DE2166463A1 - DERIVATIVES OF 7-BETA- (D-5-AMINO-5CARBOXYVALERAMIDO) -7-METHOXY-3-CEPHEM-4CARBONIC ACID - Google Patents

Description

Derivatives of 7R- (D-5-mno-5-carboxyvaleramido) -7-methoxy-3-cephem-4-carboxylic acid The invention relates to 7ß- (D-5-amino-5-carboxyvaleramido) -7-methoxy-3-cephem - carboxylic acids, substituted by a substituted methyl group in the 3-position of the "Cephem" nucleus are, and their salts, esters and amide derivatives.

Certain of these products are obtained through fermentation and others are obtained by synthetic means. The products are effective against gram-negative and gram positive bacteria.

The invention relates to a new class of antibiotic substances and a method for their preparation. In particular, these new antibiotics are products of the cephaiosporin type that contain a methoxy substituent in the 7-position of the '' Cephemlt-Xern. They are structurally related to the cephalosporin series of compounds, but unlike cephalosporin C, which contains only one D-5-amino-5-carboxyvaleramido unit in the 7-position, the present products also contain a 7-methoxy substituent ; and while acetoxymethyl is substituted for cephalosporin C at the 3-position of the ring, the products of the invention can have a wide variety of other substituents in addition to acetoxymethyl. Examples of these substituents are methyl, halomethyl, hydroxymethyl substituents, an acyloxymethyl moiety of the aliphatic, acyclic and aromatic types, carbamoyloxymethyl, N-substituted and N, N-disubstituted carbamoyloxymethyl, alkylthiomethyl-substituted heterocyclic-thiomethyl-substituted , Trialkylammoniummethyl, pyridiniummethyl substituents, nucleus-substituted pyridiniummethyl, thiouroniummethyl, amidinothiomethyl substituents, in which the two nitrogen atoms can be substituted with 1 to 7 alkyl radicals, aminothiocarbonylthiomethylcarbon-substituents, oxothiocarbonylthiomethylcarbon-, N-substituted aminothiocarbonylthiomethyl carbonethyl, N-substituted aminothiocylthiomethyl, N-substituted aminothiocarbonylthiomethyl carbonethyl methyl, alkarylsulfonylmethyl, azidomethyl, aminomethyl, amidomethyl, polyhydroxybenzyl, N-lower-alkyl-indol-3-ylmethyl and thiocyanatomethyl substituents. These products can be represented as follows: wherein R represents a hydrogen atom, a halogen radical, for example chlorine, bromine, fluorine or iodine and the like., a hydroxy radical, acyloxy radical, e.g. B. a lower alkanoyloxy group such as acetoxy, n-propionyloxy group and the like, a mononuclear and binuclear aromatic carbonyloxy group such as benzoyloxy, naphthoyloxy group and the like 4-pyridyl radical and the like, aralkanoyl oxy radical, such as, for example, phenacetyloxy, 3-phenylpropionyloxy, 2-naphthylacetoxy or hydrocinamoyloxy and the like, cycloalkanecarbonyloxy with 5 to 6 ring carbon, sulfoxy-dyno-oxy-dyloxy and methyloxy-dyno-oxy-sulfoxy and methyloxy-dyloxy-dyloxy-dyloxy-dyloxy-dyloxy-dyloxy-oxy-dyloxy-dyloxy-dyloxy-dyloxy-dyloxy-dyloxy-dyloxy-dyloxy-oxy-oxy-dyloxy-dyloxy-dyloxy-dyloxy-dyloxy-dyloxy-dyloxy-dyloxy-dyloxy-dyloxy-dyloxy-oxyloxy-dyloxy-dyloxy-dyloxy- or or α-methoxy-p-hydroxycinnamoyloxy radical and the like, a carbamoyloxy radical of the formula: -OOCNR1R2, in which R1 and R2 are hydrogen atoms, lower alkyl radicals, for example methyl, ethyl, n-propyl, tert-butyl radicals and the like, halogen lower-alkyl radicals, such as, for example, chloromethyl, 2-chloroethyl or chloro-tert.-butylre ste and the like, other alkoxycarbonyl radicals, such as ethoxycarbonyl radicals and the like, aryl radicals, for example mononuclear and binuclear aryl radicals, such as phenyl, naphthyl radicals and the like, alkarylsulphonyl radicals, for example mononuclear, alkarylsulphonyl and d-sulfonyl radicals, such as-p-alkarylsulphonyl radicals Denote benzhydryl radicals or R1 and R2 can be linked together with the nitrogen atom to which they are attached to form a mononuclear heterocycle, such as, for example, pyrrolidinyl, piperidino or morpholino ring; a thio radical of the formula: -SR3, in which R3 is a lower alkyl radical, for example an ethyl, ethyl, n-propyl radical and the like, a nitrogen-containing heterocycle, such as, for example, pyridyl radical, e.g. B. 2-, 3- or 4-pyridyl radical, a lower alkyl-substituted thiazolyl radical, such as 4-methylthiazol-2-yl or 4-ethylthiazol-2-yl radical and the like, 1,3,4-thiadiazol-2-yl radical , a lower-alkyl-substituted 1,3,4-thiadiazol-2-yl radical, such as, for example, 5-methyl-1,3,4-thiadiazol-2-yl radical and the like., 2-benzothiazolyl radical, a 4-lower-alkylpyrimidinyl radical, such as is for example 4-methylpyrimidin 2-yl radical; an ammonium radical, for example a tri-lower alkylammonium radical, such as trimethylammonium radical or triethylammonium radical and the like, or a pyridinium radical of the formula: wherein X1 is a hydrogen atom, a halogen, trifluoromethyl, cyano, carboxy, carbamoyl, N-lower alkyl and N, N-di-lower alkyl-substituted carbamoyl radical, such as, for example, N-methylcarbamoyl, thylcarbamoyl, N , N-dimethylcarbamoyl, N, N-diethylcarbamoyl or N, N-diisopropylcarbamoyl radical, carboxymethyl radical, lower alkanoyl radical, such as acetyl or propionyl radical and the like, lower alkyl radical, for example methyl, ethyl or n-propyl radical and the like. , Hydroxymethyl radical or Sulforest, ie SO2 (OH) means; a thiouronium radical of the formula: an amidinothio radical de / 2! SCM N112 for formula: -SC R5R6 in which R4, R5 and R6, which can be identical or different, denote hydrogen atoms or lower alkyl radicals, such as, for example, methyl, ethyl, n-propyl radicals and the like; an aminothiocarbon71thio radical of the formula: wherein R7 and R8, which may be the same or different, hydrogen atoms, lower alkyl radicals, e.g. B. ethyl, ethyl, n-propyl radicals and the like., Hydroxy-lower-alkyl radicals, for example 2-hydroxyethyl radical and the like., Di-lower-alkylamino-lower-alkyl radicals, such as 2- (dimethylamino) -ethyl-,, 2 - ('iäthylamino) -äthyl-, 2- (di-n-propylamino) -ethyl- or 3- (diethylamino) -propyl radical and the like., Morpholino-substituted lower alkyl radicals, e.g. B. 2-Morpholinoäthylrest and the like., N-Aryl-N-lower-alkylaminoalkylrest, for example 2- (N-phenyl-N-methylamino) -ethyl mean or the radicals R and R8 taken together with the nitrogen atom to which they are found are, with formation of a morpholino, piperidino, pyrrolidinyl or piperazino radical of the formula: in which R9 is an alkyl radical, for example an ethyl, ethyl, n-propyl, isopropyl, n-butyl, amyl, n-octyl, decyl radical and the like, or a phenyl radical, can be linked; an aroylthio, for example a mononuclear aroylthio, e.g. B. a benzoylthio radical; an oxythiocarbonylthio radical of the formula wherein 210 is a lower alkyl radical, for example an ethyl, n-propyl, isopropyl, n-butyl, n-hexyl radical and the like Cyclohexyl radical; Alkarvlsulfo nylrest, for example a mononuclear Alkarylsulfonylrest, z. B. p-tolylsulfonyl group and the like: an azido group; an amino radical, an amido radical of the formula: -NHR11 in which R11 is an acyl radical, for example a lower alkanoyl radical, such as, for example, acetyl or propionyl radical, or aralkanoyl radical, see e.g. a mononuclear aralkanoyl radical, such as, for example, 2-phenylacetylret and the like; a polyhydroxyphenyl radical, e.g. B. Dihydroxyphenylrest, such as 2, 4-iihydroyyhenylrest or an N-lower-Alkylindol-3-ylrest, z. B. N-methylindol-3-yl radical and the like. Or a thiocyanate radical. The pharmacologically acceptable salts esters and amides of the present products are also included. These include organic and inorganic salts, e.g. B. acid addition salts, metal salts, quaternary salts and amine salts derived from tertiary organic nitrogenous bases. Suitable ester and amide derivatives include the mono- and di-esters, e.g. B. those which are derived from alkanols, cycloalkanols, aromatic alcohols and aralkanols and mono- and ii-amides which are derived, for example, from ammonia, lower alkylamines, di-lower alkylamines, aralkylamines and heterocyclic amines. Examples of these derivatives and methods of making them are given in the subsection labeled Synthetic Methods.

Antibiotic 810A; The products of the above formula I essentially comprise two groups of fermentation products. One of these groups consists of a mixture of compounds from which two distinct products have been isolated and identified. These two products are characterized by the presence of an α-methoxy-p-sulfooxycinnamoyloxy or an α-methoxy-p-hydroxycinnamoyloxy unit in the 3-position of the cephem core and correspond to the following structural formula: wherein R12 is a hydroxy or sulfooxy radical, ie -OSO3H. These products are concurrently produced by culturing a new Actinomycete stainin designated MA-2837 in the culture medium of Merck & Co., Inc., Rahway, New Jersey, under controlled conditions.

A sample of this culture also became a permanent deposit into the culture collection of the Northern Utilization Research and levelopment Branch of the U. S.

Department of Agriculture in Peoria, Illinois, and received the culture number NRRL 3851. 25 other cultures were also used as producers this antibiotic mixture (Ia) identified, and this together with the culture MA-2837 are referred to below in the section referred to as the microorganisms described. The following is the antibiotic mixture (Ia), which these two Proedukte has, referred to as antibiotic 810A or simply 810A.

Antibiotics 842A: The second group of fermentation products includes 78- (D-5-amino-5-carboxyvaleramido) -3 (carbamoyloxymethyl) -7-methoxy-3-cophem-4-carboxylic acid (following formula (Ib)) and its salts. This compound has the following structural formula: This product (Ib) is also produced by a new strain of Actinomycete, and a sample of this microorganism, designated MA-2908, was added to the culture library of Merck & Co., Inc., Rahway, New Jersey. A sample of this culture has also been placed on permanent deposit with the Kaltur Collection of the Northern Utilization Research and Development Branch of the US Department of Agriculture in Peoria, Illinois. This culture was given the culture number NRRL 3802. In addition to its antibiotic activity, this product (Ib) is also an intermediate product in the production of the corresponding 3-hydroxy, 3-acyloxy and carbamoyloxy derivatives. The process for making these derivatives is described in the subsection labeled Synthetic Methods. In the following description, the product Ib, ie 7β- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid is referred to as antibiotic 842A or simply 842A.

Effectiveness A major difficulty in antimicrobial therapy is the sensitivity; of most antibiotics against enzymatic degradation. For example, penicillin G is effective against a large number gram positive and gram negative microorganisms, however, it will be in the presence broken down by penicillinase to a form ineffective against most pathogens is.

One attempt to address this problem has been development new antibiotics that have the "cephem" core property of cephalosporin C. Cephalosporin C has its own resistance to penicillinase and is effective against both gram-negative and gram-positive bacteria; However it is only moderately effective, and there are enzymes other than penicillinase that can be used Destroy effectiveness. These enzymes are called cephalosporinases. the products (I) according to the invention not only provide resistance to penicillinase, but also against the cephalosporinases. They deliver effectiveness both against gram / negative as well as gram / positive bacteria, however the extent of the Activity and the range of organisms against which they are effective are not identical.

Antibiotic 842A is opposed to by increased effectiveness gram / negative microorganisms labeled Other than Cephalosporin C, the one Has relatively little antibacterial activity, this product exerts a significant effect gram, negative effect in vivo with a effectiveness that is generally greater than is cephalothin. This effectiveness includes the effectiveness against in vivo Proteus morganii and an activity against the following gram / negative bacteria: Escherichia coli, Proteus vulgaris, Proteus mirabilis, Proteus morganii, Salmonella schottnuelleri, Klebsiella pneumoniae AD, xlebsiella pneumoniae B and Paracolobactrum arizoniae.

Antibiotic 842A represents a preferred embodiment of the invention water of generally increased gram negative potency and increased Effectiveness compared to cephalothin and greater resistance to it Gephalosporinases 842A is characterized by a lower level of toxicity and produces rapid values in the blood. Within 6 hours of administration about 100% eliminated in the urine. It is also more resistant to enzymatic Degradation as cephalosporin C and resistance to this antibiotic developed slowly and it is bactericidal. When administered orally, it protects against Infections due to Paracolobactrum arizoniae 3270, Proteus vulgaris 1810 and Salmonella schottmuelleri 3010; and when administered subcutaneously, it is 2 to 10 times more effective than cephalothin against the same infections.

Antibiotic 810A is a broad spectrum agent that an approximately balanced gram-negative and gram-positive effectiveness unfolds. In addition include effectiveness in vivo against the following gram / negative organisms: Proteus vulgaris, Proteus mirabilis, Salmonella pullorum, Escherichia coli and Klebsialla pneumoniae and in vivo effectiveness against the following gram / positive organisms: Staphylococcus aureus, Streptococcus pyogenes, and Diplococcus pneumoniae.

Of the various products that make up the antibiotic 810A, the? Ss-CD-5-amino-5-carb oxyval eramido) -3- (a-methoxy-p sulfo oxy-cinnamoyloxymethyl) -? - methoxy-3-cephem -4-carboxylic acid type corresponding to the above formula Ia, in which R is a sulfooxy radical and its salts, such as the sodium salt, represent a preferred embodiment of the invention. This product has the following structural formula: This compound has greater resistance to cephalosporinases than cephalothin and is characterized by a lower level of toxicity in mice.

It is also more resistant to enzymatic degradation than cephalosporin C and it has a bactericidal effect. When administered orally, it protects against infections it is effective against it by virtue of Proteas vulgaris and when administered subcutaneously a variety of gram / negative and gram / positive infections.

The Mikroorganismon 810A cultures: The microorganism that the Antibiotic 810A produced, was originally isolated from soil as a single colony, This colony was placed on a Stu.ch plate with the following composition: Medium A: Yeast Extract 10.0 g Dextrose 10.0 g Agar 20.0 g Distilled Water 1000.0 ml After several days of growth, the microorganism produced antibiotic 810A. This antibiotic was then reproduced in shake flasks and known from known Antibiotics due to various biological and chemical Investigations differentiated. A comparison of this data with those who came across other known antibiotics were obtained, made the best, the 810A is a new unit.

810A Taxonomy and Morphology: The Microorganism (Culture MA-2837), which produces antibiotic 810A was identified as Streptomyces griseus. The taxonomy used to make this determination is in Bergey's Manual of Determinative Bacteriology "7th Edition and in" The Actinomycetes "Volume 2, by S. A. Waksman (1961).

Using this procedure it was found that the culture consists of a strain of Streptomyces griseus which, with regard to the description, melanin production and carbon consumption, Streptomyces griseinus very closely resembles, as in International Journal fo Systemic Bacteriology "7th Edition (1957) since the group of Streptomyces griseus cultures 1959, two years after the edition was published.

However, in both Waksman and the International Journal of Systemic Bacteriology "will refer to Streptomyces griseinus as the" grisein-producing strain of Streptomyces griseus "or defined as" grisein-producing or grisein-like substance ". Streptomyces griseus (MA-2837) is a strain that differs from the classical description in Bergey and in Waksman in that he has an aerial mycelium which predominantly brownish yellow and on some media greenish yellow with a slightly different one Carbon consumption pattern is. Waksman describes on pages 111 and 133 of "The Actinomycetes "this Streptomyces griseus series as a series which many related Species and strains includes what is shown by colorless to olive-sand-colored substrate growth, Aerial mycelium that is yellowish with a thorough tint or greenish-gray or yellow-burnt or grass green until is gray, melanin is negative, straight or pliable and denotes sphorophores and oval spores produced in clusters. The following Tables compare the properties of the culture producing antibiotic 810L with the Streptomyces griseus and Streptomyces griseinus cultures.

The characterization of the parent isolate MA-2837 in comparison with Streptomycels griseus described in Bergey1 and Waksman2 and also the characterization of MA-2837 in comparison with the Streptomyces griseinus described in Waksman2 are listed in Tables I and Ia. Table I Comparison of the Cultural Characteristics of MA-2837 Culture of MA-2837 Streptomyces Streptomyces Streptomyces griseus (Bergey1) griseus (Waksman²) griseinus (Waksman²) Sporophores are mono Aerial mycelium: Abundant, straight in clusters, straight sporophores branched podially underneath powdery, water-generated spherphores. creates clumps in clumps, green. Sporophore- Spores spherical or tufts without with straight to slightly begets in clusters. to oval, 0.8 x 0.8 spirals.

curved spore chains. Spores spherical up to 1.7 µ, surface Spores rod-shaped, spores: spherical to ellipsoidal, 0.8 x 0.8 smooth 1.0 to 1.8 x 0.8 oval, diameter 0.9 µ to 1.7 µ, vegetative up to 1.0 µ to 0.9 x 1.2 µ in Ket growth: colonies of about 10-15 smooth or puckered, spores. Vegetative colorless, later after hyphae 0.9 µ wide olive to sand-colored over- (glycerine-asparagine agar- going 970X) 1 Bergey's "Manual of Determinative Bacteriology", 7th edition (1957) 2 Waksman, S.A., "The Actinomycetes", Volume 2 (1961) Table Ia Comparison of the cultural properties of MA-2837 culture medium MA-2837 S. griseus S. griseus S. griseinus (Bergey) (Waksman) (Waksman) Tomato Paste - Vegetative Growth: Oatmeal agar reverse side brown, even, spreading, aerial mycelium: middle yellow-brown to gray-yellow edge brownish-yellow Soluble pigment: yellow-brown glycerine asparagus Vegetative growth: ginagar reverse yellow-brown, even, spreading, aerial mycelium: powdery, yellow-brown to yellow Soluble pigment: light, yellow-brown-yellowish Qzapek doxagar Vegetative growth: growth sparse substrate growth (sucrose nitrate back yellowish, wrinkled, backside agar) orange, even, spreading, color-cream-colored to "spreading, les, becomes olive-brownish, aerial-aerial mycelium: powdery to sand-colored, mycelium: white to yellow-brown to yellow (thick aerial mycelium, cream-colored with different shades, powdery, light greenish but predominantly edge Water green, tinge, no soluble yellow-brown-yellowish) easily- pigment insoluble Pigment growth in the center and stronger on the edges, Soluble pigment: light, yellow-brown-yellowish Table Ia (continued) Medium MA-2837 S. griseus S. griseus S. griseinus (Bergey) (Waksman) (Waksman) Egg album agar Vegetatives Growth: reverse grayish yellow-brown, even, spreading aerial mycelium: yellow-brown to yellowish with greenish shading, margin yellowish brown, yellowish, little growth in the center, stronger growth along the edges. Soluble pigment: light yellow-brown yellowish nutrient agar Vegetative growth: abundant growth, brownish-yellow reverse almost transparent, aerial mycelium: velvety, yellow-cream-colored.

brownish yellow, edge yellowish brown aerial mycelium powdery yellow shaped, white to Soluble pigment: light light gray, no soluble brown pigment agar abundant, cream-colored, almost transparent growth, aerial mycelium powdery, white to light gray, no soluble pigment Table Ia (continued) Medium MA-2837 S. griseus S. griseus S. griseinus (Bergey) (Waksman) (Waksman) Synthetic Sparse Agar-spreading colorless growth that turns olive to sand-colored aerial mycelium: thick, powdery, water-green gelatine base Flaky cream-colored greenish-yellow Growth creamy growth is made up of or ereme-colored with a brownish bottom of the pipe. nes surface stitch Bollcome liquefaction with brown air mycelium: lacking a lichen tinge. Ra- or sparse, nutrient gelatin vegetatives Growth: liquefaction white.

agar ereme-colored Rapid liquefying aerial mycelium: pale, yellowish-gray yellow Soluble pigment: none Liquefaction of the gelatin: good Litmus milk Partly Ring, cream-colored growth. Cream-vegetative growth: ring, coagulum-colored.

brownish with rapid coagulation and aerial mycelium: low, white- Peptonization, peptonization Lich becomes alkaline Peptonization becomes alkaline Tabel Ia (continued) Medium MA-2837 S. griseus S. griseus S. griseinus (Bergey) (Waksman) (Waksman) Skimmed milk Partial ring Vegetative growth: Brownish aerial mycelium: none Soluble pigment: light brown peptonization becomes alkaline skimmed milk agar Vegetative growth: cream-colored, flat, spreading aerial mycelium: sparse, yellowish white to cream colored Soluble pigment: very light brown hydrolysis of casein Glucose agar Increased growth in the center, radiating, cream-colored to orange, jagged edge glucose liquid - copious, yellowish skin with greenish Stitch, heavily puckered Table Ia (continued) Medium MA-2837 S. griseus S. griseus S. griseinus (Bergey) (Waksman) (Waksman) Starch Agar Thin, thin growth, colorless to broad trans- spreading, cream-colored parent growth, transparent, growth, starch is hydro- strong hydrolysis aerial mycelium: greyed olive nutrient starch agar Vegetative growth: cream-colored aerial mycelium: pale yellow-brown yellow Soluble pigment: none cydrolysis good potato cut Vegetatives Growth: yellowish, wrinkled, wrinkled, wrinkled, yellowish-light brown growth, total growth, slightly white aerial mycelium: moderate, covered with white-yellowish to growth brownish yellow sem, powdery brownish, airy mycelium grayish brownish gem aerial mycelium covers with white-white with de / n the potato semen, powdered oil olive-colored medium aerial mycelium stitch Table Ia (continued) Medium MA-2837 S. griseus S. griseus S. griseinus (Bergey) (Waksman) (Waksman) Calcium malate Vegeative growth: green or gel- no soluble agar, spreading esp soluble pigments translucent and pigment that is colorless on calcium the edges of the calcium malate malate or opaque and cream-colored and succinate-Succinatmeim Focus. medium creates medium aerial mycelium: moderate, cream-colored to yellow, Margins yellow-brown yellow Soluble pigment: no tyrosine nutrient Vegetative growth: Often produced No agar produced evenly spreading, dark pigment pigment cream-colored aerial mycelium: yellowish brownish yellow with greenish shading, edges yellow-peptone-iron-yeast- Vegetative growth: extract agar cream-colored aerial mycelium: no soluble pigment: none melanin negative Table Ia (continued) Medium MA-2837 S. griseus S. griseus S. griseinus (Bergey) (Waksman) (Waksman) Production of H2S Negative Negative Negative Loeffler's blood vegetative growth: serum oblique yellow-brown turen aerial mycelium: light yellowish soluble pigment: brownish perfect Liquefaction temperature range 28 ° C good growth optimal temperature (yeast extract 37 ° C good growth 37 ° C dextrose salt 50 ° C no wax agar slant cultures) Microärophiles The surface covers aerobic growth (yeast and along the extract-dextrose- whole line of line salts-line running good 40 mm depth) Growth Reduction from negative positive positive positive nitrates to nitrites These Observations were made after three weeks of incubation at 280 a, unless otherwise is noted. The pH of the medium used in these studies was roughly neutral, d. H. 6.8 to 7.2. The physiological experiments were after expiration carried out between 7 and 22 days. The colors used for the description correspond the Definitions of Color Harmony Manual "4th Edition, 1958, Container Corporation of America.

810A Carbohydrate Use: The Streptomyces griseus Culture (MA-2837) also became various in terms of their ability to use or assimilate Carbohydrates through the growth of the microorganism in a synthetic starting medium (T. G. Pridham and D. Gottlieb, 1948) contained 1% of the carbohydrate at 28 ° C tested for 3 weeks. The following Table II gives the consumption or assimilation of these carbohydrate sources by the Streptomyces griseus culture (MA-2837). The explanation of the signs in Table II are as follows: + means good growth, + means poor growth and - means no growth on the special cabbage ehydrate.

Table II Carbohydrate M-2837 Carbohydrate MA-2837 Culture Culture Glucose + Lactose + arabinose + inositol # xylose + sucrose # maltose + rhammose t mannose + Raffinose + fructose - cellulose mannitol + The ones in the tables Properties I, Ia and II described were used to reduce Streptomyces griseus culture (MA-2837) refers to a classification of a species above that in "Bergeys's Manual of Determinative Bacteriology ", 7th Edition, pages 694-829 (1957) - 'and in 11 The Actinomycetes ", Volume 2, pp. 61-292 (1961) used.

Comparison of the culture (MA-2837) with known species shows that it is similar to Streptomyces griseus. There are morphological differences such as the color of the aerial mycelium found in Streptomyces griseus it is predominantly yellow-brown-yellow and greenish-yellow, but with regard to the considerable There is no number of similarities and only minor differences Authorization for a new species name. The microorganism (MA-2837) that thus producing antibiotic 810A was identified as a strain of Streptomyces griseus identified.

In addition to the above culture (MA-2837), there were 25 more Cultures identified as producers of antibiotic 810A. These include: three cultures from Streptomyces griseus, eleven cultures of Streptomyces viridochromogenes, five Cultures of Streptomyces fimbriatus, three cultures of Streptomyces halstedii, a culture of Streptomyces rochei, a culture of Streptomyces cinnamonensis and a culture of Streptomyces chartreusis. These Streptomyces strains are called the following cultures in the culture collection of Merck & Co., Inc., Rahway, New Jersey, identified: MA-4160, MA-4174, MA-4171, MA-4177, MA-4178, MA-4180, MA-4164, MA-4165, MA-4166, MA-4167, MA-2892, MA-3265, MA-4162, MA-4163, MA-4159, MA-4169, MA-4170, MA-4179, MA-4161, MA-4168, MA-4175, MA-4181, MA-2938, MA-4176 AND MA-4173.

