IL36281A - Cephalosporin derivatives antibiotic mixtures containing them and process for their preparation - Google Patents

Description

Tibi Q C process MBRK & CO.* IBC* C.34391 Abstract of the Disclosure Disclosed are 7β- (D-5-amino-5-carboxyvaleramido) -7-methoxy-3-cephem-4-carboxylic acids which are substitute!* in the three position' of the "cephem" necleus by a variety of alkyl/ \haloalkyl or^oxygen, sulfur and nitrogen containing substituents; and the salts,' esters and amide derivatives thereof. Certain of the products, are obtained by fermenta-tion and others are obtained by synthetic means. The products exhibit activity against gram-negative and gram-positive bacteria. ■ This invention relates to a new class of antibiotic substances and to a method for their preparation.

Specifically, these new antibiotics are cephalosporin-type products which contain a methoxy substituent at position 7 of the "cephem" nucleus. They are structurally related to the cephalosporin series of compounds but, unlike cephalo-sporin C which contains only a D-5-amino-5-carboxyvaleramido moiety' at position 7, the instant products also contain a 7-methoxy substituent; and, whereas cephalosporin C is sub-stituted by acetoxymethyl at position 3 to the ring, the products of this invention may contain a wide variety of other substituents. Illustrative of these substituents are, for example, methyl, halomethyl, hydroxymethyl , an acyloxymethyl moiety of the aliphatic, acyclic and aromatic variety, N-substituted and Ν,Ν-disubs tituted carbamoyloxymethy 1 , alky Ithiom ethyl , heterocyclic substituted thiomethyl, tri- alkylammortiummethyl , py ridiniummethy 1 , nuclear substitf¾^d pyridinium ethy 1 , . thiouroniurnmethyl , amidinothiomethyl in which the two nitrogen atoms may be substituted by from one \ to three alkyl radicals, aminothiocarbonylthiomethyl , N- substituted aminothiocarbonylthiomethyl, aroyl thiomethyl , oxythiocarbonylthiomethyl, alkary Isulfonylmethyl , azido-methyl, aminomethyl, amidomethyl, polyhy droxybenzyl. , N- lower alkyl-indol-3-ylmethyl and thiocyanatomethyl . These products can be depicted as follows: wherein R is hydrogen, halo, for example, chl ro, bromo,: , fluoro or iodo and the like, hydroxy, lower alkanoyloxy (except acetoxy) such as n-propionyloxy and the like, mononuclear and bi-nuclear aryl-carbonylox , for example, benzoyloxy, naphthoyloxy and the like, pyridyl-carbonyloxy , aralkanoyloxy such as phenacetyloxy , 3-phenylpropionyloxy , 2-naphthylacetoxy or hydrocinnamoyloxy and the like, cycloalka ne carbonyloxy containing from 5-6 nuclear carbons, for example, cyclopentanecarbonyloxy or cyclohexanecarbonyloxy and the Tike, a-me thoxy-p-s lfooxy-cinnamoyloxy or ct-methoxy-p-hydroxy cinnamoyloxy and the like; a carbamoy loxy 'radical of the formula: -OOCNR^R2 wherein 1 2 R and R are hydrogen, but at least one of them is lower alkyl, for example, methyl, ethyl, n-propyl, tertiary-butyl and the lower alkyl such as chloromethyl , 2-chloroethy1 or chloro- tertiary-butyl and the like, lower alkoxycarbonyl such as ■ ethoxycarbonyl and the like, aryl, for example, mononuclear and bi-nuclear aryl such as phenyl, naphthyl, and the like, alkarysulfonyl, for example, mono-nuclear alkarylsulfonyl such as p-tolylsulfonyl and the like and. benzhydryl or, taken together with the ni trogen atom to which they are attached, R 1 and R2 may be joined to form a mononuclear heterocycle such as pyrrolidinyl , piperidino or morpholino; a thio radical of the formula: -SR3 whereh R3 is lower alkyl such as methyl, ethyl, n-propyl and the like, a nitrogen-containing heterocycle as, for example, pyridyl such as 2-, 3- or 4-pyridyl, a lower alkyl substituted thia- zolyl such as 4-methylthiazol-2-y1 or 4-ethylthiazol-2-y1 and the like, 1 , 3 , 4-thiadiazol-2-yl , a lower alkyl substituted 1 , 3 , 4-thiadiazol-2-yl such as 5-methyl-l , 3 , 4-thiadiazol-2-yl and the like, 2-benzothiazolyl , a 4-lower alkyl-pyrimidinyl such as 4-methylpyrimidin-2-yl; an ammonium radical, for example, a pyridinium radical of the formula wherein is hydrogen ■,■halogeny, trifluoromethyl, cyano, carboxy, carbamoyl, N-lower alkyl and N,N-di-lower alkyl substituted carbamoyl such as N-methyl carbamoyl, N-ethyl carbamoyl, Ν,Ν-dimethylcarbamoyl, N,N-diethylcarbamoyl or N,N- di-isopropylcarbamoyl , carboxymethy1 , lower alkanoyl such ' as acetyl or propidnyl and the like, lower alkyl such as methyl, ethyl or n-propyl and the like, hydroxymethyl or sulfo, i.e., -S02 (OH) ; a thiouronium radical of the formula: S \ II 7 ft the formula: -SCNR'R0 herein R 7 8 w ' and R are the same or different members selected from hydrogen, lower alkyl such as methyl, ethyl, n-propyl and the like, hydroxy-lower alkyl such as 2-hydroxyethyl and the like, di-lower alkylamino-lower alkyl such as 2- (dimethylamino) ethyl, 2- (diethylamino) ethyl , 2- (di-n-propyl-amino) ethyl, or 3- (diethylamino) propyl and the like, or-pholino substituted lower alkyl such as 2-morpholinoethyl and the like, N-aryl-N- lower alkylaminoalkyl such as 2- (N-phenyl-N-methylamino) ethyl or, taken together, the R7 and R radicals may be joined with the nitrogen atom to which they are attached to form morpholino, piperidino, pyrroli-dinyl or a piperazino radical of the formula: wherein R is lower alkyl, such as methyl ethyl, n-propyl, isopropyl, n-butyl, amyl, aroylthio, for example, a mononuclear aroylthio such as benzoylthio, an oxythiocarbonylth radical of the formula: 10 OR wherein R10 is lower alkyl such as ethyl, n-propyl, iso-propyl, 'n-butyl, n-hexyl and the like or lower cyclo-alkyl, i.e., a cycloalkyl containing from 5 to 6 nuclear carbon atoms such as cyclopentyl and cyclohexyl; alkaryl-sulfonyl, for example, a iononuclear alkarylsulfonyl such as p-tolylsulfonyl and the like; azido; amino, an amido radical; of the formula: -NHR11 wherein is lower alkanoyl such as acetyl or propionyl or aralkanoyl as, for example, a mononuclear aralkanoy^ such as 2-phenylacetyl and the like; polyhydroxyphenyl, for example, dihydroxyphenyl such as 2 , -dihydroxyphenyl or an N-lower alkyl indol-3-yl such as N-methylindol-3-yl and the like or thiocyanato. Also included are the pharmacologically acceptable salts, esters and amides of the instant products. These include organic and inorganic salts as, for example, acid addition salts, metal salts, quaternary salts and amine salts derived from tertiary organic nitrogen-containing bases.

Suitable ester and amide derivatives include the mono- and di-esters s ch as are derived from alkanols, cycloalkanols aromatic alcohols and aralkanols and mono- and di-amides such as are derived from ammonia, lower alkylamines, di-lower alkylamines, aralkylamines and heterocyclic amines.

Examples of these derivatives and the methods for their preparation can be found in the sub-section entitled Synthetic Methods.

Antibiotic 810A; Essentially, the products of Formula I, supra.*, comprise two groups of fermentation products.

One of these is a mixture of compounds from which two distinct products have been isolated and identified.

These two products are characterized by the presence of an a-methoxy-p-sulfooxycinnamoyloxy or an a-methoxy-p-hydroxycinnamoyloxy moiety at position 3 of the cephem nucleus and correspond to the following planar formula: la wherein R1* is hydroxy or sulfooxy, i.e., -OSO3H. Thesre products are co-produced by cultivating under controlled conditions a new strain of actinomycete designated as MA-2837 in the culture collection of Merck & Co., Inc., Rahway, New Jersey. A sample of this culture has also been placed on permanent deposit with the culture collection of the Northern Utilization Research and Development Branch of the Ό. S. Department of Agriculture st Peoria, Illinois, and has been assigned the culture number NRRL 3851. Twenty-five other cultures have also been identified as producers of this antibiotic mixture (la) and these, together with culture MA-2837, are described infra in the section entitled The Microorganism. Hereinafter the antibiotic mixture (la) comprising these two products will be referred to as Antibiotic 810A or simply, 810A.

Antibiotic 842A; The second group of fermentation products comprises 73- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (lb, infra) and its salts. Ths compound has the following planar formula: This product (lb) is also produced by a new strain of actino- , mycete and a sample of this m croorganism, designated as MA-2908, has been placed in the culture collection of Merck & Co.', Inc., Rahway, New Jersey. A sample of this culture has also been placed on permanent deposit with the culture collection of the Northern Utilization Research and Develop-ment Branch of the U.S. Department of Agriculture at Peoria, Illinois. This culture has been assigned the culture nuiuber NRRL 3802. This* product (lb) has itself antibiotic activity and constitutes an intermediate in the preparation of the corresponding 3-hydroxy, 3-acyloxy and carbamoyloxy derivatives. The method for the preparation of these Λ derivatives is described in the subsection entitled Synthetic Methods. Hereinafter, in this specification, the product lb, i.e., 7B- (D-5-amino-5-carboxyvaleramido) -3- (carbamo loxy-methyl) -7-methoxy-3-cephem-4-carboxylic acid, will be referred to as Antibiotic 842A or, simply, 842A.

