the crude powder of the compound II prepared.  The crude powder was subjected to column chromatography with about 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 fractionate the active components.  The antimicrobial active components were collected, passed through a 500 ml Amberlite XAD-2 column.  The column was washed with water and eluted with an aqueous solution of 25% acetone. 

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



   The residue formed was subjected to column chromatography using microcrystalline cellulose (Avicel), filled with a mixed solvent of isopropanol: water (7: 3 by volume) using the mixed solvent having the above composition.  The antimicrobially active fraction obtained was collected, applied dropwise to a thin layer plate made of Avicel SF, developed with a solvent mixture of n-butanol: acetic acid: water (6: 1.5: 2.5 volume ratio) and a solution of 0.25% ninhydrin spraying was carried out in pyridine and the stain was then developed by heating.  Then the fractions with Rf 0.31 were collected.  The fractions were concentrated under reduced pressure and dried. 

 

  The residue formed was subjected to column chromatography with microcrystalline cellulose (Avicel).  The filling was prepared with a mixed solvent of isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9 volume ratio).  The obtained antimicrobial active fractions were subjected to Avicel SF thin layer chromatography with the mixed solvent having the same composition as mentioned above, using the same procedure as above.  The fractions with Rf 0.39 were collected and evaporated to dryness in vacuo. 



   The residue so formed was subjected to microcrystalline cellulose column chromatography using a mixed solvent of n-butanol: acetic acid: water (6: 1.5: 2.5 by volume) 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 1 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 Rf 0.31 were collected, concentrated and then lyophilized. 

  About 60 mg of white 745-amino-5-carboxyvaleramidof3- (1-methyl-1 H-tetrazol-5-yl) thiomethyl-7-methoxy-h 3-cephem4carboxylic acid was obtained. 



  Example 3
Preparation of 745-Ami o-5-carboxyvaleramido> 7-methoxy-345-methyl-1,3,4-thiadiazol-2-yl) thiomethyl-h 3-cephem4-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 included in 500 ml Sakaguchi bottles filled with 10 ml and sterilized at 120 "C for 20 minutes.  Each medium was then inoculated with Streptomyces oganonensis Y-G19Z strain and cultured at 30 "C for 48 hours. 

  Another culture medium, as described above, was filled in two-liter Sakaguchi bottles with 400 ml each and sterilized at 120 ° C. for 20 minutes.  Each medium was inoculated with the above-cultured broth at a 2-3% concentration, and then cultured at 30 "C for 24 hours, whereby an inoculation culture was obtained. 



   Separately, 60 liters of the culture medium, which contained 7% starch, 2% gluten flour, 2% soybean flour, 0.8% glycerol, 0.1% casamic acid, 0.01% ferrisulfate and 55 g sodium hydroxide, were combined in two 100 liter fermentors filled with 10 m] adecanol as a defoaming agent.  It was sterilized at 120 ° C. for 30 minutes and inoculated with 800 ml of the inoculation culture.  It was cultivated at 30 ° C. for 24 hours.  Then, 2-mercapto-5-methyl-1,3,4-thiadiazole was dissolved in aqueous sodium hydroxide solution and sterilized under high pressure and added to the culture broth so that the concentration thereof was 0.05% in the broth, followed by 90 hours was cultivated. 



   After the cultivation, the culture broth was adjusted to pH 2.0 and mixed with radiolite as a filter aid with stirring.  The mixture was filtered using a filter press and the filtrates were combined to give about 300 liters of the filtrate mixture. 



   The filtrate was adjusted to pH 3.0 by adding 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 aqueous 50 '/ above acetone.  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 3 liter Amberlite IRA-68 (cl-type) column.  The column was washed with 6 liters of water.  It was eluted with an aqueous solution (pH 7.2) containing 1 M sodium nitrate and 0.1 M sodium acetate.  About 5 liters of an antimicrobial active material were obtained.  The solution was adjusted to pH 3.0 and passed through a one liter Amberlite XAD-2 column. 

  After washing the column with water, the column was eluted with an aqueous solution of 50% acetone.  About 400 ml of aqueous solution containing the antimicrobial active material was obtained.  The product 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) filled with the mixed solvent of the same composition given above.  The antimicrobially active fractions obtained were then fractionated and applied drop-shaped to a thin-layer plate from Avicel SF and developed with a solvent mixture isopropanol: butanol n-butanol: acetic acid: water (21: 3: 7: 9 in volume ratio).  A pyridine solution of 0.25% ninhydrin was sprayed on and the staining developed under heating. 