These cultures became permanent deposit with the culture collection the Northern Utilization Research and Development Branch of the U.S. Department of Agriculture in Peoria, Illinois.

The assigned NRRL culture names are as follows: Streptomyces griseus: MA-4160 NRRL 3951 MA-4174 NRRL 3953 MA-4171 NRRL 3952 Stroptomyces viridochromogenes: MA-4177 NRRL 3970 MA-4178 NRRL 3971 MA-4180 NRRL 3972 MA-4164 NRRL 3966 NA-4165 NRRL 3967 MA-4166 NRRL 3968 MA-4167 NRRL 3969 MA-2892 NRRL 3962 MA-3265 NRRL 3963 MA-4162 NRRL 3964 MA-4163 NRRL 3965 Streptomyces fimbriatus: MA-4159 NRRL 3954 MA-4169 NRRL 3956 MA-4170 NRRL 3957 MA-4179 NRRL 3958 MA-4161 NRRL 3955 Streptomyces halstedii: MA-4168 NRRL 3959 MA-4175 NRRL 3960 MA-4181 NRRL 3961 Streptomyces rochei: MA-2938 NRRL 3973 Streptomyces cinnamonesis: MA-4176 NRRL 3974 Streptomyces chartreusis: MA-4173 NRRL 3975 The characterization of the isolates mentioned above in comparison with Streptomyces griseus, Streptomyces viridochromogenes, Streptomyces fimbriatus, Streptomyces halstedii, Streptomyces rochei, Streptomyces cinnamonensis and Streptomyces chartreusis are shown in Tables IIa to Ile below.

Table IIA Culture characteristics / of those producing antibiotic 810A Strains of Streptomyces griseus Medium MA-4160 MA-4174 MA-4171 Mprphology Sporophores form bushes with straight to slightly twisted chains of spores.

Spores are spherical to oval - 0.9 µ in diameter to 0.9 µ x 1.2 µ, in chains of about 10 to 15 spores tomato paste - vegetative growth: good, flat, spreading oatmeal agar yellow-brown aerial mycelium, aerial mycelium: powder aerial mycelium: powdery, yellow-brown shaped, yellow-brown powdery, yellow-brown with greenish Shading with strong green strong green tinting Tinting Soluble pigment: light brown glycerine vegetative growth: good, even, yellow brown asparagine aerial mycelium: powdery, yellow-brown with greenish shading and gray-green agar vectors Soluble pigment: light brown Czapek-Dox Vegetative growth: even, spreading, transparent agar aerial mycelium: aerial mycelium: moderate, yellow-brown moderate, yellow-brown moderate, greyish Soluble pigment: Soluble pigment: Soluble pigment Tabel IIa (continued) Medium Ma-4160 Ma-4174 MA-4171 Yeast Extract - Vegetative Growth: even, spreading, yellow-brown dextrose + aerial mycelium: good, powdery, beige Salt agar soluble pigment: light brown soluble pigment on no k no no peptone iron yeast extract agar T a b e l e IIb Cultural properties of strains producing the antibiotic 810A from Streptomyces viridochromogenes medium MA-2892 MA-3265 Ma-4162 MA-4163 morphology Sporophores are short, compact spirals that appear as side branches on aerial hyphae.

Spores are spherical to oval - 0.9 to 1.2 µ in diameter and 0.9 to 1.2 x 1.2 to 1.7 µ, chains of about 10 to 15 spores.

Tomato Paste- Vegetative Growth: Vegetative Wax- Vegetative Wax Vegetative growth: oatmeal agar backside brown tum: backside tum: backside Backside brown yellow-brown dark brown aerial mycelium: aerial mycelium: velvety, aerial mycelium: aerial mycelium: velvety, dark gray, light gray and white velvety, bluish-velvety, medium gray and white gray and white and white Soluble pigment: Soluble pigment: Soluble pigment: Soluble pigment: light brown none light brown light brown glycerine vegetative growth: Vegetative wax Vegetative wax Vegetative growth: asparagine back dark brown tum: back tum: back back brown agar dark brown dark brown Aerial mycelium: aerial mycelium: light aerial mycelium: aerial mycelium: dark gray and cream-gray cream-colored and cream-colored and g @ au-colored light gray Soluble pigment: Soluble pigment: Soluble pigment: Soluble pigment: light brown none light brown light brown Tabel IIb (continued) Medium MA-2892 MA-3265 MA-4162 MA-4163 Czapek-Dox Vegetatives Wax Vegetative Wax Vegetative Vegetative Growth: Agar tum: dark brown tum: dark brown growth: yellow brown dark brown aerial mycelium: aerial mycelium: aerial mycelium: Aerial mycelium: very sparse, light gray and white, very sparse, very sparingly soluble Pigment: soluble pigment: soluble soluble pigment: dark brown light brown pigment: light brown light brown yeast extract - vegetative wax- vegetative wax- vegetative Vegetative growth: dextrose agar tum: dark brown tum: reverse growth: dark brown brown dark brown aerial mycelium: aerial mycelium: light gray aerial mycelium: aerial mycelium: very sparse very economical very sparse Soluble pigment: Soluble pigment: Soluble pigment Soluble pigment: light brown none ment: light brown light brown Soluble dark brown Pigment on peptone-iron-yeast extract cooking Table IIb (continued) Medium MA-2892 MA-3265 MA-4162 MA-4163 Morphology Sporophores are short, compact Spirals that appear as side branches on aerial hyphae. Spores are spherical to oval - 0.9 to 1.2 µ diameter and 0.9 - 1.2 x 1.2 - 1.7 µ, chains of approx 10 to 15 spores of tomato paste - vegetative growth: reverse side is dark brown oatmeal agar Aerial mycelium: aerial mycelium: aerial mycelium: velvety, bluish-velvety, medium-velvety, medium gray velvety, dark gray gray and cream gray and cream and cream colors and cream-colored colored soluble pigment: light brown glycerine vegetative wax Vegetative wax Vegetative wax Vegetative wax asparagine: back tum: back tum: dark gray tum: back yellow agar dark brown dark brown brown Aerial mycelium: dark aerial mycelium: aerial mycelium: spär- aerial mycelium: light gray gray and cream medium gray and light grayish and cream-colored colors cream-colored soluble pigment: light brown Czapek-Dox Vegetative wax- Vegetative wax- Vegetative wax- Vegetative Wax agar do: brown tum: reverse tum: brown tum: yellow-brown brown aerial mycelium: very aerial mycelium: light aerial mycelium: very aerial mycelium: very sparse gray and creamy sparse Sparingly soluble pigment: color soluble pigment: soluble pigment: brown soluble Pigment: brown light brown light brown Table IIb (continued) Medium MA-4164 MA-4165 MA-4166 MA-4167 Yeast extract- Vegetative growth: dark brown dextrose- Aerial mycelium: very sparingly agar. Soluble pigment: light brown. Soluble pigment dark brown pept / on iron yeast extract agar Table IIb (continued) Medium MA-4177 MA-4178 MA-4180 Morphology Sporophores are short, compact spirals, which appear as side branches on aerial hyphae. The spores are spherical to oval - 0.9 to 1.2 µ diameter and 0.9 to 1.2 x 1.2 to 1.7 µ in chains of about 10 up to 15 spores of tomato paste- Vegetative growth: Vegetative growth: Vegettives Growth: oatmeal agar back side yellow-brown back side brown back side brown aerial mycelium: velvety, aerial mycelium: velvety, aerial mycelium; velvety, dark gray and dark gray dark gray and creamy white color Soluble pigment: light brown glycerine vegetative growth: Vegetative growth: Vegetative growth: A # paragin- reverse brown reverse dark brown reverse side dark brown agar aerial mycelium: aerial mycelium: aerial mycelium: dark gray dark gray mixture of light and dark gray Soluble pigment: light brown Czapek-Dox Vegetative growth: Vegetative growth: Vegetative growth: agar yellow-brown dark brown yellow brown aerial mycelium: very sparingly soluble pigment: light brown Tabel IIb (continued) Medium MA-4177 MA-4178 MA-4180 Yeast Extract - Vegetative Growth: Vegetative growth: Vegetative growth: dextrose agar dark brown brown brown Aerial mycelium: aerial mycelium: aerial mycelium: sparse-greyish very sparse very sparingly soluble Pigment: light brown soluble pigment on peptone iron dark brown yeast extract agar T a b e l l e IIc Cultural Properties of Strains Producing Antibiotic 810A of Streptomyces fimbriatus Medium MA-4159 MA-4169 MA-4170 MA-4179 MA-4161 morphology Sporophores are short, compact spirals and some loops that are sporophores occur as side branches on aerial hyphae. The spurs are ball-short hooks and shaped to oval - 0.9 µ in diameter and 0.9 x 1.2 µ with chains of loops that as about 10 to 15 spore side branches appear on aerial mycelium. Spurs in chains of less than 10 spores are spherical to oval, 0.9 µ in diameter and 0.9 x 1.2 µ Tomato Vegetative Vegetative Wax Vegetative Vegetative Vegetative Wax paste - growth: back: back growth: growth: tum: yellow-brown Hafemehl- side yellow-brown yellow-brown reverse side yellow- reverse side yellow- aerial mycelium: agar aerial mycelium: Aerial mycelium: moderate, brown brown sparse, grayish moderate, light gray light gray aerial mycelium: Aerial mycelium: moderate, light-sparse gray and cream-grayish colors Soluble pigment: light brown glycerin- vegetative vegetative wax- vegetative vegetative vegetative Wax asparagine growth: dark: dark brown growth: growth: tum: back agar light brown to light brown to gray dark brown dark brown yellow brown with gray light to grayish to gray reddish yellow-brown vectors Tabel IIc (continued) Medium MA-4159 MA-4169 MA-4170 MA-4179 MA-4161 Vegetative Czapek-Dox Vegetative Vegetative Wax Vegetative Wax Growth: Dun Vegetative Agar Growth: tum: dark brown: dark brownish growth: dark brown light to gray light to gray to gray yellow-brown light to gray aerial mycelium: aerial mycelium: moderate, air @ ycel: moderate, aerial mycelium: aerial mycelium: very very sparse gray gray very Sparse Sparse Lich Soluble Soluble pigment: I @ soluble pigment: Soluble Soluble pig pigment: brown @raun pigment: ment: brown brown light brown yeast extract Vegetative Vegetative Wax Vegetative Wax- Vegetative Vegetative Dextrose + Growth: tum: dark brown- growth: dark brown- growth: salt agar dark brownish to grayish to gray light brownish brownish to gray to gray Aerial mycelium: very sparse soluble pigment: brown soluble pigment on dark brown Peptone Iron Yeast Extract Agar T a b l l e IId cultural characteristics from strains of Streptomyces halstedii Kedium producing antibiotic 810A MA-4168 MA-4175 MA-4181 Morphology Sporophores are long, loose spirals that are known as Side branches occur on aerial hyphae. The spores are spherical to oval - 0.9 µ diameter and 0.9 x 1.2 µ - in chains of more than 10 spores of tomato paste - Vegetative growth: Vegetative growth: Vegetative growth: He @ nmehlegar back brown reverse side yellow-brown reverse side brown aerial mycelium: aerial mycelium: aerial mycelium: powdery, dark- dark gray and white, dark gray and white gray powdery soluble pigment: none Glycerin-Aspa- Vegetative growth: Vegetative growth: Vegetative growth: ragin agar reverse side brown to reverse side greyish reverse side grayish dark brown Aerial mycelium: aerial mycelium: aerial mycelium: powdery, dark powdery, predominantly dark gray, powdery gray and white and dark gray mixed with light gray and white solubles Pigment: none Table IId (continued) Medium MA-4168 MA-4175 MA-4181 Czapek-Dox Vegetative growth: Vegetative growth: Vegetative growth: Agar cream-colored backside reddish-cream-colored brown aerial mycelium: aerial mycelium: aerial mycelium: greyish cream- greyish cream-colored very sparse colors Soluble pigment: none Yeast extract Vegetative growth: Vegetative growth: Vegetative growth: Dextrose + yellow-brown yellow-brown brown salt agar aerial mycelium: aerial mycelium: aerial mycelium: grayish, scanty scanty, greyish scanty, greyish Soluble pigment: none soluble Pigment on Peptone Iron None Yeast Extract Agar Tabel IIe Cultural Characteristics of Streptomyces Species Producing Antibiotic 810A Medium MA-2938 MA-4176 MA-4173 Morphology Streptomyces rochei Streptomyces cinnamonesis Streptomyces chartreusis sporophores form Sporophores are hooks that are sporophores compact compact spirals that are loops and some loose spirals that act as pages as Side chains of spirals that appear as side branches on aerial hyphae on about 10-15 Spores - twigs appeared on aerial hyphae. The spores are spherical to oval, step. The spores are in chains of more than 0.9 µ in diameter and in chains of more than 10 spores, spherical up to 0.9 x 1.2 µ, there are 10 spores, spherical oval, 0.9 to 1.2 µ diameter to oval, 0.9 µ diameter and 0.9 to 1.2 x ser and 0.9 x 1.2 µ 1.2 to 1.7 µ tomato paste - Vegetative growth: Vegetative growth: Vegetative growth: oatmeal agar reverse reddish to yellow-brown light brown with orange to brown brown vectors aerial mycelium: medium aerial mycelium: beige with aerial mycelium: powdery, gray-pink coloring, velvety dark gray with blue-green-soluble pigment: Soluble pigment: no coloration brown Soluble pigment: brown glycerine asphalt Vegetative growth: Vegetative growth: Vegetative growth: ragin-agar back reddish to reverse dark reddish brown brown to brown aerial mycelium: aerial mycelium: beige with aerial mycelium: very sparse, medium gray pink coloration. Soluble pigment: soluble Pigment: Soluble pigment: none brown light brown Table IIe (Continued) Medium MA-2938 MA-4176 MA-4173 Czapek-Dox Agar Vegetative growth: Vegetative growth: Vegetative growth: reddish brown reverse side dark brown orange to brown aerial mycelium: aerial mycelium: moderate, beige aerial mycelium: very economical with pink coloring very sparingly soluble pigment: soluble pigment: soluble pigment: none brown light brown yeast extract Vegetative growth: Vegetative growth: Vegetative growth: Dextrose + salty yellow brown brown brown agar aerial mycelium: aerial mycelium: sparse, aerial mycelium: greyish @ reme-colored to white very sparse Soluble pigment: Soluble pigment: Soluble pigment: light brown light brown brown Soluble pigment none dark brown dark brown on peptone iron yeast extract agar The description above of the microorganisms producing antibiotic 810A is simply illustrative of the types of strains that can be used and not to limit the invention to one Organism that meets the characteristics of this particular description. The invention includes the use of other microorganisms including strains of actinomycetes, which are isolated from nature or obtained by mutation, for example those which are obtained by natural selection or those obtained by mutation means, for example X-ray irradiation, ultraviolet irradiation, nitrogen mustard gases and the like that give an identical antibiotic under suitable conditions, 842A culture: the microorganism producing antibiotic 842A is a previously unknown strain of Actinomycete.

The original isolate was based on a single colony of soil obtained on an agar slant and in a medium of the following composition cultured: Medium B: tea extract 10.0 g glucose 10.0 g phosphate buffer 2.0 ml MgSO4.7H2O 0.05 g distilled water 1000.0 ml pH 6.5 * phosphate buffer: KH2PO4 91.0 g Na2HPO4 95.0 g of distilled water 1000.0 ml It was found that after several days Growth no spore formation could be determined. The microorganism produced an antibiotic that differs from known antibiotics due to its profile in different biological trnd chemical investigations differed. Compare these data with those obtained from known antibiotics1 revealed that the 842A was a new body.

842A Taxonomy: The 842A Producing Microorganism (Culture M-2908) was identified as a new actinomycete.

The taxonomy used in this determination is in Bergey's Manual of Determinative Bacteriology ", 7th edition and in l'2he Actinomycetes", Volume 2, "Classification, Identification and Description of Genera and Species", S.A.

Waksman (1961). Using this procedure, was found that the culture belongs to the genus Streptomyces and many of the attributes of the known species Streptomyces fradiae. Biochemically it is mainly correct completely coincide with the latter. However, there are significant morphological differences. For example, the color of the Luftmycel of 5. fradiae is a sea shell-colored Pink, while culture MA-2908 is usually cream in color. Also shows vegetative growth in the MA-2908 culture pigment differences on different media used, and as indicated above, was based on standard Taxonomi e medium no formation of sp ors perceived.

Based on these differences, the culture was identified as a new Spe.cies: Called Streptomyces lactamdurans. The following Table III describes the biochemical L Properties of the Streptomyces lactamdurans species and those of the known Streptomyces fradiae. All values in Table III were obtained after 3 weeks of incubation at 280 C, except where otherwise noted. The pH of the used The medium in these investigations was roughly neutral, namely 6.8 to 7.2. The physiological Experiments were carried out after 7 and 21 days. T a b e 1 1 e III 842A biochemical comparison experiment Streptomyces Streptomyces fradiae lactamdurans aerial mycelium straight, some straight, branching twigs.

Conidia not determined rod-shaped Soluble pigment none none optimal temperature invertase negative negative reduction of negative negative nitrate gelatine positive positive liquid cellulose negative negative consumption litmus milk alkaline alkaline peptonization peptonization 842A Morphology: Sporophores were not identified when the culture was on the when Description of the cultural characteristics listed medium was grown1 itself although repeated observations were made for up to 8 weeks. However, showed colored impression slides long threads, many of which are in subunits of various sizes, generally broad in shape and about 0.9 x 1.7 / u in size were divided.

The aerial mycelium is short, straight, not very branched. IT seems about that to be the same size as the vegetative mycelium -O, 9ji 3reite. It's bright, powdery and easy to scratch off.

The vegetative mycelium is gram-positive, not acid-fast. It sticks firmly attached to the agar, and in some cases Sn is embedded in the agar. There is one some splitting into rods in shake flask cultivation, but this is not significant is. The vegetative mycelium on the shake flasks and the stationary flasks (inoculation medium 4 to 6 days, @@ o C) showed some "germs" and short, thickened, almost knot- or club-like segments on the mycelium, but these were not numerous and theirs Meaning, if at all, is not known.

Tomato paste oatmeal agar Vegetative growth - backside orange, even, dry appearance, wrinkled aerial mycelium - sparse, cream-colored, completely soluble Pigment Czapek-Dox Agar Vegetative - even, deep cream - colored aerial mycelium - powdery, creme-color @ white No soluble pigment Glycerine asparagine agar Vegetative growth - even, reverse golden yellow to orange aerial mycelium - powdery, cream-colored with Pale peach tints. Soluble pigment - pale amber in egg albumin agar Vegetative growth - flat, cream-colored to yellow aerial mycelium - powdery, cream-colored No soluble pigment Calcium-Malate-Agar Vegetative growth - flat, back side - yellow with orange Edge of aerial mycelium - powdery, white to cream-colored with a peach-colored rim. No soluble pigment. Tyrosine nutrient agar Vegetative growth - even, yellow-brown to orange aerial mycelium - sparse, cream-colored with white no soluble pigment tyrosine crystals are decomposed molasses - yeast hydrolyzate agar Vegetative growth - flat, reverse side - orange aerial mycelium - powdery, cream-colored white gein soluble pigment nutrient agar vegetative - even golden yellow aerial mycelium - powdery, cream-colored no soluble pigment litmus milk sparse ring growth - yellow-brown, vegetative growth - no aerial mycelium peptonization: alkaline reaction pH 7.3 up to 7.4 (comparison pH - 6.7) Skimmed milk N sparse ring growth - yellow-brown to orange vegetative growth - no aerial mycelium light yellow-brown soluble pigment Peptonization - alkaline reaction PE 7.2 (comparison pH - 6.6) Skimmed milk agar Vegetative growth - even, orange Aerial mycelium - moderate, cream-colored to pale coral-colored light yellow-brown soluble pigment hydrolysis of casein Gelatine base sparse, cream-colored to orange-colored, vegetative bulb growth Suspended inside the tube. No soluble pigment. Complete liquefaction Gelatine nutrient agar Vegetative growth - flat, orange aerial mycelium: sparse, powdery cream-colored gein soluble pigment liquefaction of gelatine starch nutrient agar vegetatives Growth - flat, orange-colored aerial mycelium - sparse, powdery, pink-colored to off-white No soluble pigment. Wet hydrolysis of starch. Synthetic starch agar Vegetative growth - flat, reverse side cream-colored with an orange-colored border aerial mycelium - powdery, white with a peach-colored rim, no soluble pigment. Moderate Hydrolysis of starch Löffler's blood serum agar Vegetative growth - cream-colored to orange aerial mycelium - none No soluble pigment No liquefaction Peptone Iron Yeast Extract ARar Vegetative growth - cream-colored aerial mycelium - sparse, white No soluble pigment Microaerophilic growth (yeast extract - dextrose stitch culture - 40 mm depth of the stitch) Good surface growth along the top 1/4 of the temperature stitch line - yeast extract dextrose slants Good growth at 280 C Sparse growth at 37 ° C No growth at 500 C.

Yeast extract - dextrose agar Vegetative growth - flat, golden yellow aerial mycelium - powdery, cream-colored to pale flesh-colored pink No soluble pigment Potato cut Vegetative growth - dry, even, cream-colored to orange aerial mycelium - sparse, cream-colored. No soluble pigment. Reduction of nitrates - negative.

All values were taken after 3 weeks of incubation at 280 C, except where otherwise noted. Physiological tests followed 7 and 21 days.

The morphological differences between Streptomyces lactamdurans and Streptomyces fradiae are listed in Table IV below. The observations were made on the media given in Table IV at waxing intervals of 1 Week, 3 weeks and 8 weeks. The aerial mycelium of s. Lactamdurans is short and straight with little branching. It seems about the same size as the vegetative flycel to have, d. H. O, 9 / u width. It's light, powdery, and easy to scratch off. The vegetative mycelium is gram positive, it's not acid-fast. It sticks firmly some media and is embedded in agar. There is some division in rods with shake flask growth, however this is not significant. The vegetative Mycelium from shake flasks and stationary flasks (inoculation medium for 6 days, 280 C) shows some "germs" and short, thickened, almost club-shaped segments on the Mycelium, however, these were not numerous.

All values in Table IV were after 3 weeks of incubation at 280 C, except where otherwise noted. The pH of the too The medium used in these studies was approximately neutral; H. 6.8 to 7.2. the physiological tests were carried out after? 7 and 21 days. The description The colors used are in accordance with the definitions according to "Color Harmony Manual "4, edition, 1958; Container Corporation of America. T a b e l l e IV 842A Morphological comparison of Streptomyces lactamdurans and Streptomyces fradias Medium Streptomyces lactamdurans Streptomyces fradiae Czapek-Dox Vegetatives Growth: flat, deep-cream vegetative power: colorless agar-colored aerial mycelium: Clamshell pink aerial mycelium: powdery, cream-colored white No soluble pigment Nutrient agar Vegetative growth: even, cream-colored Vegetative growth: orange to yellow to golden yellow aerial mycelium: powdery, cream-colored no soluble pigment Glycerin- Vegetative growth: flat back side - Vegetative growth: sand-colored Asparagine-golden yellow to agar orange aerial mycelium: powdery, cream-colored and pale-peach-colored Tint Soluble pigment: pale amber yeast extract - vegetative growth: even, golden yellow vegetative growth: sand-colored dextrose aerial mycelium: powdery, cream-colored agar to pale flesh-pink No soluble pigment Synthetic vegetative material Growth: flat, reverse side Vegetative growth: colorless, cream-colored starch agar with aerial mycelium: sea mucus pink, orange-colored edges aerial mycelium: powdery, white with peach-colored edges. Soluble pigment: Moderate hydrolysis of starch Tabel IV (continued) Medium Streptomyces lactumdurans Streptomyces fradiae potato Vegetative growth: dry, even, Vegetative growth: orange cut, cream-colored to orange aerial mycelium: sparse, cream-colored, no soluble pigment gelatine vegetative Growth: sparse, creamy vegetative growth: cream-colored to pale-colored to brown-orange-colored flakes suspended in the tube aerial mycelium: not soluble Pigment complete liquefaction litmus vegetative growth: yellow-brown, sparse Vegetative growth: cream-colored, milky growth ring aerial mycelium: none Peptonization: alkaline reaction, pH 7.3 to 7.4 (comparison pH - 6.7) 842A Carbohydrate Utilization: The Streptomyces lactamdurans culture (MA-2908) was also assessed for its ability to to utilize, or assimilate, various carbohydrates, studied by the microorganism in a synthetic basic medium (G. Pridham and D. Gottlieb, 1948), which contained 1% of the carbohydrate, was grown at 280 ° C for 3 weeks. Table V sets forth the recovery, or assimilation, of these carbohydrate sources the Streptomyces lactamdurans culture (M19-2908) again. The explanation of the signs in Table V are as follows: + indicates good growth, + indicates poor growth Growth and - denotes no growth on the special carbohydrate.

T a b e l l e V Carbohydrate MA-2908 Carbohydrate MA-2908 Culture Culture Glucose + rhamnose arabinose + cellulose maltose + fructose + raffinose + inositol Sucrose - Acetate + Xylose + Citrate + Mannitol + Paraffin Lactose - Glycerin Mannose -The properties described in Tables III, IV and V have been used for tracing back of Streptomyces lactamdurans Xultur (MA-2908), on a species classification about the in "Bergey's Manual of Determinative Bacteriology", 7th edition, pages 694 to 829 (1957) and in "The Actinomycetes", Volume 2, pages 61 to 292 (1961) described key is used.