ACTIVITY One major difficulty in antimicrobial therapy is the susceptibility of most antibiotics to enzymatic degrada-tion. Penicillin G, for example, is effective against a wide variety of gram-positive and gram-negative microorgan-isms but in the presence of penicillinase it is degraded to a form which is ineffective against most pathogens.

One approach to this problem has been the develop-ment of new antibiotics which contain the "cephem" nucleus characteristic of cephalosporin C. Cephalosporin C possesses an inherent resistance to penicillinase and is active against both gram-negative and gram-positive bacteria; however, l^- s only moderately active and there exist enzymes- other than penicillinase which are effective in destroying its activity. These enzymes are designated as cephalosporinases. The pro-ducts (I) of this invention demonstrate resistance not only to penicillinase but to the cephalosporinases as well. They exhibit activity against ' both gram-negative and gram-positive bacteria but the order of activity and the range of organisms against which they are effective is not identical.

Antibiotic 842A is characterized by an enhanced activity against gram-negative microorganisms. Unlike cepahlo sporin C which has a relatively low antibacterial activity, this product exhibits a significant i vivo gram-negative effect with a potency which, in general, is greater than cephalothin. This activity includes effectiveness in vivo on Proteus morga i and an effectiveness against the following gram-negative bacteria: Escherichia coli, Proteus vulgaris, Proteus mirabilis, Proteus morganii , Salmonella schottmuelleri Klebsiella pneumoniae AD, Klebsiella pneumoniae B, and Paracolobactrum arizoniae.

In addition to a generally increased gram-negative effect and an increased potency when compared to cephalothin and a greater resistance to cephalosporinases, 842A is characterized by a low order of toxicity and produces rapid blood levels. Within six hours after administration approximately 100% is eliminated in the urine. In-addition it is more resistant to enzymatic degradation than cephalosporin C and resistance to it develops slowly and it is bactericidal. Given orally it protects against Proteus vulgaris 1810, and Salmonella schottmuelleri 3010^^--. and when administered subcutaneously, it is from two to ten times more effective than cephalothin against the same in ections.

Antibiotic 81OA is a broad spectrum agent which exhibits an approximately balanced gram-negative and gram-positive effect. This includes activity in vivo against the following gram-negative organisms: Proteus vulgaris, Proteus roirabilis , Salmonella pullorum, Escherichia coli, and Klebsiella pneumoniae and in vivo activity against the fol- lowing gram-positive organisms: Staphylococcus aureus, Streptococcus pyogenes and Diplococcus pneumoniae .

Of the several products comprising the Antibiotic 810A, the 7β- (D-5-amino-5-carboxyvaleramido> -3- (a-methoxy-p-sulfooxy-cinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid species, corresponding to Formula la, supra, wherein R is sulfooxy, and the salts thereof such as the sodium salt, constitutes a preferred embodiment of this invention. This This compound has a greater resistance to cephalosporinases than cephalothin and is characterized by a low order of toxicity in mice. In addition it is more resistant to enzymatic de- gradation than cephalosporin C and it is bactericidal. Given orally it protects against infections due to Proteus vulgaris and, when administered subcutaneously, it is effective against THE MICROORGANISMS 810A Cultures : The microorganism which produces Antibiotic 810A was originally isolated as a single colony from soil. This colony was passed onto a streak plate of 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 microor .anism produced the Antibiotic 810A. This antibiotic was then reproduced in shake flasks and differentiated from known antibiotics on the basis of various biological and chemical studies. Com-parisQn of this data with that obtained via other known antibiotics established 810A as a new entity. 810A Taxonomy and Morphology; The microorganism (Culture MA-2837) which produces Antibiotic 810A has been identified as Strep omyces griseus . The taxonomy employed in this determination is described in "Bergey's Manual of Determinative Bacteriology", Seventh Edition; and in "The Actinomycetes , Vol. 2_, by S.A. Waksman (1961) . Using that procedure the culture was found to be a strain of Strepto-myces griseus which closely resembles Streptomyces griseinus in description, melanin production and carbon utilization as described in the International Journal of Systemic Bacteriology", Seventh Edition (1957) because the group of Streptomyces griseus cultures was divided in 1959 two years after publication of that Edition. However, in both Waksman and in the "International Journal of Systemic Bacteriology", Streptomyces griseinus is defined as the "grisein-producing strain of Streptomyces griseus> or as "producing grisein or grisein-like substances". Strepto- ^ myces griseus (MA-2837) is a strain that differs from the classic description in Bergey and in Waksman inasmuch as it possesses an aerial mycelium which is predominantly tannish yellow and greenish yellow on some media and with a slightly different carbon utilization pattern.

Waksman, on page 111 and 133 of "The Actinomycetes" des-cribes that Streptomyces griseus series as one which encompasses many related species and strains, as character-ized by colorless to olive-buff substrate growth, aerial mycelium that is yellowish with a greenish tint, or greenish-grey or sear or grass-green to grey, melanin negative, sphorophores straight or flexuous and produced in tufts, spores oval. The following Tables compare the characterisitcs of the culture which produces Antibiotic 810A and the Streptomyces griseus and Streptomyces griseinus cultures.

The characterization of the parent isolate MA-2837 as compared with Streptomyces griseus described in Bergey and Waksman2 and, also, the characterization of MA-2837 as compared with Streptomyces griseinus as described in Waksman2, are set forth in Tables I and la, infra.

TABLE I COMPARISON OP CULTURAL CHARACTERISTICS OP MA-28 1 Bergey' s "Manual of Determinative Bacteriology", Seventh 2 Waksman, S. A., "The Actinomycetes" , Vol. 2_ (1961).

TABLE l a COMPARISON OF CULTURAL CHARACTERISTICS OF MA-283 Medium MA-2837 S. griseus (Bergey) Tomato paste - Vegetative Growth: Reverse-oatmeal Agar brown, flat, spreading Aerial Mycelium: Center-tan to grayish-yellow Edge-tannish yellow Soluble Pigment: Tan Glycerol-aspara- Vegetative Growth: Reverse-gine Agar tan, flat, spreading Aerial Mycelium: Powdery, tannish-yellow .

Soluble Pigment: Light, tannish yellow Czapek-Dox Agar Vegetative Growth: Reverse- Gro (Sucrose nitrate yellowish orange, flat, ing, agar) spreading com Aerial Mycelium: Powdery, Aer tannish-yellow (several thi shades but predominantly wat edge-tannish yellow) men growth light in center and heavier on edges Soluble Pigment: Light, tannish yellow TABLE la (Cont.) .. S.'-.grise'us Medium MA-2837 (Bercrev) Egg Albumin Vegetative Growth: Reverse-agar grayish tan, flat, spreading Aerial Mycelium: Tan-yellow with greenish cast, edge tannish yellow, light growth in center heavier along edges.

Soluble Pigment: light tannish yellow Nutrient Agar Vegetative Growth: Reverse- Grow brownish yellow almo Aerial Mycelium: Velvety, crea tannish yellow, edge Aeri tannish yellow powd Soluble Pigment: Light ligh brown solu Agar Abundant, cream- colored almost transparent growth, aerial mycelium powdery, white to light grey. No soluble pigment.

Synthetic Agar Thin, spreading, colorless growth becoming olive- buff Aerial mycelium thick, powdery, water-green.

TABLE la (Cont.) S. griseus ' S Medium MA-2837 (Bergey) Gelatin Stab Flaky cream-colored growth Gree settling in bottom of tube. crea Complete liquefaction. face brow Rapi tion.

Nutrient Vegetative Growth: Cream Gelatin Agar Aerial Mycelium: Pale, tannish yellow Soluble Pigment: None Liquefaction of gelatin- good.

Litmus Milk Partial ring Crea Vegetative Growth: Browncoag ish rapi Aerial Mycelium: Slight, tion , whitish alka Peptonization becoming alkaline Skim Milk Partial ring Vegetative Growth: Brownish Aerial Mycelium: None Soluble Pigment: Light brown Peptonization becoming alkaline TABLE 2a (Cont.) S. grlseus ; Medium MA-2837 (Bergey) Skim Milk Agar Vegetative Growth: Cream flat, spreading Aerial Mycelium: Sparse, yellowish white to cream Soluble Pigment: Very light brown Hydrolysis of casein.

Glucose Agar Grow cent crea • oran marg Glucose. Broth Abundant, yellowish pellide with greenish tinge, much folded Starch Agar Thin, spreading Grow transparent growth ing , Starch is hydr hydrolyzed.

Nutrient Starch Vegetative Growth: Cream , Agar Aerial Mycelium: Pale tannish yellow Soluble Pigment: None >.

Hydrolysis good TABLE la (Cont.) S. griseus Medium . MA-2837 (Bergey) Potato Plug Vegetative Growth: Light Yellowish, wrinkled Grow brown growth covered with yell Aerial Mycelium: Moderate, white , powdery ish, tan aerial mycelium. whit Slight browning of aeri potato.

Calcium Malate Vegetative Growth: Plat, Gree Agar spreading, translucent solu and colorless at edges prod opaque and cream- calc colored in center. and Aerial Mycelium: Moderate, cream to yellow, edges tannish yellow Soluble Pigment: None Nutrient Vegetative Growth: Flat Dark Tyrosine Agar spreading, cream-colored prod Aerial Mycelium: Yellowish tan with greenish cast, edges tannish yellow Soluble Pigment: Very light brown Tyrosine crystale decomposed.