  The fractions with the Rf value 0.43 were collected and brought to dryness at 45-50 ° C. in vacuo.  The residue obtained was subjected to column chromatography with microcrystalline cellulose (Avicel).  The column was prepared with a mixed solvent of acetonitrile: water (7: 3 volume ratio).  The obtained antimicrobial active fraction was subjected to Avicel SF thin layer chromatography as in the above procedure.  Then the fractions with Rf 0.43 were collected and evaporated to dryness.  0.78 g of crude powder was obtained. 



   The powder was dissolved in a small amount of distilled water and developed in a Sephadex G 10 column using distilled water.  The antimicrobial 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 an Rf value of 0.43 were collected, concentrated and lyophilized.  82 mg of white 745-amino-5-carboxyvaleramidoS7-methoxy-345-methyl-1,3,4-thiadiazel-2-ylpthiomethyl-A 3-cephem4-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-ffliadiazol-2-yl) disulfide, prepared by dissolving the disulfide in water-containing methanol and sterilizing Filtration using a Millipore filter in this example instead of the solution of 5-mercapto-1, 3,4-thiadiazol-2-thioacetic acid, prepared in water using aqueous sodium hydroxide solution and sterilization at high pressure, gave 23 g of raw powder of the compound I prepared . 

  By purifying the product as indicated in Example 1, about 45 mg of 7- (5-amino-5-carboxyvaleramido 35-carboxymethylthio-1, 3,4-thiadiazol-2-yl) thiomethyl-7-methoxy-h 3-cephem4-carboxylic acid (compound I prepared) obtained. 

 

  Example 5
Using the same procedure as given in Example 2, but using a solution of bis (1-methyl-1 H-tetrazol-5-yl) disulfide, prepared by dissolving the disulfide in methanol containing water and sterilizing by filtration using a Millipore filter in this example, instead of a solution of 1-methyl-5-mercapto-1H-tetrazole, prepared by dissolving the tetrazole in water using an aqueous sodium hydroxide solution and sterilizing at high pressure, about 26 g of the crude powder of the compound II prepared was obtained. 

  By purifying the product as indicated in Example 2, about 37 mg of 745-amino-5-carboxyvaleramido) -3 (1-methyl-1 H-tetrazol-5-yl> thio methyl-7-methoxy-h 3-cephem4 -carboxylic acid (compound II) obtained. 



  Example 6
By following the same procedure as in Example 3, by inserting a solution of bis (5-methyl-1, 3,4-thiadiazol-2-yl) disulfide, prepared by dissolving the disulfide in aqueous methanol, and sterilizing by Filtration using a millipar filter in this example instead of the solution of 2-mercapto-5-methyl-1,3,4-thiadiazole, prepared in water using an aqueous sodium hydroxide solution and sterilization at high pressure, obtained about 19 g of crude powder of the compound III prepared .  By purifying the product as indicated in Example 3, about 50 mg of 7- (5-amino-5-carboxyvaleramido7-methoxy-35-methyl-1,36-thiadiazol-2-yl) thiomethyl-h 3-cephem4-carboxylic acid (Compound III) obtained. 



  Example 7
Preparation of 7- (5-amino-5-5-carboxyvaleramido) -7-methoxy-341,3,4-thiadiazol-2-yl) thiomethyl-h 3-cephem4-carboxylic acid IV. 



   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 included in 500 ml Sakaguchi bottles filled 100 ml each, sterilized for 20 minutes at 120 "C and inoculated with the Streptomyces oganonensis Y-G19Z strain.  The mixture was then cultivated at 30 ° C. for 48 hours.  Another culture medium above was filled in two-liter Sakaguchi bottles with 400 ml each and sterilized at 120 ° C. for 20 minutes.  Then inoculation was carried out with 2-3% of the culture broth prepared according to the above procedure. 

  Cultivation was carried out at 30 ° C. for 24 hours in order to obtain a seed culture. 