A comparison of the detailed properties of Streptomyces lactamdurans Culture with known species shows that culture biochemical Streptomyces fradiae is similar.

However, as indicated above, there are significant morphological differences, for example with regard to the color of the air mycelium of S. fradiae, the sea shell pink colors is compared to the cream color of the culture. Also was while the vegetative Growth in S. fradiae shows no pigment differences on different media, no sporulation found in the culture. On the basis of these differences and the properties described in the preceding tables, the Antibiotic 842A (MA-2908) producing 1-likroorganism as a new species Streptomyces lactamdurans.

The preceding description of the microorganism, which antibiotic 842A generated, serves only to explain the type of strains of the Iilkroorganisms, which can be used, and not limited to microorganisms which meet this particular description. The invention includes the use of others PEkroorgan.isrmen, including strains of actinomycetes both isolated obtained from nature as well as by mutation, for example those deliver through natural selection of those through mutation agents, such as X-ray irradiation, Ultraviolet irradiation, nitrogen mustard gases and the like, obtained strains under appropriate ones Conditions of the product 842A in vitro and in vivo studies antibiotic 810l, in vitro: The biological in vitro characterization of the antibiotic 810A took place by the Petri dish agar diffusion method. These experiments were carried out in such a way that 7 mm discs moistened with the antibiotic solution on the surface of Petri dishes were placed containing 5 ml of Difco nutrient agar and 0.2% @ yeast extract who were doused with 5 or 10 ml of vaccine 150 ml of medium each was inoculated and incubated at 25V C or 37V C for 16 hours. The method and Treatises on these attempts are in the publication: "Cross Resistance Studies and Antibiotika Identification ", applied Nicrobiology, Volume 6, pages 392 to 398 (1958).

The following Tables VI, VII and VII give the results of these Try again and again for antibacterial and crossbreeding resistance the test organisms used and the conditions applied.

Table VI 810A antibacterial spectrum, in vitro effectiveness Test organism Test conditions Zone of inhibition, diameter mm * Vaccine ** Incubation 810A ml / 150 ml Temp. ° C 5 mg / ml Escherichia coli 5 25 15 Bacillus species 5 25 22 Proteus vulgaris 5 37 29 Pseudomonas aeruginosa 5 25 7 Serratia marcescens 5 25 7 Staphylococcus aureus 5 25 26 Bacillus subtilis 5 25 33 Sarcina lutea 5 25 26 Staphylococcus aureus 5 37 19 (streptomycin-streptothricin resistant) Streptococcus faecalis 15 37 7 Alcaligenes faecalis 5 37 25 Brucella bronchiseptica 10 37 21 Salmonella gallinarum 10 25 15 Vibrio percolans 10 27 36 Xanthomonas vesicatoria 5 25 15 * 7 mm = disc size (no inhibition zone observed) ** overnight culture, diluted to a value of 60 mµ on the Lumetron colorimeter T a b e l l e VII 810A cross-breeding resistance, in vitro investigation Escherichia coli - strain ** Test conditions inhibition zone diameter mm * vaccine *** incubation 810A ml / 150 ml Temp. ° C 5 mg / ml Sensitive starting strain 5 25 15 Streptomycin-resistant 5 25 13 Streptothricin-resistant 10 25 17 OXAMYCIN-resistant 10 25 10 Pleoxidin-resistant 10 37 26 Chloramphenicol resistant 10 25 7 Chlortetracycline resistant 10 25 7 Oxytetracycline resistant 10 25 7 Neomycin resistant 10 37 20 Tetracycline resistant 10 25 7 Viomycin resistant 10 37 17 Polymyxin-resistant 10 25 13 Grisein-resistant 5 25 15 * 7 mm = slice size (no inhibition zone observed) ** Trials against a series of strains of E. coli, isolated from the same starting culture, carried out and then exposed to the individual antibiotics *** overnight culture, diluted on a value of 60 mµ on the Lumetron colorimeter Tabel VIII 810A special spectrum of activity, in vitro investigation Escherichia coli W-MB-60 Test conditions inhibition zone (with special additives as indicated in diameter) mm * Vaccine ** Incubation 810A ml / 150 ml Temp. ° C 5 mg / ml Comparison (no additives) 5 25 13 0.1 mol. Phosphate buffer - pH 5 5 25 13 0.1 mol. pH phosphate buffer - pH 7 5 25 16 0.1 mol. pHosphate buffer - pH 9 5 25 18 Human blood plasma 20% 5 25 15 cation exchange resin 5 25 12 (Dow ET 91-1%, agar concentration to 1% only reduced for the resin plate) * 7 mm disc size (no inhibition zone observed) ** Overnight culture, diluted to a value of 60 mµ on the Lumetron colorimeter antibiotic 810A, in vivo: The in vivo biological characterization shows that 810A is an antibiotic has a broad spectrum of activity and is effective against infections with three species of Proteus, two from Salmonella, a strain of Escherichia coli, two from Klebsialla, and three gram-positive organisms: Staphylococcus aureus, Streptococcus pyrogenes and Diplococcus pneumoniae protects.

Method: The method used for this characterization is like follows: White, female Swiss mice with an average weight of 20 to 33 g were infected intraperitoneally "with 3 to 20 times the number of organisms, which was calculated to be lethal for 50% of the infected control animals (LD50-: E) osis 3 to 20). At the time of infection, and again 6 hours later, it was over applied the designated path therapy. Controls the virulence of the culture and the toxicity of the antibiotic for uninfected mice was included in the experiments locked in.

7 days after infection, the experiment was deemed to have ended, and the amount of antibiotic (1) required to protect 50% of the infected and treated animals is necessary, was according to the method of Knudsen and Curtis, J. Amer.

Stat. Assoc., Vol. 42, p. 282 (1947).

Results: The results of these experiments are listed in Table IV. These data show that antibiotic 810A obtained from culture MA-2837 is a broad spectrum agent that is effective against both gram-positive and also gram: - protects negative organisms.

Although efficacy was achieved when administered orally (p.o.), the best results were obtained via the subcutaneous (s.c.) or intraperitoneal (i.p.) way. The antibiotic mixture did not kill uninfected mice, however two doses, each containing 1 mg of the product, intraperitoneally or when two doses of 18 mg each subcutaneously or orally were administered.

T a b e 1 1 e IX 810A in vivo effectiveness test organism therapy ED50 in micrograms away x two doses of Proteus vulgaris i.p. 33 s.c. 500 p.o. 12100 Proteus mirabilis i.p. 200 p.o. 5000 Salmonella schottmuelleri i.p. 419 s.c. 9000 Escherichia coli i.p. 3750 Escherichi coli i.p. .1330 Klebsiella pneumoniae i.p. 2500 Salmonella gallinarum i.p. 1670 Salmonella pullorum i.p. 625 Diplococcus pneumoniae i.p. 258 Staphylococcus aureus i.p. 927 Streptococcus pyogenes i.p. 625 Antibiotic 842A, in vitro: The biological in vitro characterization was carried out according to the disk plate agar diffusion method. This experiment was carried out in such a way that 7 mm discs, which were moistened with the antibiotic solution, on the surface were brought from Petri dishes, which were filled with 5 ml Difco nutrient agar and 0.2% yeast extract, those with 5 or 10 ml standard cell suspension (OD = 0.22 at 660 mµ) per 150 ml medium were inoculated, doused and at 250 C or 370 C for 16 hours as indicated was incubated. The method and modification of these experiments are in the publication: "Cross Resistance Studies and Antibiotic Identification, "Applied Microbiology, Volume 6, pages 392 to 398 (1958). The following tables give the results of these tests with regard to the antibacterial spectrum and the cross-breeding resistance again and lead the applied test organisms and the conditions used.

T a b e l l e X 842A antibacterial spectrum, in vitro effectiveness Test Organism Test Conditions Zone of Inhibition, Diameter, mm * Vaccine Incubation Crude 842A (1b) Free Acid Sodium Az ml / 150 ml Temp. ° C 8 mg / ml 166 µg / ml 192 µg / ml Escherichia coli 5 25 16 20 18 Bacillus sp. 5 25 7 8 7 Proteus vulgaris 5 37 21 22 24 Pseudomonas aeruginosa 5 25 7 7 7 Serratia marcescens 5 25 7 7 7 Staphylococcus aureus 5 25 7 7 7 Bacillus subtilis 5 25 16 13 18 Sarcina lutea 5 25 9 10 9 Staphylococcus aureus 5 37 7 11 10 (streptomycin-streptothricin-resistant) Streptococcus faecalis 15 37 7 7 7 Alcaligenes faecalis 5 37 22 27 7 Brucella bronchiseptica 10 37 28 24 26 Salmonella gallinarum 10 25 20 21 25 Vibrio percolans 10 27 37 30 38 Xanthomonas vesicatoria 5 25 12 19 17 * 7 mm = disc size (no inhibition zone observed) T a b e l l e XI 842A Cross-breeding resistance, in vitro investigation of Escherichia coli strain ** Experimental Conditions Zone of Inhibition, Diameter, mm * Vaccine Incubation- Rohes 842A (Ib) Free acid sodium salt ml / 150 ml Temp. ° C 8 mg / ml Acid 192 µg / ml 166 µg / mol sensitive initial strain 5 25 16 20 18 streptomycin-resistant 5 25 14 19 16 Streptothricin-resistant 10 25 13 14 18 OXMYCIN-resistant 10 25 13 13 17 Pleocidin-resistant 10 37 15 18 17 Chloramphenicol resistant 10 25 13 17 16 Chlortetracycline resistant 10 25 21 20 23 Oxytetracycline resistant 10 25 20 23 24 Neomycin resistant 10 37 18 17 17 Tetracycline resistant 10 25 16 20 20 Viomycin resistant 10 37 15 16 30 Polymycin-resistant 10 25 23 21 7 Grisein-resistant 5 25 18 21 16 ** the trials were compared to a number of E. coli strains obtained from the same starting culture were isolated, and subsequent exposure to each antibiotic carried out * 7 mm = disc size (no inhibition zone observed) T a b e l l e XIa 842A special spectrum of activity, in vitro investigation Escherichia coli W-MB-60 test conditions inhibition zone, diameter, mm * (with special Additives Vaccine Incubation- Raw 842A (Ib) Free Acid Sodium Salt As Specified) ml / 150 ml Temp. ° C 8 mg / ml 166 µg / ml 192 µg / ml Comparison (no additives) 5 25 16 20 18 0.1 mol. Phosphate buffer pH 5 5 25 19 20 15 0.1 mol. Phosphate buffer pH 7 5 25 22 29 25 0.1 mol. Phosphate buffer pH 9 5 25 21 22 24 Human blood plasma 20% 5 25 17 22 21 * Katine exchange resin (Dow ET 91) 1% 5 25 20 21 18 * (Agar concentration reduced to 1% for resin plate only antibiotic 842A, in vivo When 842A is administered subcutaneously to mice, it is generally more effective as cephalothin and about as effective as cephaloridin, ampicillin and chlormycetin regarding protection against infectin by gram-negative organisms. It is remarkably non-toxic and is rapidly excreted in the urine, being about 79% of the 842A injected subcutaneously can be recovered within 4 hours.

In in vivo studies, the antibiotic 842A protects against infections with three species of Proteus, two of Salmonella, one strain of Escherichia coli, two of Klebsialla and also against paracolobactrum arizoniae, Aerobacter aerogenes, Pasteurella multocida and Diplococcus pneumoniae E400.

: 13 The procedure used in these investigations is the same as above with respect to the in vivo characterization of 810A. described. The results of these tests are given in Table XIb below: T a b e 1 1 e XIb 842A in vivo efficacy of test organism ED50 by subcutaneous route x two doses of Proteus vulgaris 51 µg Proteus mirabilis 276iug Proteus morganii 3202 276 µg Salmonella schottmuelleri 103 K) .ebsiella pneumoniae AD 125 Klebsiella pneumoniae 3 125ng Paracolobactum arizoniae 125 Escherichia coli 200 Aerobacter aerogenic 49 Pasteurella multocida 57 µg Salmonella typhosa 34 µg Diplococcus pneumoniae E400 566 µg The antibiotics 810A fermentation: The antibiotic 810A is used during the aerobic fermentation of suitable, aqueous nutrient media under controlled conditions generated by inoculation with the Streptomyces griseus culture tA-2837. Watery Media such as those used in the manufacture of other antibiotics, are also suitable for the production of the antibiotic 810A. Such media contain sources of carbon and nitrogen that can be assimilated by the microorganism and inorganic salts.

In general, carbohydrates such as sugars, e.g. B. glucose, arabinose, maltose, xylose, mannitol and the like., And starches, such as cereals, z. B. oats, rice, cornstarch, cornmeal and the like., Either alone or in combination as assimilable carbon sources are used in the nutrient medium will. The exact amount of the carbohydrate source or sources used in the Medium depends in part on the other components of the medium, but generally the amount of sole hydrate usually varies between about 1 and 6 percent by weight of the medium. These carbon sources can be used individually or they can be several such carbon sources are combined in the medium. In general, can any proteinaceous material as a nitrogen source in the fermentation process can be used. Suitable nitrogen sources include, for example, yeast hydrolysates, Yeast autolysate, soybean flour, hydrolysates of casein, corn steep liquor, soluble Still bottoms or tomato paste and the like. The nitrogen sources are either alone or in combination in amounts ranging from about 0.2 to 6% by weight of the aqueous medium is used. Typical media used to manufacture the antibiotic 810A are those listed below.

These media and others described in the following examples: Media are only used to explain a large number of Itledien that can be used and are not intended to limit.

Medium I: Difco yeast extract 10.0 g glucose 10.0 g * phosphate buffer 2.0 ml NgSo4 7H O 0.05 g distilled water 1000.0 ml Difco agar 25.0 g * phosphate buffer: KH2PO4 91.0 g Na2HPO4 95.0 g distilled water 1000.0 ml medium II: beef extract 3.0 g * NZ amine 10.0 g dextrose 10.0 g NaCl 5.0 g distilled H 0 1000.0 ml pH adjusted to 7.2 with NaOH * an enzymatically degraded casein medium III: Dextrose 10.0 g asparagine 1.0 g K2HPO4 0.1 g MgS04 * 7H2O 0.5 g yeast extract 0.5 g * trace element gene No. 2 10.0 ml distilled H2O 1000.0 ml pH to 7.2 adjusted with NaOH * trace element mixture no. 2: FeSO4. 7H2O 1.0 g NnS04 H2O 1.0 g CuCl2. 2H2O 25.0 mg CaCl2 100.0 mg H3BO3 56.0 mg (NH4) 6MO7O24. 4H2O 19.0 mg ZnSO4. 7H2O 200.0 mg Distilled H20 1000.0 ml Medium IV: V8 juice 100.0 ml Staley's 4S Soybean Meal 20.0 g Destrose 2.0 g Agar 25.0 g Distilled H2O 1000.0 ml pH 7.9-8.0 Medium V: yeast autolysate (Ardamin) 10.0 g glucose 10.0 g * Phosphate buffer 2.0 ml MgSO4. 7H20 0.05 g Distilled H2O 1000.0 ml pH - up 6.5 adjusted using NaOH * Phosphate buffer solution: KH2PO4 91.0 g Na2HPO4 95.0 g of distilled H2O 1000.0 ml Medium VI: corn steep liquor (on a moist basis) 40.0 g destrose 20.0 g NaCl 2.5 g MgSO4. 7H2O 0.5 g polyglycol 2000 0.25%, based on the volume (added individually to each K @ lb @@ @@ flask) Distilled H2O 1000.0 ml pH - adjusted to 7.0 with NaOH Medium VII: Inoculation Production L-asparagine 5.0 g 5.0 g L-histidine 4.0 g 4.0 g DL-phenylalanine --- 2.0 g monosodium glutamate --- 1.5 g NaCl 5.0 g 5.0 g K2HPO4 2.0 g 2.0 g CaCl2 2H20 0.4 g 0.4 g MnSO4. H2O 0.1 g 0.1 g FeSO4. 7H2O 0.1 g 0.1 g ZnSO4. 7H2O 0.05 g 0.05 g MgSO4. 7H2O 1.0 g 1.0 g glycerine 10.0 g 20.0 g sucrose 2.5 g 2.5 g distilled H20 * 1000.0 ml ** 1000.0 ml * pH adjusted to 7.0 with NaOH ** pH adjusted to 7.1 with NaOH set medium VIII: meat extract 0.3% NaCl 0.5% NZ amine 1% dextrose 1 % pH 7.0 The fermentation takes place at temperatures in the range of about 20 to 57 ° C, however, it is preferred for best results to carry out the fermentation at temperatures of about 22 to 300 C. The pH that for the growth of the Streptomyces griseus culture (2837): and for the production The culture media suitable for the antibiotic 810A should range from about 5.5 to 8.0 lie.

A small fermentation batch of the antibiotic 810A is done in a simple manner Way by inoculating a suitable nutrient medium with the antibiotic-producing medium Eultur, where fermentation takes place at a constant temperature of about 24 to 28 C on a shaker for an extended period of time, such as several days, let it progress. After the incubation period has ended the nycel is removed and the supernatant is examined.

In practice, this fermentation is carried out in a sterilized flask a one-, two-, three- or four-stage seed development carried out. The nutrient medium Any suitable combination of carbon and nitrogen sources can be used for the inoculation step such as any of those described in I to VIII above Media. The inoculation flask is placed in a constant temperature chamber at around 280C shaken for a period of 1 to about 3 days, and the culture obtained is used to inoculate either a two-stage inoculation medium or the production medium Used When intermediate inoculation flasks are used, these come in handy developed in the same way, d. n., the contents of the flask are used for inoculation of the production medium used, the inoculated flasks are operated at a constant Shaken temperature for several days, and at the end of the incubation period is the contents of the flask are centrifuged to remove the mycelium. The protruding liquid or broth is then concentrated and under preparation of the antibiotic 810A purified.

Fermentation is preferred for working on a larger scale Carry out in suitable tanks, which are equipped with a stirrer and a device are provided for aeration of the fermentation medium. According to this method, the Nutrient medium is brought into the tank and heated to temperatures of up to about 1200 C sterilized. After cooling, the sterilized medium is treated with the generating Inoculated culture and allowed to ferment for several times Days, for example from 2 to 4 days, continue while the nutrient medium is stirred and / or ventilated and the temperature is maintained at about 24 to 280 o.

Changes in vaccine development and changes in the Production medium it is possible to achieve a multiple improvement in terms of To maintain production and an increase in the effectiveness of the antibiotic.

810A Experimental Method Using Proteus Vulgaris The Antibiotic 810A was easily made using elli disk and plate method from Proteus vulgaris MB-838 (ATCC 21100 and NRRL B-3361) while maintaining as a slant culture on nutrient agar (Difco) plus 0.2% yeast extract (Difco) as the test organism examined. The inoculated slants were at 3? C for 18 to 24 hours incubated and stored at refrigerator temperatures until used, being fresh slants were made every week.

The vaccine for the experimental panels is inoculated daily a 250 ml Erlenmeyer flask, the 50 ml nutrient broth (Difco) plus 0.2% refee extract (Difco) prepared with a smear of the slant culture. The piston will at 370 C on a shaker for 18 to 24 hours incubated. The liquid culture is then reduced to 40% permeability at a Wavelength of 660 mµ using a Bausch & Lomb Spectronic-20 device Uninoculated by adding a 0.2% yeast extract solution to the culture Liquid is used as a blank test for this determination. The set Broth (30 ml) is used to inoculate 1 liter of medium.

Nutrient agar (Difco) plus 0.2% yeast extract (Difco) are used as the test medium used. This medium is prepared, sterilized by autoclaving, and it is allowed to cool to 500.degree. After the medium is inoculated, each of the 10 ml are added to various sterile Petri dishes, and the medium is allowed to solidify.

Tests were carried out on these plates using the disk-platter method performed using 13 mm filter paper disks. The experimental panels were incubated at 370.degree. C. for 20 to 24 hours. The evaluations were made as mm in diameter expressed in the zone of inhibition. They were used to determine the relative effectiveness or, when compared to a purified reference standard, efficacy in, µg / ml used If such an experiment is performed quantitatively, can 2 to 4 / ng / ml antibiotic can be determined.

810A Experimental Procedure Using Vibrio percolans: There were also tests with respect to 810A using the disk plate method against Vibrio percolans (MB-1271) using 13 mm filter paper disks. The experimental panels were using Difco nutrient agar plus 2.0 g / 1 Difco yeast extract with 10 ml per plate. An overnight culture of the test organism, Vibrio percolans (MB-1272) in nutrient fluid plus 0.2% yeast extract was in sterile Saline to a suspension with 40% transmittance at one wavelength diluted by 660 mµ.

This suspension was mixed with 20 ml / l medium before the plates were poured admitted.

The test plates were kept at 40 ° C. until they were used (maximum 5 days). After the application of the test discs saturated with antibiotic the plates were incubated at 280 ° C. for a period of 8 to 24 hours. Zones of inhibition were read as mm in diameter.

Bacterial inactivation with 810A: An in vitro experiment was used for Determination of the resistance of the antibiotic 810A to bacterial inhibition in comparison with Cephalosporn G, Cephaloridiii and Cephalothin. This investigation showed that antibiotic 810A was more resistant than the latter Microorganisms is.

The degrading bacterium used in this experiment was an organism of which it is annoyed that it completely inactivates cephalosporin C, namely Alcaligenes faecalis (MB9).

(a) Production of bacterial cells: Alcaligenes faecalis (MB-9) cells were prepared as follows: The contents of a tube were mixed with a few ml of nutrient liquid, containing 0.2 ° ó yeast extract, mixed. A loopy slurry was made Spread over the surface of a nutrient agar slant and at 370 for 18 hours All slant cultures were stored at 5 ° C and incubated within 1 Used week after incubation. A loop full of surface growth from each slant culture was as / eptisch in 50 ml nutrient broth, the 0.2% yeast extract contained, transferred and incubated for 18 hours at 280 C with shaking. The culture was then centrifuged at 4000 rpm for 10 minutes. The excess material became decanted off, and the remaining cell clump was washed twice with sterile 0.1 M phosphate buffer, pH 7.5 (6.8 g potassium phosphate and 7.1 g sodium hydrogen phosphate per liter of distilled Water). The washed cells were then in one-tenth the original volume a 4 mg / ml antibiotic solution was resuspended in 0.1 M phosphate buffer. That The test mixture was then placed in a water bath set at 370 ° C. without shaking incubated for up to 4 hours. The test mixes were then run at 2000 rpm for 10 minutes centrifuged, with a clear supernatant formed, the la sterila Test tubes were decanted and immediately frozen in dry ice until it was ready for biological determination, usually within 3 hours. Comparative samples were incubated in exactly the same way but without cells.

(b) Degree of inactivation of antibiotic 810A: The protruding Liquids have been recognized for their antibacterial effectiveness in the following Ways to be tested: paper disks of 6.4 mm were with the supernatants moistened and placed on the surface of nutrient agar (0.2%) yeast extract plates, who had previously been vaccinated with the corresponding test organism.

B. subtilis s (liB-904-) test plates were made in the following manner Inoculated: 5 ml of a suspension of washed spores in 0.9% saline was added 150 ml of nutrient agar (0.2%) beef extract were added to each, of which 5 ml were then placed in Petri dishes of 15 x 100 m. All test plates were stored at 50.degree and used within 3 days. The test plates were left overnight at 250 C before measuring zones of inhibition around the test disks.

Cell-free control samples of each antibiotic were in Dilutions from 1: 1, 1: 2, 1: 4, 1: 8, 1:16 and 1:32 to a standard reference curve to obtain. Solutions of the test antibiotics were made at full strength after incubation examined in the presence of the washed bacterial cells. All samples were driven three times.

(c) Brgebnesse: The percentage inactivation was calculated by the mean of the three zones of inhibition obtained from each experiment is taken and the amount of antibiotic remaining in the test solution was determined as results from the standard curve. This value was then taken from the starting concentration (4 mg / ml) subtnbeiert and the remainder divided by the starting concentration and with Multiply 100, preserving the inactivation present. The following Tables XII and XIII give the inactivation obtained for cephalosporin G, cephalothin and antibiotic 810A under the conditions described above.

T a b e 1 1 e III Percentage inactivation after incubation with washed bacterial cells (determined on 3rd subtilis (MB-964) plates) antibiotic Incubation for 3 hours with Alcaligenes faecalis 810A 0 Cephalosporin C 99+ Cephalothin 62.5 Table XIII Percentage inactivation after incubation with washed bacterial cells (determined on V. percolans (MB-1272) plates) antibiotic 3 hours incubation with Alcaligenes faecalis 810, A 0 Cephalosporin C 99+ Cephalothin 50 The ability of antibiotic 810A and cephalosporin C, the degradation effect to withstand the Aerobacter cloacae (MB-2646) culture was also determined. This culture is gram negative and resistant to cephalosporin C. When performing of the experiment were samples of the individual mixtures of the organisms and one sample of the antibiotic mixture removed after incubation for 2 hours and applied to the remaining investigated antibiotic effectiveness. The procedure is the same examination method. as described above for Algaligenes faecalis. The source of the inactivation substance is a 1: 160 dilution of the filtrate from one carried out at 37 ° C for 18 hours Shaking culture of Aerobacter cloacae MB-2646 in nutrient broth, the O, 2'o yeast extract contained. The following table MV gives the percentage inactivation of antibiotic 810A, Cephalothin, Cephaloridin and Cephalosporin C on Vibrio percolans (MB-1272) according to this method again: T a b e 1 1 e XIV Percentage inactivation after incubation with cell-free extract (determined on V. percolans (MB-1272) plates) Antibiotic incubation for 2 hours with Aerobacter cloacae 810A 16 Cephalothin 66 Cephaloridin 96 Cephalosporin C 96 Using the same experimental procedure As described above, the following Table XV gives the relative resistance of the Antibiotic 810A against enzymatic inactivation by Aerobacter cloacae again. The initial concentration is 250 µg / ml. The results are in µg / ml expressed.