Peptone-iron- Vegetative Growth: Cream-yeast Extract colored Agar Aerial Mycelium: None Soluble Pigment: None Melanin negative TABLE la (Cont.) S. griseus Medium MA-2837 (Bergey) Production of H2S Negative ' Neg Loeffler's Blood Vegetative Growth: Tan Serum Slants Aerial Mycelium: Slight yellowish Soluble Pigment: Brownish Complete liquefaction.

Temperature 28°C - good growth Optimum temperature Range (yeast 37°C - good rowth 37°C. extract-dextrose- 50°C - no growth salts agar slants) Microaerophilic Good growth covering Aerobic Growth (yeast exsurfact and along tract-dextrose- entire stab line. salts stab-40 mm depth) Reduction of Negative Positive Pos nitrates to nitrites These observations were made after three weeks in-cubation at 28°C . except where otherwise noted. The pH of the media used in these studies was approximately neutral , that is , 6 . 8 to 7. 2 . The physiological tests were run at the end of seven and twenty-two days . The colors used in the description are in accordance with the definitions of the "Color Harmony Manual " , Fourth Edition , 1958; Container Corporation of America. 810A Carbohydrate Utilization : The Stre tomyces griseus culture (MA-2837) was also tested for its abili ty to utili ze or assimilate various car-bohydrates by growing the microorganism in basal synthetic medium (T. G. Pridham and D. Gottlieb , 1948) containing 1% of the carbohydrate at 28°C. for three weeks . Table II infra , indicates the utilization or assimilation of these carbo-hydrate sources by the Streptomyces griseus culture (MA-2837) The explanation of the symbols in Table II are as follows : + indicates good growth , ΐ indicates poor growth , and - in-dicates no growth on the particular carbohydrate .

TABLE II MA-2837 MA-2837 Carbohydrate Culture Carbohydrate Culture Glucose + Lactose + + Arabinose + Inositol Xylose + Sucrose + Maltose + Rhammose + Mannose + Raf f inose + Fructose - Cellulose - Mann i to 1 + The characterisitcs described in Tables I , la an< were used to reduce the Streptomyces griseus culture to a species classification via the keys described in "Bergey ' s Manual of Determinative Bacteriology " , Seventh Edition, pages 694-829 (1957) and in "The Actinomycetes " , Vol. 2_: pages 61-292 (1961). A comparison of the culture^ (MA-2837) with known species shows that it is similar to Streptomyces griseus . There are morphological differences as, for example, in the color of the aerial mycelium which, in Streptomyces griseus , is predominantly tannish yellow and greenish yellow but, in view of the significant number of similarities and the only minor differences there is no justification for a new species name. As a result, the microorganism (MA-2837) which produces Antibiotic 810A has been identified as a strain of Streptomyces griseus.

In addition to the foregoing culture (MA-2837) , 25 additional cultures have been identified as producers of the Antibiotic 810A. These include: three cultures of Streptomyces griseus , eleven cultures of Streptomyces viri-dochromogenes , five cultures of Streptomyces fimbriatus , three cultures of Streptomyces halstedii, one culture of Streptomyces rochei , one culture of Streptomyces cinnamonensis and one culture of Streptomyces chartreusis . These strains of Streptomyces are identified as cultures 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 in the culture collection of Merck & Co., Inc., Rahway, New Jersey. These cultures have been placed on permanent deposit with the culture collection of the Northern Utilization Research and Development Branch of the U.S. Department of Agriculture at Peoria, Illinois, The assigned NRRL culture numbers are as follows: Streptomyces griseus : MA-4160 NRRL 3951 MA-4174 NRRL 3953 ΜΑ·-43.71 NRRL 3952 Streptomyces viridochromogenes : MA-4177 NRRL 3970 MA-4178 NRRL 3971 MA-4180 NRRL 3972 MA-4164 NRRL 3966 MA- 165 NRRL 3967 MA-4166 NRRL 3968 MA-4167 NRRL 3969 A-2892 NRRL* 3962 MA-3265 NRRL 3963 MA-4162 NRRL 3964 MA- 163 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- 175 NRRL 3960 MA-4181 NRRL 3961 Streptomyces rochei: MA-2938 NRRL 3973 Streptomyces cinnamonesis: MA-4176 NRRL 3974 Streptomyces chartreusis ; The characterization of the aforementioned isolates as compared with Streptomyces griseus, Streptomyces virido-chromogenes , Streptomyces fimbriatus, Streptomyces ha ste^ , Streptomyces rochei, Streptomyces cinnamonensis and Streptomyces chartreusis are set forth below in Tables Ila-lie.

TABLE ria CULTURAL CHARACTERISTICS OF STR STREPTOMYCES GRISEUS PRODUCING ANTIBI Medium MA-4160 MA-4174 Morphology Sporophores form tufts with spore cha flexuous. Spores are spherical to ov 1.2μ - in chains of approximately 10- Tomato Paste Vegetative Growth: Good, fla Oatmeal Agar Aerial Mycelium: Aerial Myceliu Powdery; tan with Powdery; tan greenish cast strong green tone Soluble Pigment: Li Glycerol- Vegetative Growth: Goo asparagine Aerial Mycelium: Powdery, tan with g Agar gray-green Soluble Pigment: Li Czapek Vegetative Growth: Flat, spr Agar Aerial Mycelium: Aerial Myceliu Moderate; tan Moderate; ta Soluble Pigment: Soluble Pigmen TABLE Ila (cont.) I.

·»» I ' TABLE lib CULTURAL CHARACTERISTICS OP STRAINS OP STREPTOMYCES VIRIDOCHROMOGEN Medium MA-2892 MA-3265 MA- Morphology Sporophores are short, compact spirals occurring as si Spores are spherical to oval - 0.9 to 1.2y diameter an in chains of approximately 10-15 spores.

Tomato Paste - Vegetative Growth: Vegetative Growth: Vegetativ Oatmeal Agar Reverse brown Reverse tan Reverse Aerial Mycelium: Aerial Mycelium: Aerial My Velvety; dark Velvety; light Velvety g ay & white gray & white gray & Soluble Pigment: Soluble Pigment: Soluble P Light brown None Light b Glycerol-aspara- Vegetative Growth: Vegetative Growth: Vegetativ gine Agar Reverse dark . Reverse dark Reverse brown brown brown Aerial Mycelium: Aerial Mycelium: Aerial My Dark gray and Light gray Cream & cream Soluble Pigment: Soluble Pigment: Soluble P Light brown None Light b TABLE Tib (cont.) Medium MA-2892 A-3265 MA-41 Czapek-Dox Vegetative Growth: Vegetative Growth: Vegetative Agar Dark brown Dark brown Dark brow Aerial Mycelium: Aerial Mycelium: Aerial Myce Very scant Light gray & Very scan white Soluble Pigment: Soluble Pigment: Soluble Pig Dark brown Light brown Light bro Yeast Extract Vegetative Growth: Vegetative Growth: Vegetative Dextrose Agar Dark brown Reverse brown Dark brow Aerial Mycelium: Aerial Mycelium: Aerial Myce Very scant Light gray Very scan Soluble Pigment: Soluble Pigment: Soluble Pig Light brown None Light bro Soluble Pigment on Peptone- Dark brown Iron-Yeast Extract Agar TABLE lib (cont.) Medium MA-4164 MA-4165 MA-41 Morphology Sporophores are short, compact spirals occurring as side Spores are spherical to oval - 0.9 to 1.2 diameter and in chains of approximately 10-15 spores.

Tomato Paste - •Vegetative Growth: Reverse dark brown Oatmeal Agar Aerial Mycelium: Aerial Mycelium: Aerial Myce Velvety; blue- Velvety; medium Velvety; gray & cream gray & cream gray & cr Soluble Pigment: Ligh brown- Glycerol-aspara- Vegetative Growth: Vegetative Growth: Vegetative gine Agar- Reverse dark Reverse dark Dark gray brown brown Aerial Mycelium: Aerial Mycelium: Aerial Myce Dark gray & . Medium gray & Scant-gre cream cream —Soluble Pigment: Light brown- Czapek-Dox Vegetative Growth: Vegetative Growth: Vegetative Agar Brown Reverse brown Brown Aerial Mycelium: Aerial Mycelium: Aerial Myce Very scant Light gray & Very scan cream Soluble Pigment: Soluble Pigment: Soluble Pig Brown Light brown Brown TABLE Hb (cont.) Medium MA-4164 MA-4165 MA-4 Yeast Extract - -Vegetative Growths Dark brown Dextrose Agar —Aerial Mycelium: Very scant - —Soluble Pigment: Light brown Soluble Pigment Dark brown on Pe tone-Iron-Yeast Extract Agar TABLE lib (cont.) Medium MA-4177 MA-4178 Morphology Sporophores are short, compact spiral on aerial hyphae. Spores are spheric diameter and 0.9 - 1.2 x 1.2 - 1.7μ - 10-15 spores.