   Separately, 60 liters of a culture medium containing 7% starch, 2% gluten flour, 2% soybean meal, 0.8% glycerin, 0.1% casamino acid, 0.01% ferrisulfate and 55 g sodium hydroxide were mixed in two 100 liter fermentors, together with 10 ml of adecanol filled as a defoamer and sterilized at 120 "C for 30 minutes.  Then inoculated with 800 ml of the seed culture. 



  It was cultivated 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 was added to the culture broth so that the concentration of the thiadiazole was 0.05%. amounted to.  The batch was then further cultivated for 90 hours. 



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



   The filtrate was adjusted to pH 3.0, 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 aqueous 50% acetone solution.  The eluates were concentrated to 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 acetate.  About 5 liters of a solution containing antimicrobial active material was obtained. 



  The solution was adjusted to pH 3.0, passed through a one liter Amberlite XAD-2 column and washed with water. 



  About 400 ml of aqueous solution containing the antimicrobial active material was obtained.  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 of the same composition as above.  The antimicrobially active fractions were fractionated, placed in drops on an Avicel thin-layer plate, developed using a mixed solvent of isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9 in volume ratio). 

  Then a pyridine solution of 0.25% ninhydrin was sprayed on and the staining developed by heating. 



  The fractions with an Rf value of 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 prepared with a mixed solvent of acetonitrile: water (7: 3 volume ratio).  The antimicrobial active fractions were also subjected to Avicel SF thin layer chromatography as in the above procedure.  The fractions with an RF value of 0.39 were collected and 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 developed in a Sephedex G 10 column using distilled water.  The antimicrobial activity of each fraction was checked. The effective fractions were subjected to thin layer chromatography using a mixture of n-butanol: acetic acid: water (4: 1: 2 volume ratio) as described above. 



  Then the fractions with Rf 0.38 were collected, concentrated and lyophilized.  White 745-amino-5-carboxyvaleramidok7-methoxy-341,3,4-thiadiazol--2-yl) thiomethyl-h 3-cephem4-carboxylic acid was obtained. 



  Example 8 a) A culture medium which contained 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 dissolved in 500 -ml Sakaguchi bottles filled in a quantity of 100 ml each. Each bottle was sterilized at 120 "C for 20 minutes.  Each medium was inoculated with Streptomyces oganonensis Y-G1 9Z strain and cultured at 30 ° C. for 48 hours. 



   The culture medium was also filled in 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 cultured at 30 ° C. for 24 hours.  Inoculation culture was thus preserved. 



   Separately, 60 liters of a culture medium containing 7% starch, 2% gluten flour, 2% soybean meal, 0.8% glycerin, 0.1% casamino acid, 0.01% ferrisulfate and 55 g sodium hydroxide in two 100 liter fermentors were used together 10 ml of adecanol added as an anti-foaming agent, sterilized for 30 minutes at 120 "C.     It was inoculated with 800 ml of the inoculum, which had been prepared according to the above instructions.  It was cultivated at 30 ° C. for 24 hours.  

  Then, a solution of 5-mercapto-1-methyl-1H-tetrazole prepared from an aqueous sodium hydroxide solution and sterilization under high pressure was added to the culture broth so that the concentration of the tetrazole was 0.05%.  The cultivation was carried out for 90 hours. 



   After the cultivation was complete, the culture broth was adjusted to pH 2.0 and mixed with radiolite as a filter aid with stirring.  The mixture was filtered with a filter press and the filtrates obtained were combined.  100 liters of filtrate mixture containing 100 mcg / ml of 745-amino-5-carboxyvaleramidof7-methoxy-3- (1-methyl-1 H-tetrazol-5-yl) thiomethyl-A 3-cephem4-carboxylic acid were obtained. 



   b) The filtrate was adjusted to pH 3.0 and passed through a 1 2 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.  The concentrate was adjusted to pH 7.5 using aqueous sodium hydroxide solution and the compound produced was isolated. 



  Example 9
Preparation of 745-amino-5-carboxyvaleramido); 341-methyl-1 H-tetrazol-5-yl) thiomethyl-7-methoxy-h 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 dissolved in 500 ml Sakaguchi bottles filled with 100 ml each.  Each medium was sterilized at 120 "C for 20 minutes.  The sterile culture medium was inoculated with the Streptomyces oganonensis Y-G19Z strain. It was cultured at 30 ° C. for 48 hours.  The culture medium prepared above was also filled in 2 liter Sakaguchi bottles in an amount of 400 ml each. 