T a b e l l e XV Remaining antibiotic activity (yg / ml) (Initial concentration = 250 µg / ml) antibiotic test organism incubation for 2 hours with Aerobacter cloacae + 810A B. subtilis 964 190 cephalothin "140 cephaloridin "<10 810A V. percolans 12:72 210 cephalothin" 85 cephaloridin "<10 cell-free extract In view of the above data, the antibiotic is 810A apparently more stable than cephalosporin C, cephalothin and cephaloridin against inactivation by Aerobacter cloacae.

842A fermentation: The 842A antibiotic is used during aerobic Fermentation of suitable aqueous nutrient media under controlled conditions Inoculation with the organism Streptomyces lactamdurans produced. In general, can many media, which are a source of carbon and nitrogen, for manufacture can be used by the 842A. Examples are the above in connection with the Fermentation of 810A-described aqueous media and sources of carbohydrates and nitrogen. The exact amount of carbohydrate and nitrogen sources depends on the other im Fermentation medium contained components from, but is generally the The amount of carbohydrate is usually 1 to 6% by weight of the medium and the amount of. available Nitrogen, either alone or in combination, is usually in the range of about 0.2-06 to about 6 weight percent of the medium. The different ones described below Media are used to explain media used to manufacture the antibiotic 842A are suitable. These media are led.igli.ch examples of usable media and serve. not to the limit.

Medium IX: Amber Yeast No. 300 10.0 g of soluble brewery residues 20.0 g dextrose 10.0 g distilled water 1000.0 ml pH 7.0 medium 1: Staley's 4S soybean meal 30.0 g Soluble brewery residues 7.5 g Cerelose 20.0 g NaCl 2.5 g CaCO3 (afterwards pH value to 7.0) 10.0 g distilled water 1000.0 ml Medium XI: Amber yeast No. 300 10.0 g Soluble brewery residues 20.0 g Distilled Water 1000.0 m.

pH 7.0 The fermentation is carried out at temperatures in the range of about 20 to 370 C, but for best results it is preferred to carry out the fermentation at temperatures of about 24 to 320 C. The pH that for the growth of the Streptomyces lactamdurans culture (MB-2908) and for the production of antibiotic 842A should be in the range of about G, C to about 8.0 lie.

A small-scale fermentation of the antibiotic 842A is used in simply by inoculating the antibiotic in a suitable hormone medium producing culture carried out, whereby the fermentation is carried out at a constant Temperature of about 280 C for several days on a shaker lets progress. At the end of the incubation period, the mycelium is removed, and the supernatant is examined.

In practice, fermentation takes place in a sterilized flask via a one, second, three or four-stage seed development. The nutrient medium for the seeding stage can be an appropriate combination of carbon and nitrogen sources be, for example any of the media described under IX to XI above. Of the. Will germinate in a constant temperature of about 280 C chamber shaken for a period of 1 to about 3 days, and the growth obtained becomes either for inoculation of a second wedge stage or of the production medium is used. If intermediate germinating flasks are used, these are developed in practically the same way, i.

the contents of the flask are used to inoculate the production medium, the inoculated flasks are shaken at a constant temperature for several days, and at the end of the incubation period, the contents of the flasks are available for removal of the mycelium centrifuged. The supernatant liquid or broth is then concentrated and purified to obtain antibiotic 842A.

When working on a larger scale, fermentation is preferred To be carried out in suitable tanks, which are equipped with an agitator and a device are provided for aeration of the fermentation medium. According to this method, the Nutrient medium is brought into the tank and heated to temperatures up to about 1200 C sterilized. After cooling, add the sterilized medium to the production culture Deimpft, and mm lets fermentation for a period of a few days, for example 2 to 4 days, progressing while the nutrient medium is agitated and / or is ventilated and the temperature is maintained at about 280 C. Through change It is also the development of the inoculum and changes in the production medium possible @ - a multiple improvement in production and an increase in To achieve effectiveness of this antibiotic.

842A Experimental Procedure Using Vibrio percolans: There were Experiments according to the disc-plate method using 13 mm filter paper discs carried out. These experimental plates were prepared using Difco nutrient agar plus 2.0 g / l Difco yeast extract prepared at 10 ml per plate. A Overnight growth of the test organism Vibri o percolans (MB-1272) in nutrient broth and 0.2 0% yeast extract was added to a suspension containing 40% in sterile saline Thinned transmittance at a wavelength of 660 mµ.

This suspension. was in 20 ml / l medium before watering the plates admitted.

The experimental plates were kept at 4 ° C until used (maxirial 5 days). After applying the test discs saturated with antibiotic the plates were incubated at 280 C for a period of 8 to 24 hours .. The zones of inhibition were read as mm in diameter. They became determination relative efficiencies or when compared to a pure reference standard were used, the effectiveness in µg / ml. If such an attempt in quantitative Carried out this way, 1 to 2 µg / ml of antibiotic can be detected.

Bacterial inactivation with 842A: An in vitro study served to determine the resistance of the antibiotic 842A to bacterial inactivation compared to Cephalosporin C, Gephalorilin and Cephalothin.

This study showed that antibiotic 842A was more resistant than the latter is against certain microorganisms.

The degrading bacteria used in the experiment were two organisms which are known to completely inactivate cehalosporin C, viz Alcaligenes faecalis (MN-9) and Alcaligenes viscosus (MB-12).

(a) Production of bacterial cells: Alcaligenes viscosus (MN-12) and A. faecalis (MB9) cells were prepared as follows: The contents of a tube was mixed with a few ml of nutrient broth containing 0.2% yeast extract.

A slurry was dragged over the surface one Small agar slant culture distributed and incubated at 47 ° C for 18 hours. All sloping cultures were stored at 5 ° C and used within 1 week of incubation. One Loop full of surface growth from each slant culture was aseptically in 50 ml of nutrient broth containing 0.2% yeast extract, transferred and 18 hours at 28 ° C incubated with shaking. The culture was then centrifuged at 4000 rpm for 10 minutes. The supernatant liquid was decanted and the remaining cell clump became twice with sterile 0.1 M phosphate buffer, pH 7.5 (6.8 g potassium phosphate, i.e. KH2PO4 and 7.1 g sodium hydrogen phosphate per liter of distilled water).

The washed cells were then in one-tenth the original volume resuspended a 4 mg / ml solution of the antibiotic in 0.1 M phosphate buffer. That The test mixture was then placed in a water bath set at 37 ° C. without shaking incubated for 4 hours. The test mixes were then run at 2000 rpm for 10 minutes centrifuged, and a clear @@@@@ solution was produced, which in sterile Test tubes were decanted and immediately frozen in dry ice until she was ready, usually within 3 hours. Experimental samples were made in exactly the same way except for the absence of Cells incubated.

(b) Degree of inactivation of antibiotic 842A: The protruding Liquids were tested for their antibacterial effectiveness in the following way tested: 6.5 mm paper disks were moistened with the supernatant liquids and placed on the surface of nutrient agar 0.2% Hefeex @ rakt plates, which were previously had been inoculated with the corresponding test organism.

@. subtilis (MB-964) experimental plates were made in the following manner inoculated: 5 ml of a suspension of washed spores in 0.9% saline was added 150 ml each 0.2 s nutrient agar yeast extract (450 C) added, 5 ml of which were then distributed in Petri dishes of 15 x 100 mm.

All test plates were stored at 5 ° C and inside used for 3 days. The test plates were kept overnight at 250 ° C. before the measurement of the zones of inhibition around the test discs.

Cell-free control samples of each antibiotic were made in - dilutions of 1: 1, 1: 2, 1: 4, 1: 8.1: 16 and 1 32 examined to a standard reference curve to obtain. Solutions of the test antibiotics were at full strength after incubation examined in the presence of the washed bacterial cells. All samples were driven three times.

(c) Results: The percentage of inactivation was calculated by taking the mean of the three zones of inhibition obtained from each experiment and the amount of antibiotic remaining in the test solution, such as yourself is determined by the standard curve. This value was then derived from the initial concentration (4mg / ml) subtracted and the remainder divided by the initial concentration and Multiply by 100 to get the percent inactivation. The following Table XVI gives the inactivation obtained for cephalosporin C and the antibiotic 842A under the conditions described above.

CC a b e 1 1 e XVI Percentage inactivation after incubation with washed bacterial cells (determined on B. subtilis (MB-964) plates) Incubation for 4 hours organism Ceph C antibiotic 842A Alcaligenes faecalis 99+ 0 A. viscosus S12 99+ 54.4 The ability of antibiotic 842A and cephalosporin C, to withstand the degradative effects of four other cultures, was also chosen determined These cultures are: Escherichia coli 236, Proteus morganii 251, Proteus morganii 356 and Proteus mirabilis 241. Each is gram / negative and against cephalosporin C resistant. When the investigation was carried out, samples of the individual aggregates were taken of the organisms and a sample of the antibiotic taken after incubation for 4 hours and examined with regard to the remaining antibiotic effectiveness. The procedure is the same test method as above against Alcaligenes faecalis MB-9 and A. viscosus MB-12. The following table gives the proaentual inactivation from cephalosporin C and 842A to B. subtilis (MB-964) using this method again: T a b e l l e XVII culture Geph C antibiotic 842A Escherichia coli 236> 99 38 Proteus morganii 251> 99 80 Proteus morganii 356 299 69 Proteus mirabilis 241 72 5 The foregoing data shows that antibiotic 842A apparently against inactivation by A. faecalis, A. viscosus, Escherichia coli 236, Proteus morganii 251, Proteus morganii 236 and Proteus mirabilis 241 are more resistant is known as cephalosporin C.

The antibiotic obtained by the present fermentation process 842A is an amphoteric compound with an apparent isoelectric point of about pH 3.5; it is unstable at pE above 9.0, but at pH 1.5 relatively stable.

Since the antibiotics 810A and 842A and their salts the growth of different To effectively inhibit species of Salmonella, they can act as disinfectants used for various household and industrial purposes.

For example, 810A is effective against Salmonella schottmuelleri and S .; gallinarum, and 842A is effective against Salmonella schottmuelleri 3010, S. gallinarum and S. typhosa, and this property shows their usefulness as sanitary agents for household and industrial purposes.

Isolation and Purification Antibiotil.-um 810A: The Antibiotic 810A can by adsorption on an ion exchange resin, for example synthetic Anion exchange resins derived from dextrose or acrylic copolymers or non-ionic, crosslinked polymers are cleaned. The adsorbed antibiotic is made from the resin or polymer adsorbate with water or with an aqueous, alcoholic Solution of a suitable salt such as ammonium chloride, sodium chloride and the like, eluted. Examples of ion exchange resins and polymers that can be used are the DEAE-Sephadex A-25, Amberlite IRA-68, and Amberlite XAD-2 media, which are given below are described. Possibly can do that according to the above Process obtained eluate through a second and third adsorption and elution stage getting cleaned. Concentrates of all the eluates are then obtained in order to remove the to yield purified product.

810A components: The antibiotic 810A can be broken down into its components, 7ß- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxyp-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4- carboxylic acid and 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxyp-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid separated by chromatography. These include: (1) Chromatography on a highly hydrophilic anion exchange resin, such as * DEAE Sephadex A-25, developed with an ammonium bromide-formic acid system.

Different concentrates of this system can be used, however, in practice a solution of 0.5 m-ammonium bromide and 0.1 m-formic acid is used preferred.

(s) Vhromatography on a weakly basic anion exchange resin, such as @@ Amberlite IRA-68. This is a group separation, with material is abandoned in raw form at a pH of about 3 to 3.5 and initially with an acid at a pH of about 2 and then with NaCl / HCl 1 at a pH of about 1 elu-2eft.

(3) chromatography according to a non-ionic, crosslinked polystyrene polymer, such as *** Amberlite XAD-2. Elution is carried out with a suitable aqueous system, however, it is generally best to use a mixture of water and a lower alkyl ketone. Typical eluents that can be used are for example 10% methanol in water and then 50% methanol in water. Instead of the 50% methenoline-water solution, 20% acetone-in-water can be used be used.

* DEAE Sephadex A-25 is a synthetic anon exchange resin that is derived from the polysaccharide, dextran in its chloride form, d. H. with chloride counterions; pharmacia Fine Chemicals, Inc., 800 Centeni al- Avenue, Piscataway, New Market, New Jersey 08854.

** One synthetic anion exchange resin; a cross-linked acrylic copolymer, containing a weakly basic tertiary amino group; Rohm & Haas Co., Philadelphia, Pennsylvania 19105.

*** A non-ionic, crosslinked polystyrene polymer sorbent; Raw & Haas Co., Philadelphia, Pennsylvania 19105.

The individual products obtained by the above methods can can be purified by rechromatography. For example, 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) can be purified by this product as described in Method 1 above Is subjected to cleaning method, with subsequent desalination on Amberlite XAD-2 absorbent, and 7β-D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid can be obtained by rechromatography on a Sephadex-A-25 anion exchange resin containing 0.5 m-ammonium bromide and 0.05 m-acetic acid is evolved, must be purified again.

Antibiotic 842A: The antibiotic 842A can be adsorbed to an ion exchange resin, such as resins made from quaternary ammonium or sulfonic acid exchange media are built up. The adsorbed Antibiotic is made from the resin adsorbate with aqueous solutions or with an aqueous, alcoholic solution a suitable salt, e.g. 3. ammonium chloride, Sodium chloride and the like, eluted. About suitable ion exchange resins that can be used include, for example, the polystyrene resins with sulfonic acid groups in the core (45% or 53% water) or polystyrene trimethylbenzylammonium resins (43% water, known as Dowex 50 and Dowex 1, respectively. If necessary, this can be done according to the above Process obtained eluate through a second and third adsorption and elution stage further cleaned. Concentrates, all eluates, are then obtained in order to to give the purified 842A product.

Preparing the antibiotic 810A and its individual components and the antibiotic 842A can be used alone or in combination as an active ingredient can be used in any of a variety of pharmaceutical preparations. These Antibiotics and their corresponding salts can be in capsule form or as tablets, Powder or liquid solutions or as suspensions or elixirs can be used. They can be administered orally, intravenously or intramuscularly. To suitable Carriers used in the composition include, for example, mannitol, sucrose, Glucose or sterile liquids, such as. B. water, saline, glycols and oils of petroleum origin, animal, vegetable or synthetic origin, such as Peanut oil, mineral oil, or sesame oil. In addition to the carrier, the inventive Preparations also contain other ingredients, such as stabilizers, Binders, antioxidants, preservatives, lubricants, suspending agents, Viscosity agents, flavorings and the like. Furthermore, in the preparations also contain other active ingredients to make a wider range of antibiotic to deliver effectiveness.

The dose to be administered depends largely on the condition of the patient to be treated Living being and the weight of the host. The parenteral route is used for general Infections and the oral route are preferred for intestinal infactins. In general a daily dose consists of about 15 to about 175 mg of active ingredient per kg Body weight of the living being in one or more applications per day. A preferred one daily dose for the antibiotic 810A or its individual components is in Range from about 20 to 40 mg of active ingredient per kg of body weight. The preferred one daily dose for the antibiotic 842A ranges from about 40 to 80 mg of active ingredient per kg body weight.

The present preparations can be used in various unit dosage forms, for example in solid or liquid, orally ingestible dosage form will.

The preparations contain the same per unit dose. whether liquid or solid, generally from about 15 mg to about 700 mg of active ingredient on the total weight of the preparations; however, it is generally preferred to use a dosage amount ranging from about 80 mg to 320 mg. With parenteral Administration, the unit dose is usually the neat compound in one sterile water solution or in the form of a soluble powder intended for dissolution is.

A typical unit dosage form consists of mixing 120 together 810E mg antibiotic or 12G mg of any of its ingredients or a salt with 20 mg lactose and 5 mg magnesium stearate and placing the 145 mg mixture in a Gelatin capsule # 3. Similarly, using more active Ingredient and less lactose other dosage forms in gelatin capsules No. 3, and should it be necessary, more than 145 mg of ingredients together To mix, larger capsules can be used. Similarly, you can other unit doses such as compressed tablets and pills as well getting produced. The following examples serve to illustrate: Dry-filled Capsule containing 120 mg of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (αmethoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3- (cephem-4-carboxylic acid (Ic) Each capsule contains 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-sulfooxycinnamoyl) -oxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (ic 120 mg lactose 20 mg magnesium stearate capsule size Nr X 145 mg Die 7ß- (D-5-Amino-5-carboxyvaleremido) -3- (α-methoxy-psulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (a: c) is passed through a sieve for a powder with a grain size crushed a sieve with 0.25 mm (No. 60) and then lactose and magnesium stearate Put on the powder through a sieve with a mesh size of 0.25 mm (No. 60), and the combined ingredients are mixed for 10 minutes and then placed in gelatin capsules No. 3 filled.

By using 40 mg of antibiotics 842A and 100 mg of lactose instead of the 120 mg of active ingredient and 20 mg of lactose used in the preceding preparation you get a 145 mg capsule, which is also for oral administration suitable is.

250 mg (D-5-Amino-5-carboxyval erami do) - 3- («-methoxy-psulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) containing tablet per tablet 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) 250 mg of dicalcium phosphate, U.S.P. 192 mg magnesium stearate 5 mg lactose, U.S.P. 65 mg The active ingredient is mixed with the dicalcium phosphate and the lactose. The mixture is granulated with 15% corn starch paste (6 mg) and. coarsely sieved. It is dried at 450 ° C. and again passed through a sieve with a mesh size of 1.2 mm (No. 16) sifted. The magnesium stearate is added and the mixture into tablets about 13 mm in diameter.

7β- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ib) containing tablet per tablet 7β- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ib) 125 mg corn starch, U.S.P. 6 mg dicalcium phosphate 192 mg lactose, U.S.P. 190 mg The active ingredient is made with the dicalcium phosphate and the lactose and about half of the cornstarch mixed together.

The mixture is then granulated with a 15% corn starch paste (6 mg) and coarsely sieved. It is dried at 45 ° C. and passed through 1.2 mm sieves Mesh size (No. 16) sifted. The rest of the cornstarch and that Magnesia turustearate are added and the mixture becomes tablets with a Diameter of about 13 mm, each weighing 800 mg, pressed.

500 mg of 7β- (D- 5-amino-5-carboxyval erami do) -3- (α-methoxy-psulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid Parenteral solution containing (Ic) ampoule: 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) 500 mg ampoule: Diluent: Sterile water for 3 injections 2 cm The 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-psulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) can be used alone or in combination with other biologically active ingredients administered, for example with other antibacterial agents such as Lincomycin, a penicillin, streptomycin, novobiocin, gentamicin, neomycin, golistin and kanamycin.

by using an equivalent amount of 7β- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3 cephem-4-carboxylic acid (Ib) instead of the 500 mg amino-5-carboxyvaleramino) -3- (α-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) in the foregoing preparation can also be one for parenteral administration suitable preparation can be obtained.

Synthetic Methods Except for the fermentation products described above the invention includes derivatives in which the 3-carbamoyloxy- proportion of of 842A is replaced with a wide variety of substituents. In general is that replacement or substitution done by simply treating 842A with a reagent that is used to convert the 3Carbaiiioyloxy fraction into the desired Substituents R (see formula I) is capable. However, it is common in practice necessary, before replacing the 3-carbamoyloxy radical, the free amino and carboxy groups by treating 842A with a nitrogen blocking agent such as an N-lower alkoxycarbonylphthalimide and an esterifying agent to form of the corresponding 7β- (D-5-phthaloxylamino-5-carboxyvaleramino) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid diester (following compound II) to protect.

Suitable esterifying agents include phenyldiazomethane or diphenyldiazomethane and the like. Also, other nitrogen blocking agents usable in place of the N-lower alkoxycarbonylphthalimide include tertiary butyloxyazide or trihaloethoxycarbonyl halides such as trichloroethoxycarbonyl chloride and the like -Alkoxy carbonylphthalimide explains this reaction, but it should be noted that other nitrogen blocking agents, for example those mentioned above, can be used instead in an otherwise identical reaction to obtain the corresponding N-substituted derivative: wherein R¹³R14C = N2 is diazomethane or an aryl-substituted diazomethane such as phenyldiazomethane or diphenyldiazomethane and the like, and 23 and p14 are hydrogen or phenyl radicals The compound (II) thus obtained is the starting material in the following synthetic processes.

The 3-hydroxymethyl analog of 842A is obtained by treating Compound II with a nitrosyl halide, e.g. nitrosyl chloride, in a suitable solvent such as methylene chloride. The following equation explains this manufacturing process: in which R13 and R14 have the meaning given above. The diester thus obtained, preferably the di-benzyl ester (IIIa), can then be converted into the corresponding free amino acid (IV) by catalytic hydrogenation and treatment with hydrazine hydrate in a basic solution. According to this method, deblocking is most expedient when the diester reactant is the dibenzyl ester (IIIa). The following equation explains this manufacturing process: where N is the cation derived from an alkali or alkaline earth metal.

The N-mono-substituted and N, N, -disubstituted 3-carbamoyloxy analogs of 842A are easily obtained by treating the diester of 7ß- (D-5-phthaloylamino-5-carboxyval erami do) - 3- (hydroxymethyl) - 7-m ethoxy-3-c ephem-4-carbonic acid, preferably in the form of its dibenzhydryl ester (IIIb), with a carbonyl halide such as carbonyl chloride (i.e., phosgene) or carbonyl bromide, and then reacting it in this way obtained dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (haloformyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (V) with the corresponding mono-lower alkylamine, di-lower alkylamine or heterocyclic amine obtained: where COX2 is a carbonyl halide, for example carbonyl chloride (ie phosgene) or carbonyl bromide and 1 is a halogen radical, for example chlorine, bromine and the like R15 and R16 are lower alkyl radicals, such as, for example, methyl, ethyl, n-propyl, n-butyl radicals and the like, and R13 and R14, taken together with the nitrogen atom to which they are bonded, form a mononuclear, heterocyclic amine from pyrrolidine , Piperidine and / or morpholine can be combined. The product (vi) thus obtained can then be converted into the corresponding free amino acid by treatment with trifluoroacetic acid in xylene and then with hydrazine hydrate in a basic. Solution to be transferred: wherein M and R15 and R16 are as defined above.

Another method for making the N-monosubstituted derivatives of 842A is the treatment of compound III or a disalt thereof with a suitable isocyanate.

This reaction is particularly advantageous in the presence of a strong organic base, for example triethylamine and in the presence of an inert solvent, e.g. B. dimethylformamide, methylene chloride, tetrahydrofuran or acetonitrile, carried out: wherein R17 is a lower alkyl radical, halogen-substituted lower alkyl radical, e.g. B. chloromethyl, 2-chloroethyl or chloro-tert-butyl radical and the like., Lower alkoxycarbonyl radical, for example an ethoxycarbonyl radical and the like., Mononuclear and binuclear aryl radical, e.g. B. phenyl, naphthyl and the like., Mononuclear alkarylsulfonyl, z. B. p-Tolylsulfonylrest and the like., Or a Benzhydrylrest means. Typical isocyanate reactants (ie OC-NR17) that can be used are, for example, methyl isocyanate, ethyl isocyanate, tert-butyl isocyanate, chloromethyl isocyanate, β-chloroethyl isocyanate, carbethoxy isocyanate, phenyl isocyanate and benzhydryl isocyanate. The products obtained by this process can then be converted into the corresponding free acid by the method described above, de h. be converted into basic solution by treatment with trifluoroacetic acid in xylene and subsequent reaction of the intermediate product obtained with hydrazine hydrate.

The 3-acylated derivatives of the invention correspond to the formula I, in which R is a lower alkanoyloxy radical, aromatic carbonyloxy radical, aralknoyloxy radical or cycloalkanecarbonyloxy radical, are easily obtained by treating the dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvalve) - (Hydroxymethyl) -7-nethoxy-3-cephem-4-carboxylic acid (IIIb) obtained with a suitable acyl halide. The following equation explains this manufacturing process: wherein R18 is a lower alkyl radical, for example a methyl, ethyl radical and the like, mononclear and bimlkl.earen aryl radical, for example a phenyl radical, a monocyclic, nitrogen-containing heterocycle, such as, for example, 4-pyridyl radical and the like, naphthyl radical and the like, mononuclear and bi -nuclear lower alicyl radical, such as benzyl-l menaphthyl radical and the like., Or a cycloalkyl radical with 5 to 6 core carbon atoms, e.g. B. Cyclopentyl or cyclohexyl radical and the like., And 1 has the meaning given above. The product thus obtained can in turn be converted into the corresponding free acid by treatment with trifluoroacetic acid in xylene and subsequent reaction of the intermediate product obtained with hydrazine hydrate in a basic solution.

The 3-methyl analogs of 842A are obtained in a simple manner by reducing 842A or a salt thereof, for example by catalytic hydrogenation of the corresponding alkali or alkaline earth metal salt. Catalysts that can be used in this process include, for example, any Group VIII metal of the Periodic Table, such as palladium, platinum or nickel. The following equation explains this manufacturing process: 3 HOOC-CH- (CEI) (CHz) 3L! ' BOOC-Cii- (C112) NH If -r, 'CH 20 CNH2 COOH Such derivatives of 842A that are not prepared by the foregoing methods can be prepared by treating a 3-acyloxymethyl analog of 842A, for example the 3-lower alkanoyloxymethyl analog, e.g. B. 7ß- (D-5-amino-5-carboxyval erami do) "3- (ac etoxym ethyl) - 7-methoxy- 3- c ephem-4 + carboxylic acid, with the appropriate thiourea or N-substituted thiourea, triol , Dithiocarbamate, dithiocarboxylate, amine, pyridine, ring-substituted pyridine, alkali metal sulfinate, azide, polyhydroxybenzene or N-lower alkylindole. The resulting products can then be obtained by conventional means, e.g. by recrystallization from suitable solvents such as methanol and Water or by fractionation through a suitable ion exchange resin.