Tomato Paste Vegetative Growth: Vegetative Gro Oatmeal Agar Reverse tan Reverse brow Aerial Mycelium: Aerial Myceliu Velvety; dark Velvety; dar gray & white gray -Soluble Pigment: L Glycerol-aspara- Vegetative Growth* Vegetative Gro gine Agar Reverse brown Reverse dark Aerial Mycelium: Aerial Myceliu Dark gray Dark gray -Soluble Pigment: Czapek-Dox Vegetative Growth: Vegetative Gr Agar Tan Dark brown -Aerial Mycelium: -Soluble Pigment: TABLE Ti (cont.) Medium MA-4177 MA-4178 Yeas Extract Vegetative Growth: Vegetative Gro Dextrose Agar Dark brown brown Aerial Mycelium: Aerial Myceliu Scant-grayish Very scant Soluble Pigment on Peptone- Iron-Yeast Extract Agar TABLE lie CULTURAL CHARACTERISTICS OP STRAINS OP STREPTOMYCES' PIMBRIATUS Medium MA-4159 MA-4169 MA-4170 Morphology Sporophores are short, compact spirals and some loops, occu branches on aerial hyphae. Spores are spherical to oval - and 0.9 x 1.2μ - chains of approximately 10-15 spores.

Tomato Vegetative Growth: Vegetative Growth: Vegetative Growth: Paste - Reverse tan Reverse tan Reverse tan Oatmeal Agar Aerial Mycelium: Aerial Mycelium: Aerial Mycelium: Moderate; light Moderate; light Moderate; light gray gray gray & cream Soluble Pigment: Light brown.

Glycerol- Vegetative Growth: Vegetative Growth: Vegetative Growths asparagine Dark brownish- Dark brownish- Dark brownish- Agar gray gray gray TABLE lie (cont.) Medium MA- 159 MA-4165. MA-4170 Czapek-Dox Vegetative Growth: Vegetative Growth; Vegetative Growth; V Agar Dark brownish- Dark brownish- Dark brownish- gray gray gray Aerial Mycelium: Aerial Mycelium: Aerial Mycelium: A Very scant Moderate; gray Moderate; gray Soluble Pigment: Soluble Pigment: Soluble Pigment: S Brown Brown Brown Yeast Vegetative Growth; Vegetative Growth: Vegetative Growth: V Extract Dark brownish- Dark brownish- Dark brownish-Dextrose + gray gray gray Salts Agar Aerial Mycelium: Very sca Soluble Pigment: Brown Soluble Pigment on Peptone- Dark brown Iron-Yeast Extract Agar TABLE lid CULTURAL CHARACTERISTICS OP STRA STREPTOMYCES HALSTEDII PRODUCING ANTIBI Medium A-4168 MA-4175 Morphology Sporophores are long , loose spirals oc aerial hyphae. Spores are spherical t 0.9 x 1.2y - in chains of more than 10 Tomato Paste- Vegetative Growth: Vegetative Growt Oatmeal Agar Reverse brown Reverse tan Aerial Mycelium: Aerial Mycelium: Powdery; dark Dark grey & wh grey powdery Soluble Pigment: Glycerol-aspara- Vegetative Growth: Vegetative Growt gine Agar Reverse brow to Reverse greyis dark brown Aerial Mycelium: Aerial Mycelium: Powdery; dark Powdery, predo grey and white inantly dark g mixed with lig grey and white Soluble Pigment: TABLE lid (cont.) Medium MA-4168 A-4175 Czapek-Dox Vegetative Growth: Vegetative Growth Agar Cream Reverse reddish brown Aerial Mycelium: Aerial Mycelium: Greyish cream Greyish cream •Soluble Pigment: N Yeast Extract Vegetative Growth: Vegetative Growth Dextrose + Tan Tan Salts Agar Aerial Mycelium: Aerial Mycelium: Greyish; scant Scant; greyish Soluble Pigment: N Soluble Pigment Peptone-Iron- None Yeast Extract Agar TABLE tie CULTURAL CHARACTERISTICS OP STREPTOMYCES SPECIES PRODUCI Medium MA-2938 MA-4176 Streptomyces1 rochei Streptomyces cinnamones Morphology Sporophores form compact Sporophores are hooks, spirals occurring as side loops, & a few loose chains of approximately spirals, occurring as s 10-15 spores - spherical . branches on aerial hyp to oval, 0.9y diameter & Spores are in chains of 9 x 1.2μ moxe than 10 spores - spherical to oval, 0.9u diameter & 0.9 x 1.2μ Vegetative Growth: Reverse Vegetative Growth: Ta Tomato Paste - reddish-brown Oatmeal Agar Aerial Mycelium: Medium Aerial Mycelium: Beig gray with pink tint; velv Soluble Pigment: None Soluble Pigment: BrOw Glycerol-aspara- Vegetative Growth: Vegetative Growth: gine Agar Reverse reddish-brown Reverse dark reddish brown Aerial Mycelium: Medium Aerial Mycelium: Beig gray with pink tint Soluble Pigment: None Soluble Pigment: Brow TABLE He (cpnt.) Medium MA-2938 MA-4176 Czapek-Dox Agar Vegetative Growth: Vegetative Growth: Reddish brown Reverse dark brown Aerial Mycelium: Aerial Mycelium: Very scant Moderate; beige with pink tint Soluble Pigment: None Soluble Pigment: Brown Yeast Extract Vegetative Growth: Tan Vegetative Growth: Brow Dextrose + Salts Aerial Mycelium: Aerial Mycelium: Sparse Agar Grayish creamish-white Soluble Pigment: Soluble Pigment: Light brown Light brown Soluble Pigment None Dark Brown on Peptone-Iron-Yeast Extract Agar The foregoing description of the microorganisms^^^ producing Antibiotic 810A is simply illustrative of the type of strains which can be used and it should be under-stood that this invention is not limited to an organism meeting these particular descriptions. This invention includes the use of other microorganisms including strains of actinomycetes isolated from nature or obtained by mutation as, for example, those obtained by natural selection or those produced by mutating agents as, for example, X-ray irradiation, ultraviolet irradiation, nitrogen mustards and the like which, under suitable conditions will yield an identical antibiotic. 842A Culture ; The microorganism which produces Antibiotic 842A is a previously unknown strain of actino-mycete. The original isolate was obtained as a single colony from soil on an agar slant and grown in a medium having the following composition : Medium B: Yeast Extract 10.0 g.

.Glucose 10.0 g.

T>hosphate Buffer 2.0 ml.

MgS04 . 7H,0 0.05 g.

Distilled Water 1000.0 ml. pH 6.5 *Phosphate Buffers ΚΗ,ΡΟ, 91.0 g.

Na~HP0. 95.0 g.

Distilled Water 1000.0 ml.

After several days of growth it was found that no sporula-tion could be detected. The microorganism produced an antibiotic which was differentiated from known antibiotics on the basis of its profile in various biological and chemical The taxonomy employed in this determination is described in "Bergey's Manual of Determinative Bacteriology", Seventh edition; and in "The Actinomycetes " , Vol. 2, "Classification, Identification and Description of Genera and Species", S.A. Waksman (1961) . Using that procedure the culture was found to belong to the genus Streptomyces and it possesses many of the attributes of the known species Streptomyces fradiae. Biochemically it is an essentially perfect match with the latter. Morphologically, however, there are important differences. For example, the color of the aerial mycelium of fradiae is a seashell pink whereas the culture MA-2908 is usually cream colored. Also, the vegetative growth in the MA-2908 culture shows pigment differences on the various media employed, and, as stated below, no sporulation was detected on standard taxonomic media. On the basis of these differences, the culture was assigned a new species name: Streptomyces lactamdurans . Table III, infra, describes the biochemical attributes of the Streptomyces lactamdurans species and those of the known Streptomyces fradiae . All of the readings in Table III were taken after three weeks incubation at 28eC. except where otherwise noted. The pH of the media used in these studies was approximately neutral, namely, 6.8 to 7.2. The physiological tests were run at the end of seven and twenty-one days.

TABLE III 842Ά Biochemical Compariso: Test Streptomyces Streptomyces fradiae lactamdurans " aerial straight, straight, mycelium some branching branching-filaments conidia none detected rod-shaped soluble none none pigment optimum 28°C. 25°C. temperature invertase negative negative reduction of negative negative nitrate gelatin positive positive liquefaction cellulose negative , negative utilization litmus milk alkaline alkaline peptonization peptonization 842A Morphology t Sporophores were not detected when the culture was grown on the media listed in the description of culture characteristics even though repeated observations were made up to 8 weeks. However, stained impression slides showed long filaments, many segmented into sub-units of various sizes, generally, broad shaped and approximately 0.9 by 1.7 microns in size.

Aerial mycelium is short, straight, little branching. It appears to be about the same size as the vegetative myce^¾^-0.9 μ in width. It is light, powdery and scrapes off easily.

Vegetative mycelium is gram positive, not acid-fast.

It clings to, and in some media, is embedded in the agar.

There is some fragmentation into rods in shake flask growth but this is not extensive. Vegetative mycelium from shaker and stationary flasks (seed medium, 4-6 days, 28°C.) showed some "buds" and short, thickened, almost club shaped seg-ments on mycelium but these were not numerous and their significance, if any, is not known.