  After sterilizing the medium for 20 minutes at 120 ° C, the culture medium was inoculated with the culture broth obtained in the above process in an amount of 2-3%.  Cultivation was carried out at 30 ° C. for 24 hours in order to obtain a seed culture. 



   Separately, 60 liters of a culture medium containing 7% starch, 2% gluten flour, 2% soybean meal, 0.8% glycerin, 0.1% casamino acid, 0.01% ferrisulfate and 55 g sodium hydroxide were combined in 200 liter fermentors filled with 10 ml of adecanol as a defoaming agent.  Each culture medium was sterilized at 120 "C for 30 minutes.  It was inoculated with 800 ml of the inoculum prepared according to the above procedure.  Then, to each fermenter, a solution of 1-methyl-5-mercapto-1 H-tetrazole was prepared by dissolving in aqueous sodium hydroxide solution and sterilizing under high pressure so that the content was 0.05%. 



  Then the culture broth was further cultivated for 90 hours. 



   After the cultivation, the culture broth was adjusted to pH 2.0 and mixed with radiolite as a filter aid with stirring.  The mixture was filtered through a filter press and the filtrates were combined into about 100 liters of the filtrate mixture. 



   The mixture was adjusted to pH 3.0 by the addition of aqueous sodium hydroxide solution.  Then it was 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 aqueous 50% acetone.  The eluate was up to
5.5 liters concentrated.  The concentrate was adjusted to pH 3.5 with dilute aqueous hydrochloric acid solution and passed through a 3 liter Amberlite IRA-68ACI type column.  The column was washed with 6 liters of water and with aqueous solution (pH 7.2), the 1N sodium nitrate and 0.1 M
Contained sodium acetate, eluted.  About 5 liters of solution containing an antimicrobial active material were obtained.  The
Solution was adjusted to pH 3.0 and passed through a 1 liter Amberlite XAD-2 column. 

  The column was with
Washed water.  It was eluted with aqueous 50% acetone. 



   About 400 ml of aqueous solution with antimicrobial active material was obtained, which was lyophilized.  About 54 g of raw powder was obtained.  The crude powder was subjected to column chromatography.  For this purpose, 800 ml of DEAE Sephadex A-25 (acetic acid type) were used 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 eluted with aqueous 25% acetone.  The eluate was then evaporated to dryness in vacuo.  The dried product was subjected to column chromatography using a mixed solvent of isopropanol: water (7: 3 by volume) using microcrystalline cellulose (Avicel).  The column was filled with the mixed solvent of the above composition.  The fractions with antimicrobial activity were applied drop-wise on a thin layer plate made of Avicel SF and developed with a solvent mixture of n-butanol: acetic acid: water (6: 1.5: 2.5 in volume ratio). 



   A pyridine solution of 0.25% ninhydrin was sprayed on and the stain was developed by heating. 



  The fractions with the Rf value 0.31 were collected, evaporated to dryness in vacuo at 45-50 ° C. and subjected to column chromatography with microcrystalline cellulose (Avicel).  The column contained a mixed solvent of isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9 in volume ratio).  The antimicrobial active fractions were then selected and subjected to thin layer chromatography with Avicel SF.  The same procedure above was used here.  The Rf 0.39 fractions 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 cleaning of the effective component.  The purified active fractions were dried by concentration, 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 and the effective fractions were selected and subjected to thin layer chromatography as explained above. 



   A solvent mixture of n-butanol: acetic acid: water (6: 1.5: 2.5 in volume ratio) was used.  The Rf 0.31 fractions were collected, concentrated and lyophilized.  About 60 mg of white 745-amino-5-carboxyvaleramidot3- (I-methyl-1 H-tetrazol-5-yl) thiomethyl 7-methoxy-h 3-cephem4-carboxylic acid was obtained. 



  Example 10
Preparation of 7- (5-amino-5-carboxyvaleramido> 7-methoxy-3 <5-methyl-l, 3,4-thiadiazol-2-yl) thiomethyl-A 3-cephem4carboxylic 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 dissolved in 500 ml Sakaguchi bottles filled in an amount of 100 ml each. Each medium was sterilized for 20 minutes at 120 ° C. The culture medium was then inoculated with the Streptomyces oganonensis Y-G1 9Z strain. The culture was then carried out at 30 ° C. for 48 hours.