It was found that in addition to the 3-acyloxymethyl analog of 842A the 842A per se as a starting material in the reaction with thiourea and alkali azides, etc. can be used. For example, the 842A can be used with the suitable thiourea, N-substituted thiourea, N, N-disubstituted thiourea, Alkalizid, thiazole, thiadiazole, halogenating agents or alkali thiocyanate among Formation of the corresponding 3-thiouroniummethyl-, 3-azidomethyl-, 3-thiazolylmercaptomethyl-, 3-thiadiazolyl mercaptomethyl, halomethyl, and 3-thiocyanatomethyl analogs of 842A. In principle it is only necessary, i.e. the starting materials to combine to bring about synthesis, however in practice it is common expedient, di.e reaction by the application of heat, for example by heating to temperatures in the range of about 45 to 950 C for a period of about 2.5 days to a few minutes to catalyze. A preferred working temperature is in the range from about 75 to 800 C, and. good results were also obtained after heating the reactant received at 950 C for about 8 minutes. In some cases it is also appropriate to use the free amino group in the 5-amino-5-carboxyvaleramido side chain Protect the 842A by using the latter with tert-butoxycarbonylazide or a similar one Blocking agent is treated. The 7β- (D-5-N-tert-butoxycarbonylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained can then with the corresponding thiourea alkali azide, thiazole, Thiadiazole, Halogenating agents or thiocyanate reactants can be treated to the desired Sub £; tituents in the 3-position of the 842A core without the risk of any incongruous To introduce side reactins. The intermediate product thus obtained can then by treatment with a suitable reagent, such as, for example, Trifluores.sgsaure, to achieve of the desired product can be unblocked.

The 3-azidomethyl derivative obtained by the foregoing process vou 842A is converted into its corresponding 3-aminomethyl analogue by reduction. Suitable agents include, for example, molecular reduction and hydrogenation over a suitable metal catalyst such as the metals of the group VIII of the Periodic Table. Typical metals that can be used include, for example Platinum, palladium and nickel and their combinations.

The molecular reduction of the 3-azidomethyl derivative is most favorable carried out by adding zinc dust to an acidic solution of the 3-azidomethyl reactive agent is admitted.

The resulting solution is then treated with hydrogen sulphide or a mixture equivalent material for removing the soluble zinc as the solution in the form treated with a precipitate.

The product obtained in this way is the practically pure 3-aminomethyl analogue from 842A.

The products (I) according to the invention form a large number pharmacologically compatible salts with inorganic and organic bases; this includes, for example Metal salts, such as B. those that differ from alkali and alkaline earth metal hydrides, carbonates and derived from bicarbonates and salts that differ from primary, secondary and tertiary Derive amines, such as monoalkylamines, dialkylamines, trialkylamines, lower alkanolamines, di-lower alkanolamines, lower Alkylenediamines, N, N-diaralkyl-lower alkylenediamines, aralkylamines, amino-substituted lower alkanols, N, N-di-lower alkylamino-substituted lower alkanols, amino-, polyamino- and guanidino-substituted ones lower alkanoic acids and nitrogen-containing heterocyclic amines. To typical examples These salts include salts that are made up of sodium hydroxide, sodium carbonate, sodium bicarbonate, Derive potassium carbonate, potassium hydroxide, calcium carbonate and the like and d salts, which differ from amines, such as trimethylamine, triethylamine, piperidine, Morpholine, quinine, lysine, protamine, arginine, procaine, ethanolamine, morphine, benzylamine, Ethylenediamine, N, N'-dibenzylethylenediamine, diethanolamine, piperazine, dimethylaminoethanol, 2-amino-2-methyl-1-propanol, theophylline and N-methylglucamine and the like. To derive.

The above-mentioned salts can be monosalts such as, for example the monosodium salt, which can be obtained, for example, by treating one equivalent of sodium hydroxide with one equivalent of the product (I), or mixed disalts which d'jrch treatment of an equivalent of the monosalt with an equivalent of a different one Base.

These disalts can also be obtained by adding an equivalent of one Base with a divalent cation such as calcium hydroxide with a Equivalent of the product (1) is treated. There are also mixed salts and esters. such as those obtained by treating the product (I) with an equivalent Sodium hydroxide and then lactic acid equivalent can be obtained The salts according to the invention are pharmacologically acceptable, non-toxic Derivatives which are used as the active ingredient in suitable pharmaceutical unit doses Formation forms can be used. You can also use other medicines can be combined to produce preparations with a broad spectrum of activity. Further are the according to the invention Salts and also the corresponding ones Esters and amide derivatives useful as intermediates for the preparation of the by the formula I reproduced carboxylic acid product. The salts can also be used for manufacture other pharmaceutically acceptable salts can be used.

In addition to salts, the products (I) according to the invention can also be used in their corresponding mono- and diesters and mono- and diamides are converted as for example the pivaloyloxymethyl or dibenzhydryl esters or alkyl, cycloalkyl, Aryl or aralkyl esters, such as, for example, the methyl, ethyl, cyclohexyl, phenyl and benzyl esters or amides, diamides, N-lower alkylamides, N, N-di-lower alkylamides, N-aralkylamides, N, N-diaralkylamides or heterocyclic amides, such as N-methyl- and N-ethylanide, N, N-dinethylamide, N, N-diethylamide, N-benzylamide, N, N-dibenzylamide, Piperidide, pyrrolidide or morpholide and the like.

On methods of making the above-mentioned esters and amide derivatives include the reaction of the carboxylic acid product (I) or a corresponding acid halide with methanol, ethanol, cyclohexanol, phenol, benzyl alcohol or dibenzhydrol. In Similarly, the amide derivatives can be obtained by adding the appropriate Acid halide with.

Amnionia or with the corresponding alkylamine, dialkylamine, aralkylamine or heterocyclic amine is treated. These and other common methods for The person skilled in the art is familiar with the production of these esters and amides.

The following examples explain the processes by which the inventive Products can be obtained. However, the examples are only intended to illustrate and it is clear to the person skilled in the art that other functionally equivalent of the invention Products and processes for their manufacture are included. Hence each is Modification of this Syntheses leading to the formation of an identical Product leads to be regarded as an analogous method. The claimed method is unsuccessful extensive variation and modification, and therefore any slight deviation or extension is considered to be within the scope of the invention.

EXAMPLE 1 Antibiotic 810A A lyophilized test tube with Streptomyces griseus culture (MA-2837) was opened aseptically. The content was for inoculating four slant cultures of a nutrient medium with the following composition used: Medium I: Difco yeast extract 10.0 g glucose 1P, o g * phosphate buffer 2.0 ml MgSO4. 7H2O 0.05 g distilled water 1000.0 ml Difco agar 25.0 g * phosphate buffer KH2PO4 91.0 g Na2HPO4 95.0 g Distilled water 1000.0 ml The slants were prepared by distributing 14 ml / 22 x 75 mm culture test tube. The test tube was clogged with cotton wool, heated to 1200 C for 15 minutes for sterilization, and the medium was allowed to solidify in an oblique arrangement. The inoculated slants were incubated at 280 ° C for 1 month and then stored at 40 ° C until used. The culture on one of these slants was then used for inoculating with subdivisions provided 250 ml Erlenmeyer flask used the 50 ml medium TI contained the culture slant by adding 5 ml of sterile medium was scraped to stop growth and put 1 ml in each of the three germinating flasks pipetted aseptically. Medium II had the following composition: Medium II: Beef extract 3.0 g * NZ amine 10.0 g dextrose 10.0 g NaCl 5.0 g distilled Water 1000.0 ml pH adjusted to 7.2 with NaOH * An enzymatically degraded casein The germinal flask was on a rotary shaker at 220 rpm with a stroke of Shaken 5 cm for 3 days. The sprout culture was then used to inoculate 11 with Divided 2 liter Erlenmeyer flasks used containing 350 ml of medium III, using 2 to 3% inoculum. Medium III owned the following composition: Medium II: dextrose 10.0 g asparagine 190 g K2HPO4 0.1 g MgSO4. 7H2O 0.5 g yeast extract 0.5 g * trace element mixture No. 2 10.0 ml distilled Water 1000.0 ml pEf adjusted to 7.2 with NaOH * Mixture of trace elements No. 2: FeSO4. 7H2O 1.0 g MnSo4. H2O 1.0 g CuCl2. 2H2O 25.0 mg CaCl2 100.0 mg according to BO 56.0 mg (NH4) 6MO7O24 4H20 19.0 mg ZnSO4. 7H2O 200.0 mg distilled water 1000.0 ml The flasks were then on a shaker at 135 'to 150 rpm with a Stroke of 5 cm shaken at 280 ° C. for 4 days.

At the end of the incubation period, the contents of the 11 flasks united and examined. Examination of the combined, centrifuged broth showed a zone of inhibition of 22 mm (13 mm disks) against Proteus vulgaris in a standard test plate. This antibiotic was identified as 810A, d. H. a genic of antibiotics, the 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-sulfooxycinnamoyloxymethyl) -6-methoxy-3-cephem-4-carboxylic acid and 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ia) contains.

EXAMPLE 2 Antibiotic 810A A lycphilized reagent tube Streptomyces griseus $ (MA-2837) was aseptically opened and the contents were for inoculating the nutrient medium slant cultures described as medium I in example 1 used. These slant cultures were incubated at 280 ° C. for 1 week, after which they were stored at 40 C. Part of the culture was based on one of these slant cultures then to inoculate one with subdivisions equipped 250 ml vaccine Erlenmeyer flask, containing 50 ml of the medium described as medium II in Example 1 was used. This inoculated medium was at 280 C for 2 days on a rotary shaker with a Incubated stroke of 5 cm at 220 rpm. The inoculum was then aseptically centrifuged down of the mycelium washed, the supernatant poured off, in an equal Volume of saline solution resuspended, recentrifuged and again in a 0.9% igen Sodium chloride solution resuspended. The washed mycelium was then used for inoculation (2 O / o inoculum) from two graduated 250 ml Erlenmeyer flasks, each containing 50 ml of a chemically determined production medium that is used in 1200 C had been sterilized for 15 minutes. The chemically defined production medium had the following composition: Production medium: L-proline 15.0 g glycerol 20.0 g sucrose 2.5 g monosodium glutamate 1.5 g NaCl 5.0 g K2HPO4 2.0 g CaCl2 0.4 g MnCl2. 4H2O 0.1 g FeCl3. 6H2O 0.1 g ZnC12 0.05 g MgSO4. 7H2O 1.0 g distilled Water 1000.0 ml pH (not adjusted) 7.1 The production flasks were then at Shaken 220 rpm on a shaker with a stroke of 5 cm at 280 ° C. for 4 days.

Experiments were carried out at 3 and 4 days. rehearse became centrifuged and the supernatants by the disk petri dish method examined. Using 13 mm disks, these broths gave zones of inhibition against Proteus vulgaris (MB-838) of 21 mm after 3 days and 26 mm after 4 days. That Product identified as antibiotic 810A.

EXAMPLE 3 Antibiotic 810A in lyophilized test tube with Streptomyces griseus (MA-4125a) was opened aseptically and the contents in slants of the following composition: Medium IV: V8 juice 100 ml Staley's 4S soybean meal 20.0 g dextrose 2.0 g agar 25.0 g distilled water up to 1000.0 ml pH 7.9 up to 8.0 The slant cultures thus obtained were then used for inoculating various 250 ml Erlenmeyer flasks each containing 50 ml of the following medium V were used.

Medium V: yeast autolysate (Ardamin) 10.0 g glucose 10.0 g phosphate buffer 2.0 ml MgS04 71120 0.05 g Distilled Water 1000.0 ml pH using adjusted to 6.5 by NaOH.

Phosphate buffer solution: KH2PO4 91.0 g Na2HPO4 95.0 g distilled Water 1000.0 ml The germinating flasks were shaken at 280 ° C. and 220 rpm for 1 day.

The contents of the flasks were then inoculated into 39 250 ml Erlanmeyer flasks without subdivisions containing 40 ml of Me Medium VI with 3.5 ml inoculum used per piston.

Medium VI: Corn steep liquor (wet base) 40.0 g dextrose 20.0 g NaCl, 2.5 g MgSO4. 7H2O 0.5 g polyglycol 2000 0.25 vol% (for each flask individually added) Distilled water 1000.0 ml pH adjusted to 7.0 with NaOH.

The production flasks were on a rotary shaker with a Stroke of 5 cm at 220 rpm and shaken at 240 C for LCO hours, after which the Flasks were pooled, an aliquot was withdrawn for examination and the remainder was used for extraction studies. The sample was examined acidified to pH 4.0 using hydrochloric acid, filtered, on Diluted 1: 4 in phosphate buffer with pH 5.0 and put on Proteus vulgaris MB-838 plates brought using 13 mm discs. The zone of inhibition was 26.5 mm. The product was identified as antibiotic 810A but predominantly from 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-psulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid with only trace amounts of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-hydroxycinnamoyloxymethyl) -7-methoxy 3-cephem-4-carboxylic acid.

EXAMPLE 4 Antibiotic 810A A V8 medium slant of the Streptomyces griseus culture (MA-4125A) was used to inoculate 50 ml of medium V in a graduated 250 ml Erlenmeyer flask was used.

Medium V: yeast autolysate (Ardamin) 10.0 g glucose 10.0 g * phosphate buffer 2.0 ml MgSO4 7H20 0.05 g distilled water 1000.0 ml pH with NaOH to 6.5 set phosphate buffer: KH2PO4 91.0 g Na2HPO4 95.0 g distilled water 1000.0 ml. The flask was then placed on a rotary shaker at 220 rpm for 1 day at 280 C shaken. 3 ml of this vegetative inocolum were used to inoculate a sprout, the 50 ml of the following synthetic medium (Medium VII) in one with partitions equipped 250 ml Erlenmeyr flask was used.

Medium VII: germination production L-asparagine 5.0 g 5, O g L-hi stidine 4.0 g DL-phenylalanine - 2.0 g monosodium glutamate - 1.5 g NaCl 5.0 g 5.0 g K2HPO4 2.0 g 2.0 g CaCl2. 2E2 ° 0.4 g 0.4 g MnSO4. H2O 0.1 g 0.1 g FeSO4. 7H2O 0.1 g 0.1 g ZnSO4. 7H2O 0.05 g 0.05 g MgSO4. 7H2O 1.0 g 1.0 g glycerin 20.0 g 20.0 g sucrose 2.5 g 2.5 g distilled water * 1000.0 ml ** 1000.0 ml * pH with NaOH adjusted to 7.0. ** pH adjusted to 7.1 with NaOH shaken again for 1 day at 280 ° C. and 220 rpm. This synthetic germination medium was made then for inoculating different 250 ml Erlenmeyer flasks without subdivision, which contained 38 ml of the production medium VII 'with 1.5 ml inoculum per flask was used. The production flasks were shaken at 220 rpm and 240 C and their contents were then pooled and examined at 4 and 4 days of aging. In the The whole broth was tested to pH 4.0 using hydrochloric acid acidified, and the broth was then filtered and diluted to i: 4 in phosphate buffer from pH 5.0 diluted. Testing was done on Proteus vulgaris using MB-838 of 13 mm discs. There became a zone of inhibition of 25.5 mm for this fermentation broth obtained after 4 days of incubation, and the product thus obtained became identified as 810A.

EXAMPLE 5 Preparation of Antibiotic 810A and Separation into its components stage A: fermentation stage 1; The contents of a lyophilized Tube with Streptomyces griseus (MA-2837) was placed in 2 ml medium I (in example 1), and the resulting inoculum was used for inoculating slants of the same medium is used. These slants were at 280 C for 5 days or until well sporulated, and then 10 ml of Medium VIII added to the slants.

Medium VIII: meat extract 0.3 O / o NaCl 0.5% NZ amine 1% dextrose 1% - pH 7.0 The growth on each slant culture was scratched in suspension, and the suspension was used as an inoculum in Step 2 below.

Stage 2: The suspension obtained in Stage 1 was used for inoculation a graduated 250 ml Erlenmeyer flask that sterilized 50 ml Medium VIII (described in step 1) was used. The inoculated flask was then placed on a rotary shaker at 220 rpm and incubated at 280 ° C. for 48 hours.

Stage 3: The contents of an inoculation flask from Stage 2 were used for inoculation a graduated 2 liter Erlenmeyer flask used, the 500 ml of the medium identified as Medium VIII in Stage 1. The inoculated Flask was then placed on a rotary shaker at 220 rpm and 48 hours incubated at 280 ° C.

Stage 4: An inoculum made from 500 ml of the growth obtained from Stage 3 was used to inoculate a 750 liter fermentation vessel made of stainless steel, containing 467 liters of sterile medium VIII (described in step 1) was used.

The fermentation was left at a temperature of 28 ° C with agitation (130 rpm) while an air flow of 280 31 (10 cfm) was carried out for 65 hours was retained.

During the fermentation an anti-foaming agent, Polglykol 2000, added in small amounts to prevent excessive foaming.

Stage 5: An inoculum of 380 liters (10Q gallons) of the growth obtained Step 4 became the inoculation of a 5700 liter (1500 gallon) fermentation vessel stainless steel containing 4500 liters (1200 gallons) of the following Medium IX, used.

Medium IX: Corn steep liquor 4% dextrose 2 0c / pH-value with NaOH adjusted to 7.2 The fermentatin was left at a temperature of 280.degree Movement (120 rpm) proceed while an airflow of 1580 ì w55.3 cfm) during Was maintained for 30 to 36 hours. During fermentation, polyglycol became 2000 added in small amounts to prevent excessive foaming. The approach was won and the effectiveness by the disc-plate test certainly. Using 13 mm (0.5 inch) disks, this broth made one Zone of inhibition of 32.5 mm against Proteus vulgaris MB-838. if obtained after 31 hours Aging.

Step B: Isolation of Antibiotic Mixture 810A Filtered Broth 4050 liters (1075 gallons) from Level A, Level 5 was recovered after 36 hours and the pH value in the fermentation vessel from 7 to 8 by adding phosphoric acid set to 3.0. The mycelium was passed through a filter press from Plate screen type removed and discarded. The filtered broth was then passed through a 380 l (100 gallon) bed of Amberlite XAD-2 adsorbent resin at one flow rate of 38 liters (10 gallons) per minute. The used broth was examined and washed the resin bed discarded with 2 volumes of water. The antibiotic was taken from the resin bed with a 60% solution of methanol and water at a flow rate eluted at 19 liters (5 gallons) per minute. 40 fractions of 19 1 (5 gallons) each were collected and examined.

Fractions 2 to 40 were combined and the methanol by evaporation removed in vacuo. The final concentrate (158 1, 4s, 5 gallons) was made by adding Ammonium hydroxide adjusted to pE 3.5 and kept frozen.

Samples were tested against Proteus vulgaris using the disk and plate method biologically examined.

Filtered Broth: Performed on 4000 liters (1060 gallons) of filtered broth Tests showed the following zone diameters: Filtered broth Dilution Zone size 1: 2 26.8 mm 1: 4 23.8 mm 1: 8 21.1 mm Used broth and Wash Solution: 10 fractions of 380 l (100 gallons) each gave upon examination Zero without dilution. The washing water tested gave zero.

Eluate Fractins: All fractions were examined.

The zone diameters are listed in the following table: E. Eluate fractions Fraction Zone size Fraction Zone size 1 0 21 33 mm 2 28 mm 22 33 3 35 23 34 4 3LL. 24 34 5 36 25 33 6 36 26 34 7 36 27 32 8 38 28 33 9 38 29 32 10 36 30 32 11 36 31 32 12 38 32 30 13 40 33 30 14 37 34 30 15 36 35 28 16 37 36 27 17 38 37 28 18 36 38 26 19 36 39 26 20 35 40 26 Eluate composition and Eluate Concentrate: Tests were also carried out on 740 liters (195 gallons) of eluate composition and 158 liters (41.5 gallons) of Antibiotic 810A in the form of concentrate eluate.

Eluate composition: Eluate concentrate 810A dilution zone size dilution Zone size 1: 5 28.8 mm 1: 16 27.25 mm 1: 10 27.0 mm 1: 32 24.5 mm 1: 20 23.8 mm 1: 40 21.0 mm total solids test: filtered broth 119 kg 740 S (195 gallons) eluant composition 7.23 kg 158 1 (41.5 gallons) elust concentrate 7.20 kg Stage C: Adsorptin on an anion exchange resin The concentrate on stage B (78.5 liters, 20.7 gallons) was diluted to 118 liters (31 gallons) with water and turned on a 22.5 L "bed of weakly basic anion exchange resin (Ainb suffered IRA-68 resin, Chloride cycle) adsorbed at pH 4.0 and a flow of 7.6 liters (2 gallons) per minute. This was followed by washing with 45 l of water, after which the resin bed with a solution from 1 m-S sodium nitrate and 0.1 m-sodium acetate at pH 7.5 and a flow rate of 1.5 1 / min.

was eluted. Ten 19 l (5 gallons) eluate fractions were then added collected and the pH adjusted to pH 4 with the hydrochloric acid collected.

All fractions were countered by the disk-plate method Proteus vulgaris examined biologically as follows: Loading solution Eluate fractions, dilution 1:10 dilution zone size fraction zone fraction Zone size large 1: 10 28.5 mm 1 27 mm 6 25 mm 1: 20 26.5 mm 2 30 mm 7 23 mm 1: 40 24 mm 3 28.5 mm 8 .22 mm 4 26 mm 9 21 mm 5 26 mm 10 17.5 mm The used After examination, electricity gave 25 mm without dilution and the wash water gave up Examination 23 mm without dilution.

Stage D: Adsorotion on Anion Exchange Resin Fractions 1 to 10 from Stage C were combined and a 45 liter bed of Amberlite XAD-2 adsorbent at pH 3.0 and a flow rate of 5 1 / min. fed. The resin bed was washed with 90 liters of water at the same speed. The antibiotic was then made from the resin by a 25% solution of acetone and water at a Flow rate of 5 1 / min. eluted.

Sixteen 19 l (5 gallons) fractions were collected.

All fractions were countered by the disk-plate method Proteus vulgaris was examined as follows: The charge (190 liters) gave the following Zone diameter: feed solution dilution zone size 1: 5 30 mm 1: 10 27.5 to 1:20 24.2 mm The zone diameters of the eluate fractions were summarized in the following table: eluate fraction eluate fraction fraction Dilution zone size Fraction Dilution zone size a 0: 10 20a5 mm 9 1: 10 25mm 2 1:: 10 29mm 10 1: 10 26.5mm 3 0: 10 29mm 11 1: 5 26mm 4 1: 10 29 mm 12 1: 5 28 mm 5 0: 10 28 mm 13 1: 5 27.5 mm 6 1: 10 27 mm 14 1: 5 25 mm 7 1: 10 26 mm 15 1: 5 25 mm 8 1: 10 26 mm 16 1: 5 24.5 mm The above eluate fractins 2 to 16 were combined and the acetone by evaporation in vacuo to final volume removed from 17.4 liters. The 17.4 liter concentrate was replaced by ammonium hydroxide adjusted to a pH of 4.0 and freeze-dried, with- 620 g antibiotic 810A, i.e. H.

a mixture consisting essentially of 7β- (D-5-amino-5-carboxyvaleramido) -3 (α-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid and 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid was obtained.

This dry product had biological effectiveness Examination of 320 mcgXml for a 25 mm zone.

Step E: 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxyp-hydroxycinnamoyl oxymethyl) -7-methoxy- 3- c ephem-4-carboxylic acid A chromatography column of 2.5 cm in diameter was measured at a bed height of 100 cm with DEAE Sephadex A-25 anion exchange resin packed in a system containing 0.5 m ammonium bromide and 0.05 m acetic acid. This in Stage D obtained mixture of antibiotic 810A (10.0 g) was dissolved in 18 ml of a solution of 0.5 M ammonium bromide and 0.05 M acetic acid and introduced into the column.

The eluting solution was passed through the bed at a rate of 81 ml / h was pumped, and 10 ml fractions of the eluate were collected by machine. The flow of eluate was monitored by a differential refractometer. The refractometer record showed mass peaks at tubes 19, 36, 79, 109 and 206. Disc-plate experiments against. Proteus vulgaris (MB-838) were used on every third fraction performed on 13 mm disks buffered at pH 7.0. The zone diameters are listed in the following table: (Fractions 1 to 66 resulted in the investigation zero).

Fraction Zone Fraction Zone Fraction Zone diameter knife knife 69 18 mm 122 29 204 40 + 72 24 n25 28 207 40 + 75 N 128 27 210 40 + 78 y 131 26 213 40 + 81 - 124 24 216 40 + 83 35 137 21 219 40 86 37 140 20 222 38 89 38 150 18 225 35 92 38 160 20 228 32 95 40 + 170 29 231 31 98 40 +, 180 35 234 27 101 40 + 183 38 237 24 - 104 40 + 186 40 240 23 n07 40 + 189 40 + 243 19 110 40 + 192 40 + 246 17 113 m 195 40 + 249 0 116 38 198 40 + 252 0 119 33 201 40 + Fractions 80 to 133 were combined and fractions 170 to 230 became combined.

The above experiment was repeated and the fractions 82 to 130 were combined and fractions 180 to 234 were combined.

The fractions containing the first active component from the above Experiments were combined and adsorbed onto a 100 ml bed of Amberlite XAD-2 resin. The bed was washed with 1 volume of water and then with 3 volumes of 90% Solution eluted from methanol and water. The methanol was removed by evaporation in vacuo removed, and the aqueous concentrate was freeze-dried, leaving 810 ng of one Product obtained as 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid was identified. The biological effectiveness of this product is determined by the disk flattening method against Proteus vulgaris was 18 μg / ml and resulted in a 25 mn zone. Analysis based on ultraviolet adsorption indicated the following Characteristic data: UV adsorptin in 0.1 n-HCl # max 305 E1cm% 524 UV adsorption in 0.1 N NaOH # max 328 E1cm% 564 Level F: 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxyps s ul foxycinnamoylomethyl) - 7-m ethoxy- 3- c ephem-4-carboxylic acid The fractions from the two trials on Sephadex A-25, which is the second active component were combined and adsorbed onto a 100 bed of Amberlite XD-2 resin. The bed was washed with 1 volume of water and then with 3 volumes of 90% Solution eluted from methanol and water. The enriched eluates were combined, and the methanol was removed by evaporation in vacuo.