Tomato-Paste-Oatmeal Agar Vegetative growth - reverse, orange flat, dry appearing, wrinkled Aerial mycelium - sparse, cream No soluble pigment Czapek-Dox Agar Vegetative - flat, deep cream Aerial Mycelium - powdery, creamish white No soluble pigment Glycerol-Asparagine Agar Vegetative growth - flat, reverse - golden yellow to orange Aerial mycelium - powdery, cream with pale peach tones Soluble pigment - pale amber Egg Albumin Agar Vegetative growth - flat, cream to yellow Aerial mycelium - powdery, cream No soluble pigment Calcium Malate Agar Vegetative growth - flat reverse - yellow edged with orange Aerial mycelium - powdery, white to cream edged with peach No soluble pigment Nutrient Tyrosine Agar Vegetative growths - flat, tan to orange Aerial mycelium - sparse, cream with white No soluble pigment Tyrosine crystals decomposed Molasses - Yeast Hydrolysate Agar Vegetative growth - flat reverse - orange Aerial mycelium - powdery, creamish white jlo soluble pigment Nutrient Agar Vegetative - Flat to golden yellow Aerial mycelium - powdery, cream No soluble pigment Litmus Milk Sparse growth ring - ten vegetative growth - no aerial mycelium Peptonization: alkaline reaction; pH 7.3 - 7.4 (Control pH - 6.7) Skim Milk Sparse growth ring - tan to orange Vegetative growth - no aerial mycelium Light tan soluble pigment Peptonization - alkaline reaction pH 7.2 (Control pH - 6.6) Skim Milk Agar Vegetative growth - flat, orange Aerial mycelium - moderate, cream to pale coral Gelatin Stab- Sparse cream to orange flask vegetative growth .^B suspended throughout tube No soluble pigment Complete liquefaction Nutrient Gelatin Agar Vegetative growth - flat, orange Aerial mycelium - sparse, powdery cream No soluble pigment Liquefaction of gelatin Nutrient Starch Agar Vegetative growth - flat, orange Aerial mycelium - sparse, powdery, pinkish cream No soluble pigment Moderate hydrolysis of starch Synthetic Starch Agar Vegetative growth, - flat, reverse - cream edged with orange Aerial - powdery, white edted with peach No soluble pigment Moderate hydrolysis of starch Loeffler's Blood Serum Agar Vegetative growth - cream colored to orange Aerial mycelium - none No soluble pigment No liquefaction Peptone-Iron-Yeast Extract Agar Vegetative growth - cream Aerial-sparse-whitish No soluble pigment rowth Temperature - Yeast extract-dextrose slants Good growth at 28°C. ^ Sparse growth at 37eC.

No growth at 50 °C.

Yeast Extract - Dextrose Agar Vegetative growth - flat golden yellow Aerial mycelium - powdery, cream to pale flesh pink No soluble pigment Potato plug Vegetative growth - dry, flat, cream to orange Aerial mycelium - sparse, creamish No soluble pigment Reduction of Nitrates to Nitrates - Negetative All readings were taken after 3 weeks incubation at 28°C. except where noted otherwise. Physiological tests were run at 7 and 21 days.

The morphological differences between Streptomyces lactamdurans and Streptomyces fradiae are set forth in Table IV, infra. The observations were made on the media indicated in Table IV at growth intervals of one week, three weeks and eight weeks. The aerial mycelium of S_. lactam-durans is short and straight with little branching. It appears to be about the same size as the vegetative mycelium, i.e., 0.9 micrometer in width. It is light, powdery and scrapes off easily. The vegetative mycelium is gram-positive; it is not acid-fast. It clings to and in some media is imbedded in the agar. There is some fragmentation into rods in shake-flask growth but this is not extensive.

Vegetative mycelium from shaker and stationary flasks (seed medium four to six days, 28eC.) showed some "buds" and sho ck ned alm st clu - m mycelium but these were not numerous. All of the read in Table IV were taken after three weeks incubation at 28 eC. except where otherwise noted. The pH of the media used in these studies was approximately neutral, that is, 6.8 to 7.2. The physiological tests were run at the end of seven and twenty-one days. The colors used in the description are in accordance with the definitions of the "Color Harmony Manual", Fourth Edition, 1958; Container Corporation of America. 842A Morphological Comparison of a yces lactamdurans an Medium Streptomvces lactamdurans Czapek-Dox Agar Vegetative Growth: Plat, deep cream Aerial Mycelium: Powdery, creamish white No Soluble Pigment Nutrient Agar Vegetative Growth: Plat cream to golden yellow Aerial Mycelium: Powdery, cream No Soluble Pigment jGlycerol-Asparagine Vegetative Growth: Flat, reverse - golden Agar yellow to orange Aerial Mycelium: Powdery, cream with pale peach tones Soluble Pigment: Pale amber pfeast Extract - Vegetative Growth: Flat, golden yellow Dextrose Agar Aerial Mycelium: . Powdery, cream to pale flesh pink No Soluble Pigment jsynthetic Starch Vegetative Growths Flat, reverse - cream Agar edged with orange Aerial Mycelium: Powdery, white edged with peach No Soluble Pigment Moderate Hydrolysis of Starch Potato Plug Vegetative Growth: Dry, flat, cream orange Aerial Mycelium: Sparse, creamish No Soluble Pigment TABLE IV (cont.) Medium Streptomyces lactamdurans Gelatin Stab Vegetative Growth: Sparse cream to orange Vege flakes suspended throughout the tube Aerial Mycelium: No Soluble Pigment Complete Liquefaction Litmus Milk Vegetative Growth: Tan; sparse growth ring Vege Aerial Mycelium: None Peptonization: Alkaline reaction; pH 7.3 - 7.4 (Control pH - 6.7) 842A Carbohydrate Utilization; The Streptomyces lactamdurans culture (MA-2908) was also tested for its ability to utilize or assimilate various carboyhydrates by growing the microorganism in a basal synthetic medium (T.G. Pridham and D. Gottlieb, 1948) which contains 1% of the carbohydrate at 28°C. for three weeks. Table V indicates the utilization or assimilation of these carbo-hydrate sources by the Streptomyces lactamdurans culture (MA-2908). The explanation of the symbols in Table V are as follows: + indicates good growth, - indicates poor growth and - indicates no growth on the particular carbohydrate.

TABLE V MA-2908 MA-2908 Carbohydrate Cluture Carbohydrate Culture Glucose + Rhamnose - Arabinose + Cellulose - + Maltose + Fructose Raffinose + Inositol - Sucrose - Acetate + Xylose + Citrate + Mannitol + Paraffin - + Lactose - Blycerol Mannose - T e characteristics described i Tables III, IV and V were used to reduce the Streptomyces lactamdurans culture (MA-2908) to a species classification via the keys described in "Bergey's Manual of Determinative Bacteriology", Seventh Edition, pages 694-829 (1957) and in "The Actino-mycetes", Vol. 2 x pages 61-292 (1961). A comparison of the detailed characteristics of the Streptomyces lactamdurans biochemically similar to Streptomyces fradiae . indicated above, there are important morphological differ-ences as, for example, in the color of the aerial mycelium of S. fradiae which is seashell pink as compared to the cream color of the culture. Also, whereas the vegetative growth with S_. fradiae shows pigment differences on the various media, no sporulation was detected with the culture. On the basis of these differences and the characteristics described in the foregoing Tables the microorganism producing antibiotic 842A (MA-2908) was assigned the new species name Streptomyces lactamdurans .

The foregoing description of the microorganism which produces antibiotic 842A is simply illustrative of the type of strains of microorganisms which can be used and it should be understood that the present invention is not limited to organisms meeting these particular descriptions. This invention includes the use of other microorganisms, including strains of actinomycetes either isolated from nature or obtained by mutation as, for example, those obtained by natural selection of those produced by mutating agents, for example, X-ray irradiation, ultraviolet irradiation, nitrogen mustards and the like which, under suitable conditions, will afford the 842A product. 1 IN VITRO AND IN VIVO STUDIES 2 Antibiotic 810A; In vitro: The in vitro 3 biological characterization of Antibiotic 810A was 4 established by the disc-plate agar diffusion method. 5 These tests were performed by placing 7 mm. discs, 6 wet with the antibiotic solution, on the surface of ? petri plates poured with 5 ml. of Difco Nutrient 8 Agra and 0.2% Yeast Extract seeded with 5 or 10 ml. 9 of inoculum per 150 ml. of medium and incubated at 0 25eC. or 37eC. for 16 hours. The method and philosophy of these tests are described in the publication: 2 "cross Resistance Studies and Antibiotic Identification", 3 Applied Microbiology, Vol. 6_: pages 392-398 (1958). 4 The following Tables VI, VII, and VIII set forth the 5 results of these antibacterial and cross-resistance 6 tests and indicate the test organisms used and the 7 conditions employed.

TABLE VI 810A ANTIBACTERIAL SPECTRUM; In Vitro Activity Test Organism Test Conditions Inoculum** Incuba ml/150 ml Temp.

Escherichia coli 5 25 Bacillus species 5 25 Proteus vulgaris 5 37 Pseudomonas aeruginosa 5 25 Serratia marcescens 5 25 Staphylococcus aureus 5 25 Bacillus subtilis 5 25 Sarcina lutea 5 25 Staphylococcus aureus 5 37 (Streptomycin-Streptothricin- resistant) Streptococcus faecalis 15 37 Alcaligenes faecalis 5 37 Brucella bronchiseptica 10 37 Salmonella gallinarum 10 25 Vibrio percolans 10 27 Xanthomonas vesicatoria 5 25 * 7 mm = disc size (no inhibition zone observed) ** Overnight culture diluted to a reading of 60 m colorimeter.

TABLE VII 81QA CROSS-RESISTANCE; In Vitro Study Escherichia coli - Strain** Test Conditions Inoculum*** Incub ml/150 ml Temp.

Sensitive parent 5 25 Streptomycin-resistant 5 25 Streptothricin-resistant 10 25 OXAMYCIN-resistant 10 25 Pleocidin-resistant 10 37 Chloramphenicol-resistant 10 25 Chlortetracycline-resistant 10 25 Oxytetracycline-resistant 10 25 Neomycin-resistant 10 37 Tetracycline-resistant 10 25 Viomycin-resistant 10 37 Polymyxin-resistant 10 25 Grisein-resistant 5 25 7 mm = disc size (no inhibition zone observed) Tests performed versus a series of strains of the same parent culture following exposure to antibiotics" Overnight culture diluted to a reading of 60 colorimeter.