 

   The above culture medium was filled into 2 liter Sakaguchi bottles in an amount of 400 ml each and sterilized at 120 ° C. for 20 minutes.



   Then inoculated with 2-3% culture broth obtained according to the above procedure. Cultivation was carried out at 30 ° C. for 24 hours in order to obtain a seed culture.



   Separately, 60 liters of a culture medium containing 7% starch, 2% gluten flour, 2% soybean meal, 0.8% glycerin, 0.1% casamino acid, 0.01% ferrisulfate and 55 g sodium hydroxide were in 200 liter fermenters together with 10 ml
Adecanol filled and sterilized for 30 minutes at 120 "C. Then inoculated with 800 ml inoculum prepared according to the above method. It was cultivated for 24 hours at 30" C. In each fermenter, a solution of 2-mercapto 5-methyl-1, 3,4-thiadiazole prepared with aqueous sodium hydroxide solution and sterilizing at high pressure was added so that its content was 0.05% of the culture broth.

  Cultivation continued for 90 hours.



   After the cultivation was completed, the culture broth was adjusted to pH 2.0. Radiolite has been used as a filter aid
Stir added. The mixture was filtered using a filter press. The filtrates were combined to give about 100 liters of the 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, adjusted to pH 3.5 with dilute aqueous hydrochloric acid solution and poured into a 3 liter Amberlite IRA 68 (C1 type) column. The column was washed with 6 liters of water and with an aqueous solution (pH 7.2) containing 1 M
Contained sodium nitrate and 0.1 M sodium acetate, eluted. There were about 5 liters of a solution that was antimicrobially active
Contained material, won. The solution thus obtained was adjusted to pH 3.0 and poured into a 1 liter Amberlite XAD-2
Pillar filled.

  The column was eluted with 50% aqueous acetone. About 400 ml of 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 by volume) using microcrystalline cellulose (Avicel) and filled with the same mixed solvent as indicated above. The antimicrobially active fractions obtained were applied in drop form to a thin-layer plate made of Avicel SF and developed with a mixed solvent of isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9 in volume ratio).

  A pyridine solution of 0.25% ninhydrin was sprayed to stain by heating. Then the fractions with an Rf value of 0.43 were collected and evaporated to dryness under reduced pressure at 45-50 ° C. The product obtained was subjected to 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, and the Rf 0.43 fractions were collected and dried to dryness under reduced pressure.



  steams. 0.78 g of a raw powder was obtained.



   The product was dissolved in a small amount of distilled water and developed in a Sephadex G 10 column 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 an Rf value of 0.43 were collected, concentrated and lyophilized. 82 mg of white 745-amino-5-carboxyvaleramidop7-methoxy-345-methyl-1,3,4-thiadiazol-2-ylpthiomethyl-h 3-cephem4-carboxylic acid were obtained.



  Example 11 Preparation of 745-Amino-5-carboxyvaleramidof345-carboxymethylthio-1,3,4-thiadiazol-2-ylpthiomethyl-7-methoxy-d 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 placed in a 500 ml - Sakaguchi bottle filled with 100 ml each and sterilized for 20 minutes at 120 "C. Each culture medium was then inoculated with the Streptomyces oganonensis Y-G19Z strain. It was then cultivated for 48 hours at 30" C. Another culture medium above was filled in 400 ml Sakaguchi bottles with 400 ml each, and each culture medium was sterilized at 120 ° C for 20 minutes.

  Then, inoculated with 2-3% of the culture broth prepared by the above method and then cultivated for 24 hours at 30 ° C. in order to obtain a seed culture.