The aqueous concentrate was freeze-dried to give 720 mg of 7β- (D-5-amino-5-carboxiyvaleramido) -3- (α: -methoxy-psulfooxycinnamoyloxymethyl ) -7-methoxy-3-cephem-4-carboxylic acid (Ic). Analysis based on ultraviolet absorption gave the following characteristics: UV adsorption in 0.1 n-HCl #max 287 mm E1cm% 432 UV adsorption in 0.1 n-NaOH #max 280 mm E1cm% 432 Example 6 Separation of the 7ß- (D- 5-Lmino-5-carb oxyval eraini do) - 3 "(amethoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid from the antibiotic mixture 810A 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) and 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-phydroxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid containing antibiotic mixture 810A (20.0 g) from Example 5, stage D, was in 250 ml of water were dissolved and the pH value of the solution was adjusted to 3.5. This solution was through a 200 ml bed of Amberlite IRA-68 anion exchange resin (chloride cycle) given and then washed by adding 300 ml of water. The bed was then eluted with 1 liter of 1% (v / v) formic acid in water. Then were added two portions of dilute hydrochloric acid (pH 0595). The loading solution, Spent solution and washing solution and eluates 1, 2 and 3 were determined by paper electrophoresis at pH 4.0, carried out for 1 hour at 1000 volts. Direct current, analyzed. The paper chromatogram was dried, exposed to ammonia vapor around the Neutralize acid and place on a nutrient agar plate filled with Proteus vulgaris (MB-838) was incubated.

Examination after 17 hours of incubation at 37 ° C showed two Zones of inhibition in the loading material (in the direction of the anode), only a single component (the slower of the two) in the consumed Solution and the formic acid eluate and a single component in the second hydrochloric acid eluate, that corresponded to the faster component in the feed. The following table shows the total solids and the biological test data: mass volume total biological products loading units 20 g 200 ml 60,000 units Ia and Ib Used solution and washing solution 11.15 g 500 ml 7 500 n Ib formic acid eluate 4.52 g 1000 ml 20,000 U Ib first hydrochloric acid eluate --- 500 ml 1,000 second Hydrochloric acid eluate 2.10 g 500 ml 10,000 "Ia These fractions were by Adsorption on Amberlite XAD-2 resin recovered to 7β- (D-5-amino-5-carboxyvaleramino) -3- (α-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic column (Ic) abzatre: mlen, and this was replaced by a 50% solution of methanol and water eluted to give substantially pure product (Ic).

Example 7 Separation of 7β (D- 5-amino- - carb oxyval erami do) - 3- (α-m ethoxyp-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid from the antibiotic 810A The antibiotic 810A mixture of 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) and? Ss- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-hydroxycinnamoyloxymethyl) - -7-methoxy-3-cephem-4-carboxylic acid (5.0 g), which was obtained according to Example 5s Stage D, was in 20 ml of a 20% strength Solution of acetone and water dissolved and the pH adjusted to 4.0. This solution was placed on a bed of Amerlite XAD-2 adsorbent measuring 5 cm in diameter by 100 cm in height given in 20% ketone and water solution. A solution of 20% acetone and water was pumped through the bed at a rate of 880 ml / h, and 20 ml practicals were collected automatically.

Disk-Platte'nunt inquiries against Proteus vulgaris (MB-838) were carried out on every third fraction using 6mm disks. The zone diameter are listed in the following table: Fraction Zone Fraction Zone fraction zone diameter diameter diameter 1-41 0 131 0 221 18 44 12 mm 134 0 224 18 47 22 137 0 227 18 5 ° 25 140 8 - 230 17 53 39 143 11 233 17 56 31 146 13 236 15 59 35 149 13 239 15 62 3 ° 152 15 242 15 65 28 155 15 245 15 68 27 158 16 248 14 71 25 161 17 251 14 74 24 164 18 254 14 77 21 167 17 257 13 80 20 170 18 260 13 83 19 173 20 263 12 86 16 176 21 266 12 89 14 179 21 269 12 92 13 182 21 272 11 95 13 485 21 275 11 98 13 188 22 278 10 101 13 191 23 281 10 104 14 194 23 284 9 107 13 197 23 287 8 110 15 200 22 290 0 113 11 203 24 293 0 116 10 206 24 296 0 119 9 209 24 299 0 122 8 212 24 302 0 125 8 215 24 128 8 218 19 330 0 The fractions 44 to 90 were combined, acetone was evaporated in the The vacuum was removed and the aqueous concentrate was freezegetrockn.et., 3.3 g of crude 7β- (D-5-amino-5-carboxyvaleramido) -3- (a-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) were obtained.

Fractions 150-225 were combined to give similar Treatment 700 mg of 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

A repetition of the above experiment gave 3.1 g of crude 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) and 400 mg of 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxyp-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

The two amounts of 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) obtained by the foregoing method were combined, and the 6.4 g of material was placed in a bed of Amberlite XAD-2 2 adsorbent 5 cm in diameter x 100 cm high in a 5% solution of methanol and water. A 5% Methanol and water solutions were passed through the bed at one flow rate of 880 ml / h Depumpt and 20 ml fractions were automatically totaled 287 fractions were collected and every fourth fraction was disc-plate method tested against Proteus vulgaris (MB-838) using 6 mm disks. the Test results are shown in the following table. The factions 1 to 50 were not examined.

Fraction zone size Fraction zone size 51 23 mm 115 20 55 26 119 20 59 23 123 19 63 18 127 18 67 12 131 19 71 0 155 16 75 0 139 17 79 0 143 15 83 7 n47 16 87 9 151 15 91 13 155 13 95 19 159 11 99 21 163 9 103 22 167 0 107 22 171 9 111 21 287 0.

Fractions 95-159 were combined, methanol was in vacuo evaporated, and the aqueous concentrate was freeze-dried, with 700 mg of 7ß- (D- 5-Amino-5-carboxyvaleramido) -3- (α-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxwy-3-cephem-4-carbonic acid (Ic) were obtained. The ultraviolet spectrum of 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy 3-cephem-4-carboxylic acid (Ic) gave the following adsorption data: UV adsorption in 0.1 n-HCl #max 285 U% 1cm 160 UV adsorption in 0.1 n-NAOH #max 277 U% 1cm 166 at Investigation with 13 mm disks using the disk and plate method against Proteus vulgaris yielded the 7β- (D-5-amino-5-carboxyvaleramido) -3- (α-methoxy-p-sulfo oxycinnainoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) sample a 25 mm zone at 88 mcg / ml and 7β- (D-5-amino-5-carboxyvaleramdio) -3- (α-methoxy-o-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem- 4-carboxylic acid gave a 25 mm zone at 167 incg / ml.

EXAMPLE 8 Antibiotic 842A Level A: Shake flask production iA lyophilized tube of Streptomyces lactamdurans culture (MA-2908) was added aseptically opened. The tube was then used for inoculating one with partitions equipped 250 ml Erlenmeyer flask containing 50 ml nutrient medium V, -by breaking the tube in sterile gauze and removing the lump aseptically was transferred to the flask.

The medium V had the following composition: medium V: yeast autolysate (Ardamin) 10.0 g glucose 10.0 g * phosphate buffer 2.0 ml MgSO4 .7H2O 0.05 g distilled water 1000.0 ml pH 6.5 * phosphate buffer: KH2PO4 91.0 g Na2HPO4 95.0 g of distilled water 1000.0 ml This germinating flask was at 28 ° C on a Rotation Schütt-1er shaken at 220 rpm with a stroke of 5 cm for 3 days.

5 ml (10% inoculum) aliquots of this growth were then added using sterile pipettes in four germinating flasks of the second stage-of the same size, which is the same medium as described above and these flasks were then shaken as indicated above. The second stage no flasks were then aseptically combined into a flask and for inoculating 11 graduated 2 liter Erlenmeyer flasks, each containing 350 ml of Medium IX with 2 to 3% inoculum, using inoculated with sterile pipettes. Medium IX had the following composition: Medium IX: Amber yeast No. 300 lo, 0 g soluble brewery residues 20.0 g dextrose 10.0 g Distilled water 1000.0 ml pH 7.0 The production flasks were then at 28 ° C shaken on a shaker at 145 rpm with a stroke of 5 cm for 4 days. To At the end of the incubation period, the contents of 10 such flasks were combined, and a sample was centrifuged to remove the mycelium.

The presence of the antibiotic 842A d. H. of the product 7ß- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) - epheni-4-carboxylic acid (Ib) in the broth was determined by Agar diffusion tests, which were carried out with 13 mm filter paper discs soaked in the broth and placed on the surface of the test plates containing 10 ml of medium from nutrient agar (Difco) plus 0.2% yeast extract (Difoo), and with the bacterial inoculum was vaccinated. The zones of inhibition were after overnight incubation at 28 ° C in mm measured.

The investigations of the broth collected after 4 days of fermentation showed a zone of inhibition of 31.5 mm diameter with Vibrio percolans (MB-1272) inoculated plates.

Stage B: Adsorption on an anion exchange resin. The filtered Broth was adjusted to pH 7.0 and 2900 with dilute hydrochloric acid ml were to '100 ml of a stax-basic anion exchange resin with a styro Divinylbenzene matrix (Dowex 1 x 2 resin, chloride cycle) at 10 ml / in. adsorbed. The used solution was collected in 500 ml fractions. The resin column was washed with water and eluted with 3% NH4Cl in 90% methanol. The eluate was collected in 1 ml fractions.

Disc-plate tests against Vibrio percolans (MB-1272) were carried out at carried out by all political groups; the zone diameters are in the following table listed: Filtered broth. Consumed fraction. Eluate fraction Dilution Zone fraction Sun fraction Zone size size size none 26.5 mm 1. 0 1. 25 1: 2 24 2. 16 2. 29 1: 4 20 3. 23 3. 29 4. 25 4. 26.5 5. 27 5. 22 6. 27 6. 18 7. 15 8-10. 0 These tests show that about 60% of the effectiveness in the consumed fraction and about 18% in the eluates.

They also indicate that the resin capacity is only two fractions or 10 column volumes of broth. Eluate fractions 1 to 4 were combined and concentrated to remove the methanol. The fractions used up 3 to 6 were combined to give 1960 ml of solution. An 1860 ml portion of the solution was adjusted with dilute sodium hydroxide from pH 7.2 to 8.0 and on strongly basic anion exchange resin with a styrene-divinylbenzene matrix (Dowex 1 x 2 resin, chloride cycle) at 14 ml / min. ad-.

sorbed. The solution used was in four equal fractions and tests showed that it was 5% effectiveness. The column became washed with water and eluted with 5% aqueous sodium chloride. The eluate was poured into 50 ml fractions; mmelt and examined. The investigations showed that 90% of the effectiveness was in the cuts 3 to 16, so these combined became.

Stage C: Ads2ffu-onan a cation exchange resin. A 50 ml portion of a Concentrate prepared according to stage B, was diluted to 500 ml, the pH value adjusted from 8.8 to pH 2.0 with dilute hydrochloric acid and added to 25 ml a strong acidic cation exchange resin of the sulfonate type with a styrene-divinylbenzene matrix (Dowex 50 x 2 resin, hydrogen cycle) at 2.5 ml / min. adsorbed. The column was washed with 25 ml of water, then eluted with 2% pyridine until the pH of the column effluent rose to pH 7 (54 ml). Investigations of the consumed Fraction and the eluate showed 9% effectiveness in the fraction used and 90% in the eluate. The eluate was identified as the pyridinium salt of antibiotic 842A identified, The 842A product is amphoteric with an apparent isoelectric Point at about pH 3.5. The product is unstable above pH 7, but at pH 1.5 stable. The eluate thus obtained was adjusted to pH 8.0 with dilute sodium hydroxide and concentrated under vacuum to remove the pyridine. The product thus obtained was identified as the monosodium salt of antibiotic 842A. The molecular weight is 468 based on the 'empirical formula.

Analysis for C16H21N4SO9Na: calc .: C 41.0%; H 4.5%; N 12.0%; S 6.8; O 30.8%; Na 4.9% found: C 39.31%; H 4.76%; N 11.16%; S 6.46%; O 34.12%; Na 4.19%.

In in vitro studies this product inhibited, i. H.

the antibiotic 842A stimulates the growth of the following graninegative bacteria: Escherichia coli, Proteus vlugaris, Alcaligenes faecalis, Brucella bronchiseptica, Salmonella gallinarum, Vibrio percolans and Xanthomonas vesicatoria.

The product also inhibits the growth of the following gram positive Bacteria: Staphylococcus aureus, Sarcina lutea and Bacillus subtilis.

Also, in in vivo studies in mice revealed the antibiotic 842A has the following effects. Administration was by subcutaneous injection. After the trial period ends, usually 7 days after administration, was the amount of product that was required by 50% of the mice before the otherwise protect fatal injection (ED50), calculated: ED50 by subcutaneous route x two Dosages Proteus vulgaris 51 µg Proteus mirabilis 276 µg * Proteus morganii 3202 276 µg Salmonclla schottmuelleri 103 µg Klebsiella pneumoniae AD 125 Klebsiella pneumoniae B 125 µg Paracolobactrum arizoniae 125 Escherichia coli 200 Aerobacter aerogenes 49 Pasteurella multocida, 57 g Salmonella typhosa, 34 g Diplococcus pneumoniae E400 566 µg * cephaloridin and cephalothin failed in terms of protection 4000 mg x 2 dosages In addition to the above-mentioned in vivo studies of the The product was a clinical isolate of Proteus morganii 356, which was opposed to cephalosporins is resistant and can degrade cephalosporin C, used in a mouse protection test, which was carried out in the same manner as stated above.

The ED50 values for these experiments are as follows: infection Antibiotic ED50 by subcutaneous route x 2 doses (mean of two trials) Proteus morganii 356 842A 273 µg Proteus morganii 356 Cephalothin 20,000 Proteus morganii 356 Cephaloridin 9 270 Example 9 Antibiotics 842t Shake flask production: The inoculum was prepared as described in Example 8.

Two second stage germinators were put together and the broth used to make 62 250 ml Erlenmeyer flasks (at a ml / flask) each containing 50 ml of medium X contained- to inoculate. The medium X had the following composition: Medium X: Staley's 4S soybean meal 30.0 g Soluble brewery residues 7.5 g Cerelose 20.0 g NaCl 2.5 g CaCO3 (afterwards pH value to 7.0) 10.0 g distilled water 1000.0 ml The flasks were at 28 ° C on a shaker at 220 rpm with a stroke of 5 cm shaken for 5 days. At the end of the incubation period, the contents became of 60 of these flasks combined, and a sample for removal of the mycelium centrifagi ert.

The presence of the antibiotic 842A was determined according to the in Method described in Example 1 via agar diffuser investigations, which were carried out on 13 mm filter paper test disks were carried out. After 4 days of incubation As a result, examination of the broth revealed a zone of inhibition of 33 mm against Vibrio percolans (MB-1272).

Example 10 Antibiotic 842A Fermentation: Stage 1: A lyophilized Streptomyces lactamdurans culture tubes (MA-2908) were used for inoculation of 50 ml of sterile medium V is used in a 200 ml Erlei) meyer segmented flask.

Medium V: yeast autolysate (Ardamin) 10.0 g glucose 10.0 g * phosphate buffer 2.0 ml MgSO4. 7H2O 0.05 g Distilled Water 1000.0 ml pH using NaOh adjusted to 6.5 * Phosphate buffer: KH2PO4 91.0 g Na2HP4 9.50 g distilled Water 1000.0 ml The inoculated flask was placed on a rotary shaker at 220 rpm brought with a stroke of 5 cm and incubated at 28o C for 72 hours.

Step 2: an inoculum of 10.0 ml of the obtained vegetative Growth was then used to inoculate a 2 liter Brlenmeyer compartmentalized flask containing 50 ml of the sterilized medium V described above was used. The inoculated Flask was then placed on a rotary shaker at 220 rpm and 48 hours incubated at 28 ° C.

Stage 3: The contents of the inoculum cob was then used to inoculate a 190 1 (50 gallons) stainless fermentation vessel that is 160 liters of the same Medium V described above was used. The inoculated medium was incubated at 28 ° C for 48 hours with agitation while an air flow of 85 1 / min. (3 cfm) was retained by the fermentation broth. During the fermentation period Small amounts of Polygrfol 2000 were added to regulate the foaming.

Step 4: An inoculum of 43 liters of the obtained growth was then for inoculating a 750 l (200 gallon) stainless steel fermentation vessel used, the 467 liters of a sterile medium III of the following composition contained: Medium XI: Amber yeast No. 300 10.0 g Soluble brewery residues 20.0 g of distilled water 1000.0 m pH 7.0 The fermentation was left at one temperature of 28 ° C with agitation while an air flow of 280 1 / min. (10 cfta) was maintained for 72 hours.

During the fermentation, an anti-foam agent, Polyglycol 2000, was added in small amounts to prevent excessive foaming. The approach was obtained and the effectiveness determined by disk-plate tests. the Fermentation broth was then through diatomaceous earth at a PH value of?, 8 filtered, and the product thus obtained was identified as 842A according to that in Example 1 identified the procedure described. Slice-plate tests at a 1:10 dilution gave a zone of inhibition of 21.5 mm against Vibrio percolans (MB-1272).

B e i 5 p i 'e 1 11 Relinung, monosodium salt of 7B- (5-amino-5-carboxyvaleramino) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid Adsorption on coal: four fermentation batches, which were obtained according to Example 8, were each to 100 ml of a strongly basic ion exchange resin with a Styrene-divinylbenzene matrix (Dowex 1 x 2 resin, chloride cycle) adsorbed and with 1% aqueous sodium chloride eluted.

-The eluate was collected in 50 ml fractions and examined.

Eluate fractions from all four batches were diluted to pH 5 with Adjusted hydrochloric acid and combined to give 4,300 ml of solution. 4200 ml of this solution was stirred with 42 g charcoal (Darco G-60) for 1/2 hour. the Charcoal was filtered off and washed with water. The filtrate and the washing solution showed no activity. The charcoal cake was twice with a 1 liter amount of 60%, aqueous acetone eluted by stirring the mixture for 1/2 hour and filtering each time became. The eluates were: concentrated under vacuum to 108 ml and 100 ml, respectively. try showed that the first eluate contained 76% of the activity, 18 times as potent as was the starting material and that the second eluate was 17% potency and 14 times as effective as the starting material. The two concentrates were combined and further concentrated to 61 ml and from pH 4 to pH 5 with diluted Sodium hydroxide set. This concentrate contained 40 ng / ml dry solids and gave a 25 mmw zone against MB-1272 at a dilution of 1: 100 (400 mcg / ml). The product was known as the monosodium salt of the 7β- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ia) identified.

EXAMPLE 1 12 cleaning; Monosodium salt of 7 [beta] (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid Adsorption on gel: A 22 ml portion of that obtained in accordance with Example 8, Stage B above Eluate was adjusted to pH 7.0 with dilute sodium hydroxide and on a 388 ml column containing Biogel P-2 chromatographed The column was with Water develops, and the material flowing out is recorded with a differential refractometer and 5 ml fractions automatically collected and biologically examined. The, iological Efficacy occurred in fractions 47 to 63, while 1triumchloride in fractions 62 to 72 occurred.

Fractions 50 to 60 were pooled and re-examined and concentrated to dryness to give 1058 mg of residue which was identified as the Monosodium salt of 7β- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ia) was identified. After testing with Vibrio percolans, this product was found a 25 mm zone at 8 mcg / ml.

B e i 5 p i e 1 1) 7β- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid Modified Fermentation Procedure: Stage A: Slants A lyophilized Streptomyces lactamdurans culture tubes (MA-2908) were opened aseptically and the organism in a medium of the following composition transferred: Medium 1% residual molasses 1% National brewer's yeast 2.5% Difco-Agar pH 7.0 water to make up the volume The slants were 7 days at 28 ° C incubated. When stored in the cold, the slants were more stable for more than 13 weeks.

Room B: Sprouting stages: Two-stage system First rhyme: The first Germ culture is directly from the S culture of stage A to 40 ml of 1% primary, dried Yeast N.F., pH 7.0 (obtained from Yeast Product Corporation) into a compartmentalized 250 ml Erlenmeyer flasks inoculated. The flasks were then placed on a rotary shaker at 200 rpm with a stroke of 5 cm at 28 ° C for a time frame of 2 to 3 Days shaken.

Second germination: A 2.5% inoculum from the first stage of germination was made placed in a flask containing a 2% Fleischmann S-150 yeast autolysate, pH 7.0 contained. The growth at this stage is characteristically light, and the Incubation, which was done as in the first stage, was not extended beyond 48 hours.

Stage C: Production medium The production medium contained per liter Distilled water: 30 g soluble brewery residues, 7.5 g primary, dried Yeast N.B. and 0.25 56 V / V Mobilpar-S defoamer. The medium comes with a small amount of concentrated NaOH Solution adjusted to pH 7.0, , distributed in Erlenmeyr flasks and autoclaved for 15 or 20 minutes at 121 ° C. To After cooling, the medium received a 2.5% strength inoculum of the non-culture obtained in stage B. The incubation time can vary between about 50 hours and 100 hours, however an incubation period of about 72 hours is preferred. The volume of the medium in each flask can vary between 30 and 50 ml, but have been routinely used 40 ml used. The amount of inoculum can vary between 1 to 5%, however an amount of 2.5% is generally used in practice ~ Level D: Investigation After the fermentation had ended, the cells were centrifuged off and the broth with Phosphate buffer, pH 7.0 diluted. The concentration of 7β (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid in the fermentation fractures was rsucl-ungs method according to the biological standard disc certainly. The test organism used was Vibrio percolans (ATCC 8461). Rushed "paper disks are dipped into the diluted broths and applied to the surface brought from agar-containing Petri dishes with the test organism Vibrio percolans (ATGrJ 8461) had been inoculated. There were also petri dishes on these Brought disks that had previously been immersed in standard solutions, which known concentrations of 842A. The slices were monitored at 28 ° Incubated C and the diameter of the zones of inhibition - recorded. The concentration an 842A and the fermented broth are obtained by interpolating from the standard curve Obtain the zone diameter with the known concentrations of the 842A standard solutions to relate. According to this procedure, it was calculated that Streptomyces lactamdurans MB-2908 produced 78.6 µg / ml 842A in the modified fermentation process.

B e i 5 p i e 1 14 disodium salt of 7β- (D-5-amino-5-carboxyvaleramido) -3- (N, N-dimethylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid Step A: 7β- (D-5-amino-5-carboxyval eramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid 2.005 g (4.27 mmol) of 7 [beta] (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid are in a solution of 10% aqueous dipotassium hydrogen phosphate; (26 ml) solved. The mixture is then filtered and the solid obtained with 10%, aqueous dipotassium hydrogen phosphate (2 ml).

To the filtrate is added 10 ml of acetone. The solution becomes light cloudy and has a pH of 8.45. 50% aqueous solution was added to this solution Tripotassium phosphate, adjusted to pH 9.18, was added. 1.492 g (6.69 mmol) of N-ethoxycarbonylphthalnide in 5.0 ml of acetone are then added over a period of 5 minutes. The pH is made by adding proportions of 50% aqueous tripotassium phosphate over the next 1.5 hours set to 9.1. The mixture is then used for removal. of acetone evaporated and the pH adjusted to 2 to 2.5 with 2.5 N hydrochloric acid.

The mixture is extracted with four washes with ethyl acetate (25 ml), and the combined pale yellow extracts are washed with 25 ml of water, with anhydrous sodium sulfate, dried overnight and evaporated, leaving 2.356 g of a.

yellow foam were obtained. Nuclear Magnetic Resonance and Electrophoresis confirm that the product obtained in this way 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) - Is 3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Stage B: Dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid A solution of 0.653 mg of 7β- (D-5-phthaloylamino-5-carboxy valeramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid in methanol is treated with a small excess of freshly made diphenyldiazomethane. After standing overnight at room temperature, the solution is evaporated and obtained 1.053 g residue, which as the dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid is identified.

Stage C: Dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyval eramido) - 5 (hydroxymethyl) -7-methoxy-3-cephem-4-carboxylic acid 0s5 g of the in stage B obtained dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid are dissolved in 5 ml of purified dioxane containing 1.5 equivalents of pyridine. A solution of 1.3 equivalents of nitrosyl chloride in methylene chloride is added and stir the solution in an ice bath for 1 hour. The one obtained in this way The product is a solution of the dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (hydroxymethyl) 7-methoxy-3-cephem-4-carboxylic acid, which is used directly in the following step.

Stage D: Dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (chlorocarbonyloxymethyl) 7-methoxy-3-cephem-4-carboxylic acid An excess of phosgene is stirred into a Solution of the 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (hydroxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained in step C blown in, and the mixture is allowed to stand overnight at room temperature. The excess Phosgene is removed by allowing dry nitrogen through the solution for several Hours is blown through. The product so obtained is called the dibenzhydryl ester of 713- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (chlorocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid identified.

Stage E: Dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (N, N-dimethylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid The D: i.benzhyyl ester of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (chlorocarbonyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid obtained after stage D) is cooled in an ice bath and 2.5 equivalents of dimethylamine are added.