TABLE VIII 810A SPECIAL EFFECTS SPECTRUM? In Vitro Study Escherichia coli W-MB-60 Test Conditions (with special addition noted) Inoculum** Incuba ml/150 ml Temp.

Control (no additions) 5 25 0.1 M Phosphate Buffer r PH 5 5 25 0.1 M Phosphate Buffer - pH 7 5 25 0.1 M Phosphate Buffer - pH 9 5 25 Human Blood Plasma 20% 5 25 Cation Exchange Resin 5 25 (Dow ET 91-1%; agar concentration reduced to 1% for resin plate only) 7 mm disc size (no inhibition zone observed) Overnight culture diluted to a reading of 60 colorimeter.

Antibiotic 810A; In vivo: The in vivo biologica^^ characterization indicates that 810A is a broad spectrun* antibiotic which protects against infection with three species of Proteus , two of Salmonella , one strain of Escherichia coli/ two of Klebsiella and three gram-positive organisms : Staphylococcus aureus , Streptococcus pyogenes and Diplococcus pneumoniae.

Methods ; The method employed in this characterization is as follows: Female white Swiss mice, average weight 20-23 grams, were infected intraperitoneally with 3-20 times the number of organisms calculated to be lethal for 50% of the infected control animals (3-20 LDJQ doses) . At the time of infection and again six hours later, therapy was given by the designated route. Controls of the virulence of the culture and the toxicity of the antibiotic for uninfected mice were included in the tests. Seven days after infection the test was considered complete and the amount of the antibiotic (I) that would be required to protect 50% of the infected and treated animals was cal-culated by the method of Knudsen and Curtis; J. Amer. Stat. Assoc. Vol. 42: page 282 (1947).

Results : The results of these tests are listed in Table IV, infra. This data indicates that the antibiotic 810A obtained from culture MA-2837 is a broad spectrum agent, protecting against both gram-positive and gram-negative organisms.

Although effective when given orally (p.o.) the best results were obtained via the subcutaneous (s.c.) or intraperitoneal (i.p.) route. The antibiotic mixture did not kill uninfected mice when two doses containing 1 mg. each of the product were administered intraperitoneally or when two doses of 18 mg. each were administered sub-cutaneously or orally.

TABLE IX 810A In Vivo Activity Route of ED50 in Micro- Test Organism Therapy grams x Two Doses 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* . 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 in vitro biological characterization was established by the discplate agar diffusion method. These tests were performed by placing 7 mm. discs, wet with the antibiotic solution, on the surface of petri plates poured with 5 ml. of Difco Nutrient Agar and 0.2% Yeast Extract seeded with 5 or 10 ml. of standard cell suspension (OD = 0.22 at 660 κΐμ) per 150 ml. of medium and incubated at 25eC. or 37eC. for 16 hours as indicated. The method and philosophy of these tests are described in the publication : "Cross Resistance Studies and Antibiotic Identification", Applied Microbiology, Vol. : pages 392-398 (1958) . The following Tables set forth the results of these antibacterial spectrum and cross-resistance tests, and indicate the test organisms used and the conditions employed.

TABLE X ANTIBACTERIAL SPECTRUM ; IN VI Escherichia coli 25 16 Bacillus sp. 25 7 Proteus vulgaris 37 21 Pseudomonas aeruginosa 25 7 Serratia marcescens 25 7 Staphylococcus aureus 25 7 Bacillus subtilis 25 16 Sarcina lutea 25 9 Staphylococcus aureus 37 7 (Streptomycin-Streptothricin- resistant) Streptococcus faecalis 37 7- Alcaligenes faecalis 37 22 Brucella bronchiseptica 37 28 Salmonella gallinarum 25 20 Vibrio percolans 27 37 Xanthomonas vesicatoria 25 12 * 7 mm = disc size (no inhibition zone observed) TABLE XI 842A CROSS RESISTANCE; IN VITRO ST Escherichia coli - Strain* Test Conditions t N H I B I T Inoculum Incubation Crude 842A ml/150 ml Temp. °C. 8 mg/ml Sensitive parent 5 25 16 Streptomycin-resistant 5 25 14 Streptothricin-resistant 10 25 .13 OXMYCIN-resistant 10 25 13 PleOcidin-resistant 10 37 15 Chloramphenicol-resistant 10 25 13 Chlortetracycline-resistant 10 25 21 Oxytetracycline-resistant 10 25 20 Neomycin-resistant 10 37 18 Tetracycline-resistant 10 25 16 Viomycin-resistant 10 37 15 Polymycin-resistant 10 25 23 Grisain-resistant 5 25 18 * * Tests performed versus a series of strains of E. coli isolat culture following exposure to the individual antibiotics * 7 mm = disc size (no inhibition zone observed) TABLE XZa 842A SPECIAL EFFECT SPECTRUM; IN VITR Escherichia coli W-MB-60 Test Conditions I N H I B I T (with special addition noted) Inoculum Incubation Crude 8 2A ml/150 ml . Temp. °C. 8 mg/ml Control (no additions) 5 25 16 0.1 M Phosphate Buffer -pH 5 5 25 19 0.1 M Phosphate Buffer -pH 7 5 25 22 0.1 M Phosphate Buffer -pH 9 5 25 21 Human Blood Plasma 20% 5 25 17 I ♦Cation Exchange Resin 5 25 20 0 I (Dow ET 91)1% * (Agar concentration reduced to 1% for resin plate only) Antibiotic 842A; In Vivo When 842A is given subcutaneously to mice it is generally more active than cephalothin and approximatel equal to cephaloridine , ampicillin and Chlorom cetin in protecting against infection from gram-negative organisms. It is remarkably nontoxic and is rapidly excreted in the urine with approximately 79% of the subcutaneously injected 842A recovered within four hours .

In ill vivo studies Antibiotic 842A protects against infection with three species of Proteus , two of Salmonella, one strain of Escherichia coli , two of Klebsiella and also against paracolobactrum arizoniae, Aerobacter aerogenes m Pasteurella multocida and Diplococcus pneumoniae E400.

The method employed in these studies is the same as described above with regard to the in_ vivo characteriza-tion of 810A. The results of these tests are described below in Table Xlb: TABLE Xlb 842A In Vivo Activity ED5Q by Subcutaneous Test Organism Route X Two Doses Proteus vulgaris 51 yg.

Proteus mirabilis 276 Hg.

Proteus morganii 3202 276 yg.

Salmonella schottmuelleri 103 yg.

Klebsiella pneumoniae AD 125 yg.

Klebsiella pneumoniae B ^25 Paracolobactrum arizoniae 25 yg! Escherichia coli 200 Va Aerobacter aerogenes Pasteurella multocida 5 Salmonella typhos a 24 yg Diplococcus pneumoniae E400 c c THE ANTIBIOTICS 810A Fermentation; The Antibiotic 810A is produced during the aerobic fermentation of suitable aqueous mediums under controlled conditions via inoculation with the Streptomyces griseus culture MA-2837. Aqueous mediums such as those employed for the production of other antibiotics are also suitable for producing Antibiotic 810A. Such mediums contain sources of carbon and nitrogen assimilable by the microorganism and inorganic salts.

In general, carbohydrates such as sugars, for example, glucose, arabinose, maltose, xylose, mannitol and the like and starches such as grains, for example, oats, rye, corn starch, corn meal and the like can be used either alone or in combination as sources of assimilable carbon in the nutrient medium. The exact quantity of the carbohydrate source or sources utilized in the medium depend in part upon the other ingredients of the medium but, in general, the amount of carbohydrate usually varies between about 1% and 6% by weight of the medium. These carbon sources can be used individually or several such carbon sources may be combined in the medium. In general any proteinaceous material may be used as a nitrogen source in the fermentation process. Suitable nitrogen sources include, for example, yeast hydrolysates , yeast autolysate, soybean meal, hydrolysates of casein, corn steep liquor, distiller's solubles or tomato paste and the like. The sources of nitrogen, either alone or in combination, are used in amounts langing from about 0.2 to 6% by weight of the aqueous medium. Typical of the mediums which are suitable for the preparation of Antibiotic 810A are those listed below. These mediums and others described in the examples which follow are merely illustrative of the wide variety of media which may Medium I : Difco Yeast Extract 10.0 g- Glucose 10.0 g.

♦Phosphate Buffer 2.0 ml .

MgSO^ · 7H20 0.05, g.

Distilled Water 1000.0 ml . Difco Agar 25.0 g- ♦Phosphate Buffer: KH2 P0¼ 91.0 g- Na2HPC¼ 95.0 g- Distilled Water 10Q0.0 ml .

Medium II: Beef Extract 3.0 g. *NZ Amine 10.0 g. Dextrose 10.0 g. NaCl 5.0 g.

Distilled H20 1000.0 ml . pH adjusted to 7.2 with NaOH *an enzymatic digested casein Medium III: Dextrose 10.0 g.

Asparagine 1.0 g.

K2HPO4 0.1 g.

MgSOij · 7H20 0.5 g.

Yeast Extract 0.5 g.

♦Trace Element Mix No. 2 10.0 ml.

Distilled H20 1000.0 ml. pH adjusted to 7.2 with NAOH ♦Trace Element Mix No. 2: FeSOt» · 7H20 1.0 g.

MnSOi, · H20 1.0 g.

CUCI2 * 2H20 25.0 mg.