   Separately, 60 liters of a culture medium containing 7% starch, 2% gluten flour, 2% soybean meal, 0.8% glycerin, 0.1% casamino acid, 0.01 ferrisulfate and 55 g sodium hydroxide were combined in 200 liter fermentors 10 ml of adecanol filled as a defoaming agent. Each medium was sterilized for 30 minutes at 120 ° C. and inoculated with 800 ml of the inoculation culture prepared according to the above method. Subsequently, cultivation was carried out 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 water-containing methanol and sterilizing by filtration using a Millipore filter was then added to each fermenter. added in such an amount that the content in the culture broth was 0.05%. The cultivation was carried out for 90 hours. After the cultivation, the culture broth was adjusted to pH 2.0 and radiolite as a filter aid was added with stirring. The mixture was filtered using a filter press. The filtrates were combined. About 100 liters of a filtrate mixture were obtained. The filtrate was adjusted to pH 3.0 by the addition of an aqueous sodium hydroxide solution. The solution was 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. After the insoluble matter formed was removed, water was added to the concentrate to obtain 10 liters of solution. The solution was adjusted to pH 3.5 with dilute aqueous hydrochloric acid solution, passed through a 3 liter Amberlite IRA-68 (CI type) column. 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 active 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 aqueous 50% acetone.

  About 400 ml of aqueous solution containing antimicrobial active material was obtained. About 23 g of crude powder was obtained by lyophilizing the aqueous solution. 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 antimicrobial active fractions were collected and placed in 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 in drop form to a thin-layer plate made from Avicel SF and developed with a solvent mixture of isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9 in volume ratio). Then a pyridine solution of 0.25% ninhydrin was sprayed on and the staining developed by heating. Then the fractions with Rf 0.39 were collected, evaporated to dryness under reduced pressure.

  The residue was subjected to column chromatography filled with microcrystalline cellulose (Avicel). A mixed solvent of isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9 in volume ratio) was used. The antimicrobial active fractions were collected and subjected to thin layer chromatography on an Avicel SF plate. The mixed solvent had the same composition as the above mixture. The procedure was as described above. The Rf 0.39 fractions were collected and evaporated to dryness in vacuo.



   The concentrate was purified by column chromatography using microcrystalline cellulose and a mixed solvent 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 on a Sephadex G-10 column 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 in volume ratio) was used. The fractions with Rf 0.32 were collected, concentrated and lyophilized. About 45 mg of white 745-amino-5-carboxy-valeramidof345-carboxymethylthio-1,3,4-thiadiazol-2-yl) thio-methyl-7-methoxyA 3-cephem4-carboxylic acid were obtained.



  Example 12 Preparation of 7- (5-Amino-5-carboxyvaleramidot7-methoxy 3 (1 1, 3,4-thiadiazol-2-ylhiomethyl-A 3-cephem-4-carboxylic acid.



   A culture medium containing 1% starch, 1% glucose, 1.5% soybean meal, 0.5% yeast extract, 0.1% dicalim hydrogen phosphate, 0.05% magnesium sulfate and 0.3% sodium chloride was dissolved in 500 ml Sakaguchi bottles were filled with 100 ml each and sterilized for 20 minutes at 120 ° C. Then each culture medium was inoculated with the Streptomyces oganonensis Y-Gl9Z strain. Subsequently, cultivation was carried out at 30 ° C. for 48 hours. Another culture medium above was filled in 2 liter Sakaguchi bottles with 400 ml each and sterilized at 120 ° C. for 20 minutes. Each culture medium was inoculated with the culture broth prepared above.



  Cultivation was carried out for 24 hours at 30 ° C. in order to obtain a seed culture. Separately, 60 liters of culture medium containing 7% starch, 2% gluten flour, 2% soybean meal, 0.8% glycerol, 0.1% casamino acid, 0.01 % Ferrisulfate and 55 g sodium hydroxide contained, filled in 200 liter fermentors together with 10 ml adecanol as a defoaming agent and sterilized at 120 "C for 30 minutes. Each culture medium was then inoculated with 800 ml of the inoculation culture and then cultivated at 30 ° C. for 24 hours.

  Then, a solution of 2-mercapto-1,3,4-thiadiazole prepared with aqueous sodium hydroxide solution and sterilizing at high pressure was 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 complete cultivation, the culture broth was adjusted to pH 2.0 and mixed with radiolite as a filter aid with stirring. The mixture was filtered using a filter press. The filtrates were combined and about 100 liters of filtrate mixture were obtained. The filtrate was adjusted to pH 3.0 by adding an aqueous sodium hydroxide solution and packed in a 12 liter Amberlite XAD-2 column. The column was washed with 30 liters of water and eluted with 30 liters of aqueous 500 / above acetone. The eluate was concentrated to 5.5 liters, adjusted to pH 3.5 with dilute aqueous hydrochloric acid and then added to a 3-liter Amberlite-IRA-68Cl type column. The column was washed with 6 liters of water.