The mixture is stirred at room temperature for 1 hour and the excess Amine hydrochloride filtered off. The product so obtained is called the dibenzhydryl ester of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (N, N-dimethylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid identified.

stage f: disodium salt of 7ß- (d-5-phthaloylamino-5-carboxyvaleramido) -3- (n, n-dimethylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid The dibenzhydryl ester of 7β- (D-5 phthaloylamino-5-carboxyvaleramido) -3- (N, N-dimethylcarbamo; yloxyme) obtained in step E thyl) -7-m ethoxy-3-c epheiii-4-c carboxylic acid is dissolved in 3.5 ml of anisole and treated with 10 ml of trifluoroacetic acid at room temperature for 10 minutes. The trifluoroacetic acid and the anisole are then removed under reduced pressure while the temperature is kept below 40 C, and the residue is taken up in 25 ml of chloroform and treated with 20 ml of water containing 0.120 g of sodium bicarbonate. The mixture is stirred for 0.5 hours at room temperature and the organic Phase separated and washed with water. The combined aqueous phase becomes then washed twice with methylene chloride and lyophilized, with the disodium salt de 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (N, N-dimethylcarbamoyloxymethyl) -7-methoxy-3-ce 4-carboxylic acid is obtained.

Step G: Disodium salt of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (N, N-dimethylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid The disodium salt of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (N, N-dimethylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained in step F is dissolved in water.

1 equivalent of hydrazine hydrate is then added and the mixture is left to read Stand mixture overnight at room temperature. The aqueous solution is made with ethyl acetate extracted before lyophilization, and the disodium salt of 7β- (D-5-amino-5-carboxyval eramido) -3- (N, N-dimethylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

B e i s i e l 15 disodium salt of 7ß- (D-5-amino-5-carboxyvaleramido) -3-pip eri dino carbonyloxym ethyl) -7-m ethoxy-- 5- c ephem-4-carboxylic acid Level A: disodium salt of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (N, N-dimethylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid With replacement of an equivalent amount of piperidine instead of the dimethylamine according to Example 14, stage E, and following the procedure described there thus becomes the dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (piperidinocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtain.

Step B: Disodium salt of 7β- (d-5-amino-5-carboxyvaleramido) -3- (piperidinocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid Using the dibenzhydryl ester of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (piperidinocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained in step A instead of the dibenzhyryl ester of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (N, N-dimethylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid given in Example 14, Step F and under, reworking the deesterification method described there is thus the disodium salt of 7ß- (D-5-phthaloylamino- 5- carb oxyval erami do) - 3- (pip eridino carbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained, after Treatment with hydrazine hydrate according to that described in Example 14, stage G Method the disodium salt of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (pipeidinocarbonylxoymethyl) -7-metho-3-cephem-4-carboxylic acid results.

For example, the 1 16 disodium salt of 7β- (D-5-amino-5-carboxy-valeramido) -3- (pyrrolidinylcarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid Stage A: Dibenzhydryl ester of 7β- (D-5-phthaloylamino-5-carboxyval ramido) -3- (pyrrolidinylcarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid Using an equivalent amount of pyrrolidine instead of the dimethylamine according to Example 14, stage E, and following the procedure described there thus the dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxy; yvaleramido) -3- (pyrrolidinylcarbonyloxymethyl) -7-methoxy-3-c ephemeral-> carboxylic acid obtained.

Step: Disodium salt of 7ß- (D-5-amino-5-carboxy; yvaleramido) -3- (pyrrolidinylcarbonyloxymethyl) -7-methoxy-3cephem-4-carboxylic acid Using the dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyval eramido) -3-pyrrolidinylcarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid instead of the 7ß- (D-5-phthaloylamino-5-carboxyval eramido) -3- (N, N-dimethylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid and by reworking the deesterification method described there, the Disodium salt of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (pyrrolidinylcarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained, after treatment with H-drazinhydrat according to the in Example 14, stage G, method described the disodium salt of 713- (D-5 amino-5-carboxyvaleramido) -3- (pyrrolidinylcarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid results.

For example, 17 disodium salt of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (morpholinocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid Level A: Dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (morpholinocarbonyloxymethyl) 7-methoxy-3-cephem-4-carboxylic acid With replacement of an equivalent amount of morpholine instead of the dimethylamine genäss example 14, stage E, and with reworking of the process described there, the dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (morpholinocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid is thus obtained obtain.

Step B: Disodium salt of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (morpholinocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid Replacing the dibenzhydryl ester of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3-morpholinocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained in step A instead of the dibenzhydryl ester of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (N, N-dimethylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid given in Example 14, Step F and by reworking the deesterification method described there is thus the disodium salt of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (morpholinocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained, after treatment with hydrazine hydrate according to that in Example 14, step G, method described the disodium salt of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (morpholinocarboyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid results.

For example, 18 disodium salt of 7β- (D-5-amino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid Stage A: Dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3cephou - '% - carboxylic acid Excess acetyl chloride is converted to those in Example with stirring 14, stage C, obtained dibenzhydryl ester of 7β- £ D-5-phthaloylauiino-5-carboxyval eramido) -3- (hydroxymethyl) -7-methoxy-3-cephem-4-carboxylic acid was added. One solves the mixture stand for several hours at room temperature.

The mixture is then concentrated in vacuo to remove excess acetyl chloride and remove solvent. The residue obtained in this way is the dibenzhydryl ester the 7ß- (D-5 Phthaloylamino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-metho = g- 3-c ephemeral 4-carboxylic acid.

The dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid can also be achieved by using ketene instead of acetyl chloride and otherwise under Reprocessing of the process described in stage A can be obtained.

Step B: Disodium salt of 7ß- (D-5-phthaloylamino-5-carboxy valeramido) -3- (acetoxymethyl) -7-methoxy-3-cephem-4carboxylic acid The dibenzhydryl ester of 7ß- (D-5-Phthaloylauino-5-carboxyvaleramido ) - gi- (ac etoxymethyl) -7-methoxy-3 cephem-4-carboxylic acid is dissolved in dioxane, and the mixture is the de-esterification method described in Example 14, stage F subjugated. In this way a residue is obtained which is the disodium salt of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid having.

Stage C: Disodium salt of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid The disodium salt of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (aceotxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained in step 3 is dissolved in methylene chloride and the solution with 5% sodium bicarbonate solution extracted. The resulting mixture is then acidified to pH 2 with sulfuric acid and extracted with ethyl acetate. After drying, the extract is concentrated in vacuo and the residue dissolved in water. 1 equivalent of hydrazine hydrate is then added, and the mixture is allowed to stand overnight at room temperature. The aqueous solution is with ethyl acetate before Lyophilize extracted, and you receives the disodium salt of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (acetoxymethyl) -7 m ethoxy- 3- c ephem- #carboxylic acid.

In the same manner as that described in Example 18, all 3-acyloxy-substituted derivatives of the invention can be obtained. For example, by using the corresponding acyl halide instead of the acetyl chloride according to Example 18, stage A, and following the procedure described in stages A, B and G of this example, all of the corresponding 3-alkanoyloxy, 3-aromatic carbonyloxy, 3-aralkanoyloxy and 3-cycloalkanecarbonyloxy substituted derivatives of the invention can be obtained. The following equation and Table XVIII illustrate this procedure. and the resulting products.

Table XVIII Example X R19 19 Cl -C2ii5 20 Br Daft 21 cl 22 -cII2-- 23 Cl -Cfi2 < 24 cy 25 cl 26 Cl .CtI2wCE 27 Br B ice P ie 1 28 disodium salt of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (N-methylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid Step A: Dibenzhydryl ester of 7ß- (D -5-Phthaloylamino-5-carboxyvaleramido) -3- (N-methylcarbamoyloxyinethyl) 7-methoxy-3-cephem-4-carboxylic acid 250 mg of the dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- ( hydroxymethyl) -7-methoxy-3-cephem-4-carboxylic acid are suspended in 5 ml of dimethylformamide.

The reaction mixture is placed under nitrogen and ultrasonic waves emotional. 0.20 ml of triethylamine and 0.625 ml of methyl isocyanate are then added, and after the mixture has dissolved, it is left to stand for 0.5 hours.

Ethyl ether is added to the mixture and crave centrifugation the ether is decanted. An extra amount of ethyl ether becomes the oily one Residue is added and the solidification of the product is promoted by scratching. The solid obtained is made up of a hot mixture.

Methanol and isopropanol (180 mg) recrystallized in two yields, and the combination of these two crops are in turn recrystallized to to obtain a product which as the dibenzhydryl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (N-methylcarbamoyloxymethyl) -7-methoxy-3-cephem-4carboxylic acid was identified.

Stage B: disodium salt of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (N-methylcarbamoyloxymethyl) -7-methoxy- 3-cephem-4-carbonic acid The dibenzhydryl ester of 7β- (D-5-thaloylamino-carboxyvaleramido ) -3- (N-methylcarbmaoyloxymethyl ) -7-methoxy-3-cephem-4-carboxylic acid is dissolved in 3.5 ml of anisole and treated with 10 ml of trifluoroacetic acid at room temperature Treated 10 minutes. The trifluoroacetic acid and anisole are then reduced under reduced pressure Pressure is removed while the temperature is kept below 40 ° 9, and the The residue is taken up in 25 ml of chloroform and.

treated with 20 ml of water containing 0.120 g of sodium bicarbonate. The mixture is stirred for 0.5 hour at room temperature, and the organic phase separated and washed with water. The combined, aqueous phase is then twice washed with ethylene chloride and lyophilized, the disodium salt of 7β- (D-5-phthaloylamino-5-carboxyvaleraini do) - 3- (N-methylcarb amoyloxymethyl) - 7-m ethoxy-3-cephem-4-carboxylic acid was obtained.

Step c: Disodium salt of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (N-methylcarbonyloxymethyl) -7-methoxy 3-cephem-4-carboxylic acid The disodium salt of 7β- (D-5-phthaloylamino-5-carbonylvaleramido) -3- (N-methylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained in step B is dissolved in water. One equivalent of hydrazine hydrate is then added and one let the mixture stand at room temperature overnight. The aqueous solution is extracted with ethyl acetate before lyophilization, and the disodium salt is obtained of 7β- (V-5-amino-5-carboxyvaleramido) -3- (N-methylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Following the procedure described in Example 28, all 3- (N-mono-substituted carbamoyloxymethyl) derivatives of the invention can be obtained. By using the corresponding isocyanate instead of the methyl isocyanate according to Example 28, stage A, and following the process described in stages A, B and C of this example, all of the corresponding 3-carbamoylo; yethyl-substituted derivatives of the invention can be synthesized will. The following equation explains the reaction according to Example 28, stages A, B and C and, together with Table IX, explains the starting materials of this process and the products obtained thereafter: Table XIX -o = C = NR3 | R17 Base | p1 -s 8 4 = NCH2ai2Cl -CH2CH2C1 NaHCq3 -Na 3β 0 = C = NCH2C1 -aI2Cl KHCO 3 -K 31 O = C = NC (CH3) 3 -C (CH3) 3 NaHCO3 -Na 32 0 = C = NC2EI5 -C2H5 KHOO3 - 33 - O = C =} 1C (CH3) 2CH2C1 -C (CH3> 2CH2C1 NaHCO3 -Na 34 0 = C = NCOOC2H5 -COOC2 Hs NaHCO3 -Na 35 O = C = NS02-CH3 -so N aHCO 3 -Na 36 O = C = Ne t3 NaEICO 3 -K 37 O = C = DICHXl c} '- ffi \ ½iD) 2 NaHCO3 3, -Na 3 ice T he 1 38 7ß- (D-5-amino-5-carboxyvaleramido) -3-- (pyridiniummethyl) -7-methoxy-3-cephem-4-carboxylic acid A solution of 1.0 g of 7ß- (D- 5-Amino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid is brought to pH 2.5. 8 ml of pyridine are added and the solution is heated to 60 ° C. for 2 hours. The reaction mixture is then lyophilized and the residue is dissolved in water and passed through a polystyrene-trimethylbenzylammonium anion exchange resin (43% H2O). The resulting mixture of 7β- (D-5-amino-5-carboxyvaleramido) -3- (pyridi niummethyl) -7-methoxy-3-cephem-4-carboxylic acid is diluted with water and selected fractions are lyophilized, whereby practically pure 7β- (D-5-amino-5-carboxyvaleramido) -3- (pyrid. Niuamethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Using an equivalent amount of trimethylamine and triethylamine instead of pyridine in the preceding process and otherwise with reworking of the process described there is thus 7β- (D-5-amino-5-carboxyvaleramido) -3- (trimethylammoniummethyl) -7-methyl-3-cephem-4-carboxylic acid and 7β- (D-5-amino-5-carboxyvaleramido) -3- (triethylammoniummethyl) -7-methoxy-3-cephem-4-carboxylic acid obtain.

All of the 5-pyridiniummethyl derivatives of the invention can be obtained by using the appropriate mononuclear substituted pyridine in place of the pyridine reactant mentioned in the previous example. The following equation and the associated table explain this manufacturing process and the products obtained from it: Table XX Ex. X¹ Ex. X¹ 39 3-F 52 3-CON (C2H5) 2 40 3-Cl 53 3-CH2COOH 41 3-Br 54 3-COCH3 42 3-I 55 4-COCH3 43 4-CF3 56 2-CH3 44 3-COOH 57 2-C2H5 45 4-COOH 58 3-CH3 46 3-CONH2 59 4-C2H5 47 4-CONH2 60 3-CH2OH 48 3-CONHCH3 61 4-CH2OH 49 4-CONHC2H5 62 4-CH2CH2CH3 50 4-CONHCH (CH3) 2 63 3-SO3H 51 4-CON (CH3) 2 64 3-CN Example 1 65 S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] -isothiourea A solution of 1.0 g 7β- (D -5-Amino-5-carboxyvalera-mido) -3- (acetoxymethyl) -7-methoxy-3-cepehm-4-carboxylic acid and 1.0 g of thiourea in 25 ml of water are kept at 37 ° C. for 5 days. 200 ml of acetone are added and the mixture is cooled in an ice bath. The product obtained is then filtered and fractionated by polystyrene-trimethylbenzylate monion anion exchange resin (43% H2O). Selected fractions are lyophilized and the crude product is then recrystallized from a mixture of methanol and water to give practically pure S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3- cephem-3-ylmethyl] isothiourea.

After replacing an equivalent amount of N-methylthiourea, N-ethylthiourea, N-propylthiourea, N, N-dimethylthiourea, N, l-diethylthiourea, and N , N-dipropylthiourea in place of that listed in your previous procedure @@ Thiourea and otherwise reworking the procedure described there becomes in this way N-methyl-S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7 methöxy- 3 c epliem-3-ylm ethyX7-i s othi oharnsto ff, N-ethyl-E (D- 5-amino-5- carboxyval crami do) -4- carb oxy- 7-m ethoxy-3-cephem-3-ylmethyl] isothiourea, N-propyl-S [7β- (D-5-amino-5-carboxyval erami do) -4-carboxy-7-methoxy-3-c ephein- 3-ylmethylj-isothiourea, N, N-dimethyl-S- [7β- (D-5-amino-5-carboxyvaleramino) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] -isothiourea, N, N-diethyl-S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] isothiourea and N, N-dipropyl-S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] isothiourea obtain.

B e i 5 p i e 1 66 7ß- (D-5-Amino-5-carboxyvaleramido) -3- (ethylthiomethyl) -7-methoxy'-3- cephem-4-carboxylic acid A mixture of 0.654 g of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid and 0.37 ml of ethanethiol in one Mixture of one part acetone and one part water (10 ml) is at room temperature stirred and 2.0 ml of a 10% sodium hydroxide solution were added with stirring. That The mixture is then heated in a sealed tube for 100 hours and the resulting The mixture is concentrated in vacuo. The residue is dissolved in water and filtered through Polystyrene-trinethylbenzylammonium anion exchange resin (43% H20) fractionated. Selected fractions are combined and lyophilized, whereby 7β- (D-5-amino-5-carboxyvaleramido) -3- (ethylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid is obtained.

Using an equivalent amount of methanethiol, propanethiol, pyridine-2-thiol, Pyridine-3-thiol, pyridine-4-thiol, benzothiazole-2-thiol, 4-methylpyrimidine-2-thiol and 2-methyl-574-thiadiazole-5-thiol in place of that in the foregoing procedure specified ethane thiol and otherwise reworking the one described there Process, in this way 7ß- (D-5-Aamino-5-carboxyvaleramido) -3- (methylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 7B- (D-5-amino-5-carboxyvaleramido) -3- (propylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 7ß- (D-5-amino 5-carboxyvaleramido) -3- (2-pyridylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 7ß- (D-5-amino-5-carboxyvaleramido) -3- (3-pyridylthiomethyl) -7-methoxy-3cephem-4-carboxylic acid, 7E- (D-5-amino-5-carboxyvaleramido) -3- (4-pyridylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 7β- (D-5-amino-5-carboxyvaleramido) -3- (2-benzothiazolylthiomethyl) -7-methoxy-3-c ephemeral-4-carboxylic acid, 7β- (D-5-aminocarboxyvaleramido ) -3- (4-methylpyrimidin-2-ylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid and 7ß- (D-5-amino-5-carboxyvaleramido) -3- (2-methyl-3,4-thiadiazol-5-ylthiomethyl) -7-methoxy- 3- c ephemous carbonic acid obtained.

a3 e i s p 1 e 1 67 S- [7 [beta ]- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] -N, N-dimethyldithiocarbamate A solution of 6.82 g of 7β- (D-5-Amino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid and. 2.86 g of sodium N, N-dimethyldithiocarbamate in 60 ml of water is applied for 24 hours Heated to 50 ° C. The product is lyophilized and then passed through a polystyrene trimethylbenzylammonium Anion exchange resin (43% H2O) fractionated. Selected actions are then lyophilized, whereby S- [7β- (D-5-amino-5-carboxyval gave do) -4- carboxy-7-m ethoxy-3-c ephem-3-ylmethyl] -N, N-dimethyldithiocarbamate.

Using an equivalent amount of the following reactants: Sodium N-methyldithiocarbamate, sodium N, N-diethyldithiocarbamate, sodium N, N-di-n-propyldithiocarbamate, Nftrium4T-methyl-'N- (2-dimethylaminoethyl carbamate, sodium N-ethyl-N- (2-diethylaminoethyl) -dithiocarbamate, Sodium N- (2-di-n-propylaminoethyl) dithiocarbamate, sodium N-methyl-N- (2-morpholinoethyl ) -dithiocarbamate, sodium-N-ynethyl-N- (3-diethylaminopropyl) -dithiocarbamate, sodium-N-phenyl-N- (2-methylaminoethiyl) dithiocarbamate, sodium N, N-tetramethylene dithiocarbamate, Sodium N, N-pentamethylene dithiocarbamate, sodium N, N-bis (2-hydroxyethyl) dithiocarbamate and the sodium salt of 4-methyl-piperazinodithiocarboxylate in place of that in that previous method indicated sodium dimethyldithiocarbamate, the sodium bicarbonate is omitted, but otherwise the one described there procedure is reworked, S- [7ß- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy 3-cephem-4-ylmethyl] -N-methyldithiocarbamate, S- [7β- (D-5-amino-5-carboxyvaleranido ) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] -N, N-diethyldithiocarbamate, S- [7β- (D-5-amino-5-carboxyval erami do) -Xcarboxiy-7-m ethoxy- 3-c eph cm- 3-ylm ethyl N, N-di-n-propyldithiocarbamate, S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] -N-methyl-N- (2-dimethylaminoethyl) dithiocarbamate, S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] -N-ethyl-N- (2-diethylaminoethyl) dithiocarbamate, S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-c ephem-3-ylmethyl] -N- (2-di-n-propylaminoethyl) -di thi, ocarbamate, S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] -N -methyl-N- (2-morpholinoethyl) -dithiocarbamate, S- [7β- (D-5-amino-5-carboxyvaleramido) 4-.Rarboxy-7-1l ethoxy-3-c ephem-3-ylmethyl] -N-m ethyl N- (3-diethylaminopropyl) dithocarbamate, S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] N-phenyl-N- (2-methylaminoethyl) dithiocarbamate, S- [7β- (D-5-amino-5-carboyval erami do) -4- carboxy- 7-n ethoxy- 3- c ephem-3-ylmethyl] -N, N-tetramethylene diocarbamate, S- [7ß- (D-5-amino-5-carboxyval eramido) -4- carboxy- 7-m ethoxy- 3- c ephem-3-ylmethyl] -N, N-pentamethylene tiocarbamate, S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carbox-7-methoxy-3-cephem-3-ylmethyl-N , N-bis- (20-hydroxyethyl) -dithiocarbauiat and f7ß- (D-5-amino-5-carboxyval eramido ) -4-carboxy-7-niethoxy-3-cephem-3-ylmethyl] -4-methyl-piperazinodithiocarboxylate obtain.

B e i s p i e l 68 7ß- (D-5-Amino-5-carboxyvaleramido) -3- (benzylithiomethyl) 7-methoxy-3-cephem-4-carboxylic acid A mixture of 0.654 g of 7ß- (D-5-amino-5-carboxyvaleramido) - 3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid, 0.504 g of sodium bicarbonate and 0.414 g of thiobenzoic acid in 5.0 ml of water is added to 50 ° C overnight under a nitrogen atmosphere. The product is through Addition of acetone precipitated and crystallized from a mixture of alcohol and water, wherein 7β- (D-5-amino-5-carboxyvaleramido) -3- (b enzoylthiometh;) -7-methoxy-3-cephem-4-carboxylic acid is obtained.

Using an equivalent amount of potassium ethyl xanthate, potassium n-propyl xanthate, Potassium isopropylxanthate, potassium n-butylxanthate, potassium n-hexylxanthate, potassium cyclopentylxanthate and potassium cyclohexyl xanthate in place of those given in the foregoing process Thiobenzoic acid and otherwise reworking the process described there, in this way 7ß- (D-5-amino-5-carboxyvaleramido) -3- (äthoxythio carbonyltlii omethyl) - 7-m ethoxy-3-c ephem-4-carboxylic acid, 7β- (D-5-amino-5-carboxyvaleramido) -3- (n-propoxythiocarbonylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid , 7ß- (D-5-amino-5-carboxyvaleramido) -3- (isopropoxythiocarbonylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 7ß- (D-5-amino-5-carboxyvaleramido) -3- (n-butoxythicarbonylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 7ß- (D-5-amino-5-carboxyvaleramido) -3- (n-hexyloxythiocarbonylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 7β- (D-5-amino-5-carboxyvaleramido) -3- (cyclopentyloxythiocarbonylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid and 7β- (D-5-amino-5-carboxyvaleramido) -3- (cyclohexyloxythiocarbonylthiomethyl) -7-ynethoxy-3-cephem-4-carboxylic acid obtain.

B e i 5 p i e 1,69 7β- (D-5-amino-5-carboxyvaleramido) -3- (toluene-p-sulfonylmethyl) -7-methoxy-3-cephem-4-carboxylic acid A mixture of 0.654 g of 7ß- (D-5-amino-5-carboxyvaleramido- 3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid .and 1.0 g of sodium toluene p-sulfinate in 5.0 ml of water is heated at 50 ° C for 24 hours. The mixture is concentrated in vacuo and from a mixture of methanol and water crystallized, with 7β- (D-5-amino-5-carboxyvaleramido) -3- (toluene-sulfonylmethyl ) -7-methoxy-3-c ephem-4-carboxylic acid is obtained.

For example 1 70 7β- (D-5-hmino-5-carboxyval eramido) - 3- (azidomethyl) -7-methoxy-3-cephem-4-carboxylic acid A mixture of 2.0 g of 7β- (D-5-amino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid and 1.0 g of sodium azide is dissolved in 10 ml of water and heated at 50 ° C. overnight. The mixture is then lyophilized and crude 7β- (D-5-amino-5-carboxyvaleramido) -3- (azidemethyl ) -7-methox; y-3-cephem-4-carboxylic acid instead of the treatment of 7ß- (D-5-amino-5-carboxyvalerami do) - 3- (ac etowme ethyl) - 7-methoxy 3- c ephem-4-carbonic acid with sodium azide it is also possible to use the 842A yourself in an otherwise analogous process, to get an @ identical product. The following example illustrates this production Procedure: 100 mg of 7β- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxiymethyl) -7-methoxy-3-cephem-4-carboxylic acid in a 0.5 M phosphate buffer solution (5 ml, by adding 3.5 g sodium dihydrogen phosphate and 3.4 g of disodium phosphate in 100 ml of water and then adding a sufficient amount Amount of hydrochloric acid to bring the pH to 5) heated in the presence of 20 mg of sodium azide at 95 ° C. for 8 minutes. Preparative thin-layer chromatography revealed 20 mg of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (azidomethyl) -7-methoxy-3-cephem-4-carboxylic acid, from treatment determined by infrared and nuclear magnetic resonance tuning this material with 1.0 ml of trifluoroacetic acid at 0 ° C for 5 minutes and then Quenching in a large volume of ether and evaporation of the solvent 5 resulted 15 mg of 7β- (D-5-amino-5-carboxyvaleramido) -3- (azidomethyl) -7-methoxy-3-cephem-4-carboxylic acid in the form of a white powder. This material possessed biological activity against different bacterial strains. This product was characterized by the following data: Thin-layer chromatography: carried out on cellulose plates in a 75%, aqueous acetonitrile solvent; the Rf value for this product was 0.7; the Rf value for the disodium salt of 842A was 0.3.

Ultraviolet determination: # Maximum was 263 millimicrons of molecular Extinction coefficient was about 3400 (theoretical value: 8000); Infrared determination in Nujol was 1780 cm-1, indicating the presence of a ß-lactam ring; the Product also gave a positive ninhydrin test for the presence of an α-aminoadipoyl site chain num prop ...

Biological @@ such: The disk zone sizes in the following Ta @@@ le are expressed in mm for 100 γ / ml antibiotic.