CaCl2 100.0 mg.

H3BO3 56.0 mg.

(NHt,) 6M0702i> · 4H20 19.0 mg.

ZnSOt» · 7H20 200.0 mg.

Distilled H20 1000.0 ml.

Medium IV: V8 Juice 100.0 ml .

Staley's 4S Soybean Meal 20.0 g.

Dextrose 2.0 g- Agar 25.0 g- Distilled H20 to 1000.0 ml . pH 7.9-8.0 1< Medium V: Yeast Autolysate (Ardamine) 10.0 g.

Glucose 10.0 g.

*Phosphate Buffer 2.0 ml.

MGSO4 * 7H20 0.05 g.

Distilled H20 1000.0 ml. pH - adjust to 6.5 using NaOH *Phosphate Buffer Solution: KH2 PO^ 91.0 g.

Na2HPOit 95.0 g.

Distilled H20 1000.0 ml.

Medium VI; Corn Steep Liquor (wet basis) 40.0 g.

Dextrose 20.0 g.

NaCl 2.5 g.

MgSOt, · 7H20 0.5 g.

Polyglycol 2000 0.25% by volume (add to each flask individually) Distilled H20 1000.0 ml. pH - adjust to 7.0 with NaOH *pH adjusted to 7.0 with NaOH **pH adjusted .to 7.1 with NaOH Medium VIII: Meat Extract 0.3% NaCl 0.5% NZ Amine 1 % Medium V: Yeast Autolysate (Ardamine) 10.0 g.

Glucose 10.0 g.

*Phosphate Buffer 2.0 ml.

MGSOij * 7H20 0.05 g.

Distilled H20 1000.0 ml. pH - adjust to 6.5 using NaOH *Phosphate Buffer Solution: Medium VI ; Corn Steep Liquor (wet basis) 40.0 g.

Dextrose 20.0 g.

NaCl 2.5 g.

MgSO^ · 7H20 0.5 g.

Polyglycol 2000 0.25% by volume (add to each flask individually) Distilled H20 1000.0 ml. pH - adjust to 7.0 with NaOH *pH adjusted to 7.0 with NaOH **pH adjusted -to 7.1 with NaOH Medium VIII: Meat Extract 0.3% NaCl 0.5% NZ Amine 1 % Dextrose 1 % pH 7.0 The fermentation is carried out at temperatures ranging from about 20°C. to 37°C. ; however, for optimum results it is preferable to conduct the fermentation at temperatures of from about 22°C. to 30°C, The pH of the nutrient mediums suitable for growing the Streptomyces griseus culture (MA-2837) and producing Antibiotic 810A should be in the range of from about 5.5 to 8.0.

A small scale fermentation of Antibiotic 810A is con-veniently carried out by inoculating a suitable nutrient medium with the antibiotic-producing culture and permitting the fermentation to proceed at a constant temperature of from about 24-28°C. on a shaker over an extended period as, for example, for several days. At the end of the incubation period the mycelium is removed and the supernatant liquid is assayed. In practice this fermentation is conducted in a sterilized flask via a one, two, three or four stage seed development. The nutrient medium for the seed stage may be any suitable combination of carbon and nitrogen sources as, for example, any one of Mediums I-VIII described above. The seed flask is shaken in a constant temperature chamber at about 28°C. for a period of from one to about three days and the resulting growth is used to inoculate either a second stage seed or the-production medium. Intermediate stage seed flasks, when used, are developed in essentially the same manner, that is, the contents of the flask are used to inoculate the production medium, the inoculated flasks are shaken at a constant tem-perature for several days and at the end of the incubation period the contents of the flask are centrifuged to remove the mycelium. The supernatant liquid or broth is then con-centrated and purified to afford the Antibiotic 810A.

For larger scale work, it is preferable to conduct the fermentation in suitable tanks provided with an agitator^_^ and a means of aerating the fermentation medium. According to this method, the nutrient medium is .made up in the tank and sterilized by heating at temperatures of up to about 120 °C. Upon cooling, the sterilized medium is inoculated with the producing culture and the fermentation is permitted to proceed for a period of several days as , for example , from two to four days while agitating and/or aerating the nutrient medium and maintaining the temperature at about 24-28°C.

Through changes in inoculum development and changes in pro-duction medium it is possible to achieve a several-fold im-provement in production and increase the potency of the antibiotic. 810A Assay Procedure Using Proteus Vulgaris; Antibiotic 810A was conveniently assayed by a disc-plate procedure using Proteus vulgaris MB-838 (ATCC 21100 and NRRL B-3361) maintained as a slant culture on nutrient agar (Difco) plus 0.2% yeast extract (Difco) as the test organism. The in-oculated slants are incubated at 37°C. for 18-24 hours and stored at refrigerator temperatures until used, fresh slants being prepared each week.

The inoculum for the assay plates is prepared each day by inoculating a 250 ml. Erlenmeyer flask containing 50 ml. of nutrient broth (Difco) plus 0.2% yeast extract (Difco) with a scraping from the slant. The flask is incubated at 37eC. on a shaking machine for 18-24 hours. The broth culture is then adjusted to 40% transmittance at a wavelength of 660 my, using a Bausch & Lomb Spectronic 20 by the addition of a 0.2% yeast extract solution to the growth. Uninoculated broth is used as a blank for this determination. The adjusted broth (30 ml.) is used to inoculate 1 liter of medium.

Nutrient agar (Difco) plus 0.2% yeast extract (Dif is used as the assay medium. This medium is prepared, sterilized by autoclaving and allowed to cool to 50 °C.

After the medium is inoculated, 10 ml. is added to each of several sterile petri dishes and the medium is allowed to solidify.

Assays were run on these plates by the disc-plate pro-cedure using 0.5 inch filter paper discs. The assay plates were incubated for 20-24 hours at 37°C. Assays are ex-pressed as mm. diameter zone of inhibition. They were used to determine relative potencies or, when compared with a purified reference standard, the potency in yg./ml. When such an assay is performed in a quantitative fashion from 2 to 4 pg./ml. of antibiotic can be detected. 810A Assay Procedure Using Vibrio Percolans; Assays were also run on 810A by the disc-plate procedure against Vibrio percolans (MB-1272) using 0.5 inch filter paper discs. The assay plates were prepared using Difco nutrient agar plus 2.0 g./liter Difco yeast extract at 10 ml. per plate. An overnight growth of the assay organism, Vibrio percolans (MB-1272) in nutrient broth plus 0.2% yeast extract was diluted in sterile saline solution to a sus-pension having 40% transmittance at a wave length of 660 my. This suspension was added at 20 ml./liter of medium prior to pouring the plates.

The assay plates were held at 4°C. until used (5 day maximum) . Following the application of antibiotic-saturated assay discs the plates were incubated at 28°C. for a period of from 8 to 24 hours. Zones of inhibition were read as Bacterial Inactivation With 810Ά; An in vitro study was designed to determine the resistance of 810A, to bacterial inactivation as compared sporin C, cephaloridine and cephalothin. This study showed that Antibiotic 810A is more stable than the latter against certain microorganisms.

The degradative bacterium used in this study was an oiganism known to completely inactivate cephalosporin C, namely, Alcaligenes faecalis (MB-9) . (a) Preparation of Bacterial Cells t Alcaligenes faecalis (MB-9) cells were prepared as follows: the contents of an L-tube were mixed with a few ml. of nutrient broth containing 0.2% yeast extract. A loop- ful of the slurry was spread over the surface of a nutrient agar slant and incubated for 18 hours at 37 °C. All slants were stored at 5°C. and used within one week after incubation. A loopful of the surface growth from each slant culture was asceptically transferred to 50 ml. of nutrient broth containing 0.2% yeast ex- tract and shake incubated fx 18 hours at 28°C. The culture was then centrifuged at 4000 rprr for ten min- utes. The supernatant was decanted and the residual pellet of cells was washed twice with sterile 0.1 M phosphate buffer, pH 7.5 (6.8 g. of potassium phosphate and 7.1 g. of sodium hydrogen phosphate per liter of dis- tilled water ) . The washed cells were then resuspended in one-tenth original volume of a 4ng./ml. solution of antibiotic in 0.1 VL phosphate buffer. The test mixture was then incubated without shaking in a water bath set at 37°C. for up to four hours. The test mixtures were then centrifuged at 2000 rpm. for 10 minutes and this produced a clear supernatant which was decanted into sterile tubes and immediately frozen^^" in dry ice until ready for bioassay, usually within three hours . Controls were incubated in exactly the same manner, except for the absence of cells. (b) Extent of Antibiotic 810A Inactivatio ; The super-natants were tested for antibacterial activity in the following manner: 1/4 inch diameter paper discs were moistened with the supernatants and placed on he surface of nutrient agar-yeast extract (0.2%) plates that had been previously seeded with the appropriate test organism. B. subtilis (MB-964) assay plates were seeded in the following manner: 5 ml. of a suspension of washed spores in 0.9% saline was added to each 150 ml. of nutrient agar-yeast extract (0.2%) of which 5 ml. was then dispensed into 15 x 100 mm. petri plates. All assay plates were stored at 5°C. and used within three days. Assay plates were incubated overnight at 25°C. before measurement of zones of inhibition around the test discs .

Cell-free controls of each antibiotic were assayed at 1:1, 1:2, 1:4, 1:8, 1:16 and 1:32 dilutions in order to obtain a standard reference curve. Solutions of test anti-biotics were assayed at full strength after incubation in the presence of the washed bacterial cells. All samples were run in triplicate. (c) Results : Percents of inactivation were calculated by taking the average of the three zones of inhibition obtained for each test and determining the amount of anti-biotic remaining in the test solution as shown by the the standard curve. This value was then subtracted from the starting concentration (4 mg. /ml.) and the remainder Tables XII and XIII demonstrate the inactivation obtained for cephalosporin C, cephalothin and Antibiotic 810A under the conditions described above.