  Elution was carried out with an aqueous solution (pH 7.2) which contained 1 M sodium nitrate and 0.1 M sodium acetate. 5 liters of solution containing antimicrobial active material were obtained. The solution was adjusted to pH 3.0, placed in a 1 liter Amberlite XAD-2 column. The column was washed with water and eluted with 50% acetone. About 400 ml of aqueous solution containing antimicrobial active material which was lyophilized was obtained.



   The product obtained was subjected to column chromatography with a solvent mixture of n-butanol: acetic acid: water (4: 1: 2 in volume ratio) using microcrystalline cellulose (Avicel) as a filling, the solvent mixture having the same composition given above being used, to select the antimicrobial active fractions.



   The fractions were applied dropwise to a thin layer of Avicel SF and developed with a mixed solvent of isopropanol: n-butanol: acetic acid: water (21: 3: 7: 9). A pyridine solution of 0.25% ninhydrin was sprayed on and stained by heating. Then the fractions with Rf 0.39 were collected, evaporated to dryness under reduced pressure to 45-50 "C. Then the fractions were subjected to thin layer chromatography with Avicel SF in the manner described above to find the fractions with Rf 0.39 The fractions were evaporated to dryness to give 0.92 crude powder.

  The powder was dissolved in a small amount of distilled water and subjected to column chromatography with distilled water using Amberlite CG-50 (H type) to select the antimicrobial active fractions. The fractions were then concentrated and lyophilized. The product was dissolved in a small amount from distilled water and developed in a Sephadex G-10 column using distilled water. The antimicrobial activity of each fraction was checked. The effective fractions were subjected to thin layer chromatography in the manner already explained above. A mixed solvent of n-butanol: acetic acid: water (4: 1: 2 by volume) was used to collect the fractions with the Rf value of 0.38.

  The fractions were concentrated and lyophilized, and 75 mg of white 745-amino-5-carboxyvaleramidof7-methoxy-341,3,4-thiadiazol--2-yl> thiomethyl-A 3-cephem4-carboxylic acid were obtained.



   Medicaments which contain compounds of the formula A can be prepared as follows: a) Dry-filled capsules with 120 mg 7 <5-Amino-5-carboxyvaleramidof7-methoxy-341-methyl-1H-tetrazol-5-yl) thiomethyl-A 3-cephem4-carboxylic acid per capsule 745-amino-5-carboxyvaleramidof7-methoxy-341-120 mg methyl-1 H-tetrazol-5-yl) thiomethyl A 3-cephem-4-carboxylic acid lactose 20 mg magnesium stearate 5 mg capsule No. 3 145 mg
7- (5-Amino-5-carboxyvaleramido) -7-methoxy-3- (1-methyl1H-tetrazol-5-yl) thiomethyl-h 3-cephem-4-carboxylic acid was triturated to a powder No.60, and lactose and magnesium stearate were passed through a No. 60 sieve.

  The combined ingredients were mixed together for 10 minutes and then filled into dry gelatin capsules No. 3.



  b) tablets with 150 mg of 7- (5-amino-5-carboxyvaleramido) -7-methoxy-3- (1-methyl-1 H-tetrazol-5-yl) thiomethyl-h 3-cephem4carboxylic acid per tablet 745-amino- 5-carboxyvaleramido) -7-methoxy-3- (1 - 150 mg methyl-1 H-tetrazol-5-yl) thiomethyl A 3-cephem4-carboxylic acid dicalcium phosphate JP 115 mg magnesium stearate 3 mg lactose J.P. 39 mg
The active component is mixed with the dicalcium phosphate and lactose. The mixture becomes 15%
Grain starch paste (4 mg) granulated and coarsely sieved, dried at 40 ° C. and sieved again through a No. 16 sieve.