Organism disodium salt 7ß- (D-5-Amino-5-carboxyvon 842A valeramido) -3- (azidomethyl) - (100 γ / ml) 7-methoxy-3-cephem-4-carboxylic acid (100 / ml) Escherichi a coli W-MB-60 18 21 Pseudomonas stutzeri MB-1231 22 20 Vibrio percolans MB-1272 18 16 (5 γ / ml) For example, 1 71 7β- (D-5-amino-5-carboxyvaleramido) -3- (aminomethyl) -7 methoxy-3-cephem-4-carboxylic acid The crude sample of 7β- (D-5-amino-5-carboxyvaleramido) -3- (azidomethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained in Example 70 is hydrogenated over platinum oxide in an acetic acid demolishing methanol solution. Of the The catalyst is filtered off and the solution concentrated. A solution of the residue is then replaced by a polystyrene-trimethylbenzylammonium anion exchange resin (43 % H2 /) fractionated, and selected fractions are pooled and lyophilized, wherein 7β- (D-5-amino-5-carbo: yval eramido) -3- (aminomethyl) 7-methoxy-3-c ephem-4-carboxylic acid is obtained.

The acylation of 7β- (D-5-amino-5-carboxyvaleramido) -3- (aminoinethyl) -7-methoxy-3-c ephemeral 4-carboxylic acid with acetyl halide, propionyl halide and 2-phenylacetyl halide gives the corresponding N-acylated derivatives: 7ß- (D-5-amino-5-carboxyvaleramido) -3- (acetamidomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 7β- (D-5-amino-5-carboxyvaleramido) -3- (propionamidomethyl) -7-methoxy-3-cephem-4-carboxylic acid and 7β- (D-5-amino-5-carboxyvaleramido) -3- (2-phenylacetamidomethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Instead of the catalytic process given above, 7β- (D-5-amino-5-carboxyvaleramido) -3- (azidomethyl) -7-methoxy-3-cephem-4-carboxylic acid can also be reduced to the corresponding amine by molecular hydrogenation. That the following example explains this manufacturing process.

7β- (D-5-amino-5-carboxyvaleramido) -3- (azidomethyl) -7-methoxy-3-cephem-4-carboxylic acid was dissolved in a solution of 90% acetic acid and 10% water. The mixture was cooled to 0 to @ 0 n and 30 mg of zinc dust was added. After 10 minutes it was the mixture is filtered and treated with hydrogen sulfide to add soluble zinc remove. The resulting mixture was then diluted with a large volume of water and lyophilized, whereby practically pure 7β- (D-5-amino-5-carboxyvaleramido) -3- (aminomethyl) -7-methoxy-3-cephem-4-carboxylic acid was obtained. This product was characterized by the following data: Thin-layer chromatography: carried out on cellulose plates in a 75 aqueous acetonitrile solution; the Rf value for this product was 0.2, the Rf value for the disodium salt before 84.2A was 9.3.

Ultraviolet determination: # maximum was 263 m; the molecular extinction coefficient was around 3500 (theoretical value: 8000); found the infrared determination in Nujol 1780 cm-1, indicating the presence of a β-lactam ring; the product also yielded a positive ninhydrin test, which shows the presence of a γ-aminoadipoyl side chain indicates.

Biological Trial: The slice zone sizes are in the following Table expressed in mm for equal concentrations of the antibiotic organism Disodium salt 7ß- (D-5-amino-5-carboxyvaler of 842A amido) -3- (aminomethyl) -7-methoxy-3-cephem-4-carboxylic acid Vibrio percolans MB-1272 18 20 (5 γ / ml) Salmonella gallinarum MB-1287 27 21 (100 γ / ml) Pseudomonas stutzeri MB-1231 21 21 (100 γ / ml) B e i s p i e l 72 7β- (D-5-amino-5-carboxyvaleramido) -3- (2,4-dihydroxybenzyl-7-methoxy-3-cephem-4-carboxylic acid A mixture of 0.654 g of 7β- (D-5-amino-5-carboxyvaleramido) 3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid, 1.1 g resorcinol and 10 ml water were heated at 50 ° G for 2 days.

The reaction mixture is then lyophilized and crude is obtained 7β- (D-5-amino-5-carboxyvaleramido) -3- (2,4-di-hydroxybenzyl) -7-methoxy-3-cephem-4-carboxylic acid.

For example, 73 7β- (D-5-amino-5-carboxyvaleramido) -3- (N-methylindol-3-ylmethyl) -7-methoxy-3-cephem-4-carboxylic acid A solution of 0.655 g of N-methylindole in 5 ml of acetone becomes a solution of 0.654 g of 7β- (D-5-amino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid given in 5 ml of water at 50 ° G. The mixture is heated for 48 hours, and so is the solvent is then removed in vacuo and the residue rubbed with ether, with crude 7β- (D-5-amino-5-carboxyvaleramido) -3- (N-methylindol-3-ylmethyl) -7-methoxy-3-c ephcm-4-carboxylic acid is obtained.

For example, 74 disodium salt of 7β- (D-5-amino-5-carboxyvaleramido) -3- (hydroxymethyl) -7-methoxy-3-cephem-4-carboxylic acid Stage A: Dibenzyl ester of 7ß- (D-5-phthaloylamino-5-carbxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3 c ephem-4-carb onic acid A solution of 0.653 mg of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Example 14, step A) in methanol is freshly prepared with a small amount cm phenyldiazomethane treated. After standing overnight at room temperature, the Solution evaporated to give 1.053 g of residue, which is the dibenzyl ester of 7β- (D-5-phthaioylamino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid was identified.

Stage B: Dibenzyl ester of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (hydroxymethyl) -7-methoxy-3-cephem-4-carbonic acid 0.5 g of the dibenzyl ester of 7β- (D-5-phthaloylamino-5-carbonylvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained in step A are dissolved in 5 ml of pure dioxane containing 1.5 equivalents of pyridine. One A solution of nitrolsyl chloride (1.3 equivalents) in methylene chloride is added and stir the solution in an ice bath for 1 hour. The product thus obtained is a solution of the dibenzyl ester of 7ß- (D-5-Phthaloylamino-5-carboxyvaleramino) -3- (hydroxymethyl) -7-methoxy-3-cephem-4-carboxylic acid, which is used directly in the following stage.

Step C: Disodium salt of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido ) - 3- (hydroxm ethyl) -,? - m ethoxy-3-cephem-4-carboxylic acid A solution of 0.5 g 10% palladium-on-charcoal is added together with 0.5 ml of glacial acetic acid to that in stage B. obtained dibenzyl ester of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3-hydroxymethyl) -7-methoxy-3-cephem-4-carboxylic acid admitted. The mixture is applied at 2.8 kg / cm2 (50 lb. per square inch) with agitation Hydrogenated for 2 hours, the catalyst is filtered off through diatomaceous earth and the Solvent removed by concentration in vacuo. The residue is dissolved in methylene chloride dissolved and the solution extracted with a 5% 9gen sodium bicarbonate solution. The received The mixture is then acidified to pH 2 with sulfuric acid and extracted with ethyl acetate. After drying, concentrate the extract in vacuo to give a residue is known as the disodium salt of 7ß- (D-5-phthaloylamino-5-carboxyva'leramido ) -3- (hydroxymethyl) -7-methoxy-3-cephen-4-carboxylic acid is identified.

Step D: Disodium salt of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (hydroxymethyl) -7-methoxy-3-cephem-4-carboxylic acid e The disodium salt of 7ß- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (hydroxymethyl) -7-methoxy-3-cephem-4-carboxylic acid, obtained in step F is dissolved in water, 1 equivalent of hydrazine hydrate is then added and the mixture is allowed to stand overnight at room temperature. The aqueous solution is extracted with ethyl acetate before lyophilizing, and the disodium salt of 7ß- (D-5-Ainino-5-carboxyval erami do) - 5- (hydroxymethyl) is obtained ) -7-m ethoxy-3-cephem-4-carboxylic acid e.

EXAMPLE 1 75 Purification Monosodium salt of 7β- (D-5-amino-5-carbo; '-yvaleramino) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid 1.0 g of the monosodium salt of 7β- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid prepared according to Example 12 were dissolved in 20 ml of 1% aqueous n-butanol and chromatographed on a column, which contained 2530 ml Sephadex G "10, a modified dextran gel in the form of a bead. The column was developed with 1% aqueous n-butanol at 10 ml / min, and 10.5 ml fractions were collected automatically. The flowing liquid became recorded on a recording refractometer and the fractions became biological examined. The biological activity occurred in fractions 99 to 122, and these were combined and concentrated to dryness, 670 ml of product being obtained which is predominantly the monosodium salt of 7ß- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid contained.

The column was then chromatographed on a mixture Calibrated by Blue Dextran 2000 and sodium chloride under identical conditions. Blue Dextran 2000 was in fractions 85 to 93 and sodium chloride in fractions 14V to 155 determined what indicates that the biologically active product of impurities none of this type are severed.

B e i s p 1 e 1 S6 7β- (D-5-amino-5-carboxyval erami do) - 3-m ethyl 7-methoxy-3-cephem-4-carboxylic acid A 10% palladium on carbon catalyst was in 80 ml Suspended water and treated with hydrogen. The catalyst was then filtered and resuspended in 50 ml of water, and to 2667 g of this mixture was added 1.0 g of the sodium salt of 842A in 10 ml of water. The resulting mixture was shaken at room temperature for 22 hours.

The catalyst was filtered off and washed once with 50 ml of water. The combined washings and filtrates were then concentrated to dryness, and a 52.8% yield of 7β- (D-5-amino-5-carboxyvaleramido) -3 methyl-7-methoxy-3-cephem-4-carboxylic acid was obtained (528 mg).

The 7β- (D-5-amino-5-carboxyvaleramido) -3-methyl-7-methoxy-3-cephem-4-carboxylic acid thus obtained was compared to starting material using thin layer chromatography.

Silica gel plates were used, the top phase being four Parts n-butyl alcohol, one part acetic acid and four parts water. That Hydrogenolysis product gave two spots with a higher flow rate (Rf value) as the starting material. The spots were identified by inhydrin and ultraviolet fluorescence determined. The following table shows the observed differences between the Sodium salt of 842A and the 7β- (D-5-amino-5-carboxyvaleramido) -3-methyl-7-methoxy-3-cephem-4-carboxylic acid product again.

Measurement of the sodium salt of 842A 7β- (D-5-amino-5-carboxyvaleramido) -3-methyl-7-methoxy-3-cephem-4-carboxylic acid Biological activity - 5 µg / ml resulted in a 25 mm zone inactive at 50 µg / ml (<2% initial seediness against Vibrio percolans activity) (MB-1272) thin-film Rf 0.1 ninhydrin (positive) Rf 0.25 inhydrin (positive) Chromatography Ultraviolet (positive) Ultraviolet (positive) ~ 15 ug / spot Rf 0.35 ninhydrin (positive) ultraviolet (negative) ultraviolet # max 266 mµ, E1cm1% 142 # max 265 mµ E1cm1% 100 Example 1 77 S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-yl-methyl] thiouroium 100 mg of antibiotics 842A were treated with 26 ng thiourea at 95 ° C for 8 minutes 5 nil of a 0.5 m phosphate buffer solution, which is obtained by adding 3.5 g of sodium dihydrogen phosphate and 3.4 g of disodium phosphate in 100 ml of water and subsequent addition of sufficient Hydrochloric acid to bring the pH to 5 was obtained. Electrophoresis of the solution at pH 7 revealed the thiouronium compound to be immobile Unit, while 842A has an Rf value of about 0.2. The mixture became by absorption to a polystyrene nucleus sulfonic acid cation exchange resin (hydrogen cycle, Dowex 50) cleaned by removing the phosphate buffer. The elution took place under Use of a 0.1 N pyridine solution. The p) r'rn "i'ert was with @ n-sodium hydroxide adjusted to 8 and evaporated in vacuo to remove residual pyridines.

Lyophilization gave a tan powder which was classified as S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-yl-methyl] -thiouronium was identified. Diose's product was characterized by the following data: Thin-layer chromatography: carried out on oil-cellulose plates in a 25%, aqueous acetonitrile solvents; the Rf value for this product was 0.15; the Rf value for the disodium salt of 842A was 0.3.

Ultraviolet determination: # Maximum was 263 µg; the molecular extinction coefficient was about 5700 (theoretical value: 8000); found the infrared determination in Nujol 1780 cm-1, indicating the presence of a β-lactam ring; the product also yielded a positive ninhydrin test, as the presence of an α-aminoadipoyl side chain indicates electrophoresis: the introduction of a positive charge into the product has been made by electrophoresis at 1000 volts using a 0.05 m phosphate buffer confirmed at pH 7; the Rf value for the product was 0f01; the Rf value for that The disodium salt of 842A was 0.4.

Biological Examination: The slice zone sizes are in the following Table expressed in mm with the same sonic concentrations of the antibiotic.

Organism disodium salt S- [7β- (D-5-amino-5-carboxy of 842A valeramido) -3-carboxy-7 (100 γ / ml) methoxy-3-cephem-3-ylmethyl] thiouronium (100 γ / ml) Vibrio Percolans O1272 235 27 Salmonella gallinarua MB-1287 27 23 Pseudomonas Stutzeri MB-1231 22 22 Proteus vulgaris MB-838 27 17 Example 78 7β- (D-5-Amino-5-carboxyvaleramido) -3- (4-methylthiazol-2-ylmercaptomethyl) -7-methoxy-3-cephem-4 -carboxylic acid Step A: 7β- (D-5-N-tert-buthoxycarbonylamino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid 1.0 g of the disodium salt of 842A was dissolved in 31.6 ml of 5% di-basic sodium phosphate dissolved, and 20.8 ml of acetone was added with stirring. The pH of the solution became then adjusted to 9.5 to 9.6 with sodium hydroxide and 1.0 ml of tert-butoxycarbonylazide was added with stirring. Stirring was done for 4 to 6 hours at Xauntenperatur continued while the pH was between 9.0 and 9.6 with the addition of sodium hydroxide was retained. The reaction mixture was then stirred overnight, after which the pH had dropped to 8.5. The pH was restored with sodium hydroxide Set 9.6 and stirring continued for a further 3 hours while maintaining the pH was held between 9.0 and 9.6.

The resulting mixture was then. with half a volume of ethyl acetate extracted and the extract discarded. Half a volume of ethyl acetate was added and the aqueous layer was washed to pH with concentrated hydrochloric acid 2.5 set in an ice bath to keep the temperature below 5 ° C. After separation of the ethyl acetate, the aqueous solution was twice with a Hold volumes of ethyl acetate extracted at pH 2.5. Based on ultraviolet regulations 50% of the starting ultraviolet absorbent was present in the extracts and 22% in the used, aqueous phase. The extracts were combined and taken to dryness concentrated, whereby 645 mg of 7β- (D-5-N-tert-butoxycarbonylamino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid with a tenth of the biological effectiveness of the starting material was obtained.

Thin layer chromatograms were performed using Analtech silica gel G.F. plates and the upper phase from a 4: 1: 4 n-butanol, acetic acid, water system carried out. The product had an Rf value of 0.54 based on ultraviolet determination and was ninhydrin-negated. The starting material had an Rf value of 0.1 and was ultraviolet and ninhydrin positive.

Removal of the tert-butoxycarbonyl protecting group from a 10 mg sample of 7β- (D-5-N-tert-butoxycarbonylmino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid was made by dissolving the material in 0.2 ml of trifluoresetic acid and allowing it to stand the solution carried out at room temperature for 5 minutes. The solution then became concentrated to dryness at room temperature.

Thin layer chromatography as described above gave one Stain for this product with an Rf value of 0.1 that is ultraviolet and ninhydrin positive was.

Step B: 7β- (D-5-amino-5-carboxyvaleramido) -3- (4-methyl-thiazol-2-ylmetcaptomethyl) -7-methoxy-3-cephem 4-carboxylic acid 100 mg of 7β- (D-5-N-tert-butoxycarbonylamino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid in 5 ml of a pH 7 buffer (a C, 5 M solution of a mixture of 3.5 g of sodium dihydrogen phosphate and 3.4 g disodium phosphate in 100 ml water), the 50 mg 2-mercapto-4-methylthiazole is heated at 95 ° C for 8 minutes.

Thus, 7β- (D-5-N-tert-butoxycarbonylamino-5-carboxyvaleramido) -3- (4-methylthiazol-2-ylmercaptomethyl) -7-methoxy-3-cephem040carboxylic acid obtained after treatment with 0.2 ml of trifluoroacetic acid 7ß- (D-5-amino-5-carboxyvaleramido) -3- (4-methylthiazol-2-ylmercaptomethyl) -7-methoxy-3-c ephemous-4-carboxylic acid provided This product was characterized by the following data; Thin-layer chromatography: carried out on cellulose plates in a 25%, aqueous acetonitrile solvent; the Rf value for this product was 0.6 The Rf value for the disodium salt of 842h was 0.3.

Ultraviolet determination: # Maximum was 269 mµs the molecular extinction coefficient was around 2000 ' (theoretical value: 8000); Infrared determination in Nujol was 1780 cm-1, indicating the presence of a β-lactam ring; the product also gave a positive ninhydrin test indicating the presence of an α-amir dipoyl side chain indicates.

Biological examination: The disk zone sizes in the following Tables are expressed in mm for the same sonic concentrations of antibiotic.

Organism disodium salt 7ß- (D-5-amino-5-carboxyvon 842A valeramino) -3- (4-methyl- (100 # / ml) thiazol-2-ylmercaptomethyl) -7-methoxy-3-cephem-4-carboxylic acid (100 γ / ml) Pseudomonas c ', tutzeri' H3-1231 22 13.5 Escherichia coli W-MB-60 18 14 Vibrio 1 percolans MB-1272 23 20 Example 79 7β- (D-5-amino-5-carboxyvaleramido) -3- (1,3,4-thiadiazol-2-ylmercaptomethyl) -7-methoxy-3-cephem-4-carboxylic acid With replacement of 2-mercapto-1,3,4-thiadiazole instead of 2-mercapto-4-methylthiazole according to Example 78, stage B, and otherwise reworking that described there In this way 7β- (D-5-amino-5-carboxyvaleramido) -3- (1,3,4-thiadiazol-2-ylmercaptomethyl) -7-ynethoxy-3-cepheni-4-carboxylic acid is processed obtain.

7β- (D-5-amino-5-carboxyvaleramido) -3- (thiocyanatomethyl) -7-methoxy-3-cephem-4-carboxylic acid 100 mg of antibiotic 842A were added to 5 ml of a 0.5 M buffer solution, consisting of 3.5 g of sodium dihydrogen phosphate and 3.4 g of disodium phosphate in 100 ml of water were added and the pH of the mixture was brought to pH 5 by adding hydrochloric acid. 20 mg of sodium thiocyanate were added and the mixture was at 95 ° for 8 minutes G heated.

In this way 7β- (D-5-amino-5-carboxyvaleramido) -3- (thiocyanatomethyl) -7-methoxy-3-cephem-4-carboxylic acid obtain.

Ex. 1 81 7 [beta] (D-5-amino-5-carboxyvaleramido) -3- (chloromethyl) -7-methoxy-3-cephem-4-carboxylic acid Stage A: Dibenzhydryl ester of 72- (D-5-N-tert-butoiycarbonylamino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid To a solution of 15.0 g of 7β- (D-5-N-tert-butoxycarbonylamino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid 5.5 g of diphenyldiazomethane in 70 ml of ether are added to 500 ml of ethyl acetate. The mixture is heated to 40 ° C. with stirring and then after g Hi'-grooves with additional 5.5 g of diphenyldiazomethane treated in 0 µl of ether. After 3 hours the solvent will removed in vacuo and replaced by a mixture of 500 ml of methanol and 20 ml of water. The methanol-water solution is extracted four times with hexane and then evaporated in vacuo.

The residue is dissolved in ethyl acetate and dried over sodium sulfate and evaporated in a vacuum, - with the Di-Bei stalk 80 benzhydryl ester of 7β- (D-5-N-tert. -Butoxycarbonylanino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid is obtained.

Stage B: Dibenzhydryl ester of 7β- (D-5-N-tert-butoxycarbonylamino-5-carboxyvaleramido) -3- (chloromethyl) -7-methoxy-3-cephem-4-carboxylic acid 1 mmol dibenzhydryl ester of 7β- (D-5-N-tert-butoxycarbonylamino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid is dissolved in 5 ml of methylene chloride and the mixture is cooled to 0 ° C. 1 milol collidine is added, and then a solution of 0.6 mmol of phosphorus pentachloride is added dropwise added in 5 ml of methylene chloride. The mixture is then placed in an ice bath for 1 hour stirred at 0 ° C and the solution obtained with sodium bicarbonate, dilute hydrochloric acid and a saturated sodium chloride solution. The mixture becomes dry evaporated and then chromatographed on a cooled silica gel column isolated using chloroform as the eluant. The product thus obtained is the dibenzhydryl ester of 7ß- (D-5-N-tert-botoxycarbonylamino-5-carboxyvaleramido) -3- (chloromethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Step C: Trifluoroacetic acid salt of 7β- (D-5-amino-5-carboxyvaleramido) -3- (chloromethyl) -7-methoxy-3-cephem-4carboxylic acid A solution of 1 mmol of dibenzhydryl ester of 7β- (D-5-N-tert-butoxycarbonylamino-5-carboxyvaleramido) -3- (chloromethyl) -7-methoxy-3-cephem-4-carboxylic acid 13 ml of anisole are poured onto 6.5 ml of cold (0 ° C.) trifluoroacetic acid with stirring. After 5 minutes, the solution is kept at 0 ° (while stirring in 1800 ml Poured ethereal solution.

The resulting solid precipitate is then collected and dried, wherein 7β- (D-5-amino-5-carboxyvaleramido) -3- (chloromethyl) -7-methoxy-3-cephem-4-carboxylic acid trifluoroacetate is obtained.

The new compounds of the invention have been identified as compounds with the 5-Amino-5-carboxyvaleramido radical in the ß-configuration with respect to the cephem nucleus described. Although this is based on information currently available and as a Assuming correctly, the connections are not intended to be based on this particular configuration should be specified if it is proven to be incorrect on the basis of further findings became.

The organisms mentioned in the present description are in deposited in the culture collection of the American Type Culture C'ollection, where it is under the following ATCC designations are available: Escherichia coli W-MB-60 ATCC 9637 Proteus vulgaris MB-838 ATCC 21100 Alcaligenes faecalis MB ATCC 212 Alcaligenes viscosus MB-12 ATCC 337 Vibrio percolans MB-1272 ATCC 8461 Bacillus subtilis MB-964 ATCC 6633 Several materials with trade names are also used in the description bez ei without. These have the following composition and are of the following References available: Amber Yeast No. 300: A fraction of autolyzed brewer's yeast; Amber Laboratories, Juneau, Wisconsin.

Nobel par-S: an oil-based defoamer (composition unknown); Mobil Oil Company, 150 E. 42nd Street, New York, New York.

Polyglycol 2000: A Defoaming Agent; Polypropylene glycol polymer with an average molecular weight of 2000; Dow Chemical Company, Midland, Michigan.

Biogel P-2: a gel filtration medium; a spherical polyacrylamide crosslinked with methylene-bis-acrylamide; Bio-rad Laboratories, Richmond, California.

Dowex 50: A polystyrene with a sulfonated sulfonic acid cation exchange resin in the core; Dow Chemical Company, Midland, Michigan.

Analtech G.F. Plates; Silica gel with calcium sulfate binder and a fluorescent one Indicator, 250 micrometers thick; Analtech Inc., 100 South Justison Street, Wilmington, Delaware, 19801.

Claims (2)

P a t e n t a n s p r ü c h e 1. Derivatives of 7β- (D-5-amino-5-carboxyvaleramido) -7-methoxy-3-cephem-4-carboxylic acid of the general formula in which R denotes hydroxyl, a radical of the general formula -O-CO-R18 in which R18 is lower-alkyl aryl, a mononuclear, N-containing heterocycle, aralkyl or cycloalkyl is an amidinothio radical of the general formula: in which R4, R5 and R6 represent hydrogen or lower alkyl radicals; a thio radical of the general formula -SR³, in which R3 is a lower alkyl radical or a heterocycle of pyridyl, alkyl-substituted thiazolyl-, 1,3,4-thiadiazol-2-yl, alkyl-substituted 1,3,4-thiadiazol-2-yl, 2 -Benzothiazolyl- or 4-lower-alkylpyrimidin-2-yl radical; an aniinothiocarbonylthio radical of the general formula: wherein R7 and R8 are hydrogen, lower alkyl, hydroxy-lower alkyl, di-lower-alkylamino-lower-alkyl, morpholino-lower-alkyl, N-aryl-N-lower-alkylaminoalkyl radicals or together with the nitrogen atom that they are bound, a heterocycle of norpholino, piperidino, pyrrolidinyl or a piperazino radical of the formula: represent in which R9 is a lower alkyl or phenyl radical; Aroylthio; an oxythiocarbonylthio radical of the formula wherein R10 represents a lower alkyl or lower cycloalkyl radical; Alkarylsulfonyl; Azido; Polyhydroxyphenyl; N-lower alkylindol-3-yl; Tri-lower alkylammonium or pyridinium of the general formula: wherein X¹ is hydrogen, halogen, trifluoromethyl, cyano, carboxy, carbamoyl, N-lower alkylcarbamoyl, N, N-di-lower alkylcarbamoyl, carboxymethyl, lower alkanoyl, lower alkyl, Hydroxymethyl radical or Sulforest, a radical of the general formula -O-CO-NR15R16, in which R15 and R16 are lower alkyl radicals or together with the nitrogen atom to which they are attached, a mononuclear heterocycle of pyrrolidine, piperidine or morpholine, or a radical of general formula -O-CO-NHR17, in which R17 is a lower alkyl, halo-lower alkyl, lower alkoxycarbonyl, aryl, alkarylsulfonyl or benzhydryl radical and salts', esters and amides of these compounds, 2. Medicines, characterized by a content of a compound according to claim 1.