  The magnesium stearate is added and the mixture is pressed into tablets approximately 0.8 cm in diameter. C) Parenteral solution with 500 mg of 7- (5-amino-5-carboxyvaler amido) -7-methoxy-3- (1-methyl- 1 H-tetrazol-5-yl) -thiomethyl A 3-cephem4-carboxylic acid per ampule 7- (5-amino-5-carboxyvaleramido) -7-methoxy-3- (1 - 500 mg methyl-1 H-tetrazol-5 -yl) thiomethyl A 3-cephem-4-carboxylic acid
The active compound (50.0 g) is placed in 150 ml of sterile water for injections. The solution obtained is adjusted to pH 8.0 by adding dilute sodium hydroxide solution. The volume of the solution is made up to 200 ml. The solution is filled into 100 ampoules, lyophilized and fused.


    

Claims (5)

 PATENT CLAIMS 1. Process for the preparation of 7-methoxycephalosporin derivatives having the formula (A) EMI1.1  wherein R2 represents a heterocyclic group containing nitrogen as ring heteroatom and M represents a hydrogen atom, alkali metal, alkaline earth metal, heavy metal or bases which form quaternary salt or amine salt, characterized in that Streptomyces oganonensis ATCC 31167 is grown in a culture medium which contains nutrients with the addition of a heterocyclic thiols with the formula R2SH, wherein R2 has the meaning given above, or a salt of the heterocyclic thiol or a compound having the formula R2-S-S-R2, in which R2 has the same meaning already mentioned.  2. The method according to claim 1, characterized in that R2 is a 1-methyl-1 H-tetrazol-5-yl group and M represents a hydrogen atom.  3. The method according to claim 1, characterized in that R2 represents a 5-carboxymethylthio-1, 3,4thiadiazol-2-yl group and M represents a hydrogen atom.  4. The method according to claim 1, characterized in that R2 represents a 5-methyl-1,3,4-thiadiazol-2-yl group and M represents a hydrogen atom.  5. The method according to claim 1, characterized in that R2 is a 1, 3,4-thiadiazol-2-yl group and M is a hydrogen atom.  The present invention relates to a process for the preparation of 7-methoxycephalosporin derivatives having the formula (A) EMI1.2  wherein R2 represents a heterocycle-containing heterocycle containing nitrogen and M represents a hydrogen atom, alkali metal, alkaline earth metal, heavy metal or bases which form quaternary salt or amine salt, characterized in that Streptomyces oganonensis ATCC 31167 is grown in a culture medium which is nutrient with the addition of a heterocyclic thiol with the formula R2SH, in which R2 has the meaning given above, or a salt of the heterocyclic thiol or a compound having the formula R2-S-S-R2, in which R2 has the same meaning already mentioned.  As a nitrogen-containing heterocyclic group, which is indicated by R2 in the general formula above, the following are mentioned for clarification: a carboxymethylthio-1,3thiadiazol-2-yl group, a 1-methyl-1 H-tetrazol-5-yl group, a 5-methyl-1,3,4-thiadiazol-2-yl group, a 1, 3,4-thiadiazol-2-yl group, etc.  The cationic group represented by M which forms the salt of cephalosporin means an inorganic group or an organic group. As the inorganic group, an alkali metal such as sodium, potassium, etc .; a calcareous metal such as calcium, magnesium, barium, etc .; as well as a heavy metal such as iron, copper, zinc, etc.; bases which form quaternary salts or amine salts, for example triethylamine, diethanolamine, piperidine, morpholine, etc., serve as the organic radical. Practical examples of compounds of this invention are 745-amino-5-carboxyvaleramidof345-carboxymethylthio-1, 3,4thiadiazol-2-yl> thiomethylj-methoxy-A 3-cephem4-carboxylic acid, 75-amino-5-carboxyvaleramido> 3- (1 -methy1-1 H-tetrazol-5-yl) thiomethyl-7-methoxy-h 3-cephem4-carboxylic acid, 745-amino-5-carboxyvaleramidof7-methoxy: 345-methyl-1,3,4-thiadiazol-2-yl ) thiomethyl-A 3-cephem4-carboxylic acid, 745-amino-5-carboxyvaleramidof7-methoxy-341, 3,4-thiadiazol-2yl) thiomethyl-A3-cephem4-carboxylic acid, and the salts of these compounds.    Some 7-methoxy-3-heterocyclothiomethylcephalosporins are described in British Patent 1,321,412 (1970) as compounds obtainable by chemical synthesis, but no practical physical and chemical properties of these compounds are given in this British patent. ** WARNING ** End of CLMS field could overlap beginning of DESC **.