TABLE XII Percent Inactivation After Incubation With Washed Bacterial Cells (Assayed on B. subtilis (MB-964) Plates) TABLE XIII Percent Inactivation After Incubation With Washed Bacterial Cells (Assayed on V. percolans (MB-1272) Plates) The ability of Antibiotic 810A and cephalosporin C to withstand the degradative effect of the culture Aerobacter cloacae (MB-2646) was also determined. This culture is gram-negative and resistant to cephalosporin C. In con- ducting the assay individual mixtures of the organisms te and one of the antibiotic mixtures were sampled after tv. hours incubation and assayed for residual antibiotic activity. The procedure is the same assay method as des- cribed above with Alcaligenes faecalis . The source of the inactivating substance is a 1:160 dilution of the filtrate of an 18 hour 37°C. shake culture of Aerobacter cloacae MB-2646 in nutrient broth containing 0.2% yeast extract.

Table XIV, infra, indicates the percent inactivation of Antibiotic 810A, cephalothin, cephaloridine and cephalo- sporin C on Vibrio percolans (MB-1272) via this method: TABLE XIV Percent Inactivation After Incubation With Cell-Free Extract (Assayed on V. percolans (MB-1272) Plates) Using the same assay procedure as described above, Table XV, infra, indicates the relative resistance of Antibiotic 810A to enzymatic inactivation by Aerobacter cloacae . The starting concentration is 250 yg./ml.

Results are expressed in yg./ml.

TABLE XV Antibiotic Activity Remaining (Starting Concentration = 250 *Cell-free extract In view of the foregoing Antibiotic 810A is , apparently, more resistant than cephalosporin C, cephalothin and cephaloridine to inactivation by Aerobacter cloacae . 842A Fermentation: Antibiotic 842A is produced during the aerobic fermentation of suitable aqueous nutrient mediums under controlled conditions via inoculation wi the organism S treptomyces lactamdurgns . In general, many media which are a source of carbon and nitrogen may be used for the production of 842A. Illustrative of these are the aqueous mediums and carbohydrate and nitrogen sources des-cribed above in connection with the fermentation of 810A. The exact amount of the carbohydrate and nitrogen sources will depend upon the other ingredients comprising the fer-mentation medium but, in general, the amount of carbohydrate is usually about 1% to 6% by t^eight of the medium and the amount of available nitrogen, either alone or in combination is usually in the amount from about 0.2% to about 6% by weight of the medium. The several mediums described below are il-lustrative of these which are suitable for the preparation of Antibiotic 842A. These media are merely typical of the media which may be employed and are not intended to be limitative.

Medium IX; Amber Yeast #300 10.0 g.

Distiller's Solubles 20.0 g.

Dextrose 10.0 g.

Distilled Water 1000.0 ml. pH 7.0 Medium X: Staley's 4S-Soybean Meal 30.0 g.

Distiller's Solubles 7.5 g.

Cerelose 20.0 g.

NaCl 2.5 g.

CaC03 (after pH to 7.0) 10.0 g.

Distilled Water 1000.0 ml.

Medium XI: Amber Yeast #300 10.0 g.

Distiller's Solubles 20.0 g.

Distilled Water 10C0.O ml. pH 7.0 fermentation is carried out at temperatures ranging from about 20°C. to 37°C. but for optimum results it is preferable to conduct the fermentation at tempera-tures of from about 24°C. to 32 °C. The pH of the nutrient mediums suitable for growing .the Streptomyces lactamdurans culture (MB-2908) and producing Antibiotic 842A should be in the range of from about 6.0 to about 8.0.

A small scale fermentation of Antibiotic 842A is conveniently carried out by inoculating a suitable nutrient medium with the antibiotic-producing culture and permitting the fermentation to proceed at a constant temperature of about 28°C. on a shaker for several days.

At the end of the incubation period the mycelium is re-moved and the supernatant liquid is assayed.

In practice this fermentation is conducted in a steril-ized flask via a one, two, three or four stage seed develop-meht. The nutrient medium for the seed stage may be any suitable combination of carbon and nitrogen sources as, for ex-ample, any one of Mediums IX-XI described above. The seed flask is shaken in a constant temperature chamber at about the production medium. Intermediate stage seed flasks, when used, are developed in essentially the same manner, that is, the contents of the flask are used to inoculate the production medium, the inoculated flasks are shaken at a constant tem-perature for several days and at the end of the incubation period the contents of the flasks are centrifuged to remove the mycelium. The supernatant liquid or broth is then con-centrated and purified to afford Antibiotic 842A.

For larger scale work, it is preferable to conduct the fermentation in suitable tanks provided with an agitator and a means of aerating the fermentation medium. According to this method, the nutrient medium is made up in the tank and sterilizedby heating at temperatures of up to about 120eC. Upon cooling, the sterilized medium is inoculated with the producing culture and the fermentation is permitted to proceed for a period of several days as, for example, from two to four days while agitating and/or aerating the nutrient medium and maintaining the temperature at about 28eC. Through changes in inoculum development and changes in production medium it is also possible to achieve a several-fold im-provement in production and increase the potency of this antibiotic. 842A Assay Procedure Using Vibrio Percolans: Assays were run by the disc-plate procedure using 0.5 inch filter paper discs. The assay plates were prepared using Difco nutrient agar plus 2.0 g./l. Difco yeast extract at 10 ml. per plate. An overnight growth of the assay organism, ibrio percolans (MB-1272) in nutrient broth and 0.2% yeast extract was diluted in sterile saline solution to a sus-pension having 40% transmittance at a wave length of 660 TrniT. This suspension was added at 20 ml. /liter of medium prior to pouring the plates .

The assay plates were held at 4°C. until used (5 day maximum). Following the application of the antibiotic-saturated assay discs the plates were incubated at 28°C. for a period of from 8 to 24 hours. Zones of inhibition were read as mm. diameter. They were used to determine relative potencies or, when compared with a purified reference standard, the potency in yg./ml. When such an assay is performed in a quantitative fashion from 1 to 2 yg./ml. of antibiotic can be detected.

Bacterial Inactivation With 842A: An in_ vitro study was designed to determine the resistance of Antibiotic 842A to bacterial inactivation as compared with cephalosporin C, cephaloridine and cephalothin. This study showed that Antibiotic 842A is more stable than the latter against certain microorganisms .

The degradative bacteria used in the study were two organisms known to completely inactivate cephalosporin C, namely, Alcaligenes faecalis (MB-9) and Alcaligenes viscosus (MB-12) . (a) Preparation of Bacterial Cells; Alcaligenes viscosus (MB-12) and A. faecalis (MB-9) cells were prepared as follows: tho contents of an L-tube wcro mixed with a few ml. of nutrient broth containing 0.2% yeast extract. A loopful of the slurry was spread over the surface of a nutrient agar slant and incubated for 18 hours at 37eC. All slants were stored at 5eC. and used within one week after incubation. A loopful of the surface growth from each slant culture was asceptically transferred to 50 ml. of nutrient broth containing /^**N . 042% yeast extract and shake incubated for 18 hours at 28°C. The culture was then centrifuged at 4000 rpm. for ten minutes. The supernatant was decanted and the residual pellet of cells was washed twice with sterile 0.1 M phosphate buffer, pH 7.5 (6.8 g. of potassium phosphate, i.e., KH2P04 and 7J. g. of sodium hydrogen phosphate per liter of distilled water) . The washed cells were then resuspended in one-tenth original volume of a 4 mg./ml. solution of antibiotic in 0.1 M phosphate buffer. The test mixture was then incubated without shaking in a water bath set at 37°C. for four hours.

The test mixtures were then centrifuged at 2000 rpm. for 10 minutes and this produced a clear supernatant which was decanted into sterile tubes and inmediately frozen in1 dry ice until ready for bioassay, usually within three hours. Controls were incubated in exactly the same manner, except for the absence of cells. (b) Extent of Antibiotic 842A Inactivation ; The super-natants were tested for antibacterial activity in the follow-ing manner: 1/4 inch diameter paper discs were moistened with the supernatants and placed on the surface of nutrient agar-yeast extract (0.2%) plates that had been previously seeded with the appropriate test organism. B. subtilis (MB-964) assay plates were seeded in the following manner: 5 ml. of a suspension of washed spores in 0.9% saline was added to each 150 ml. of a 2% nutrient agar-yeast extract (45eC.) of which 5 ml. was then dispensed into 15 X 100 mm. petri plates. All assay plates were stored at 5°C. and used within three days. Assay plates were incubated overnight at 25 °C. before measurement of zonefe of inhibition around the test discs.

Cell-free controls of each antibiotic were assayed at 1:1, 1:2, 1:4, 1:8, 1:16 and 1:32 dilutions in order to obtain a standard reference curve. Solutions of test antibiotics were assayed at full strength after incubation in the presence of the washed bacterial cells. All samples were run in triplicate. (c) Results : Percents of inactivation were calculated by taking the average of the toee zones of inhibition obtained for each test and determining the amount of antibiotic remaining in the test soltuion as shown by the standard curve. This value was then subtracted from the starting concentration (4 mg./ml.) and the remainder divided by,the starting concentration and multiplied by 100 to obtain the percent of inactivation. The following Table χν I demonstrates the inactivation obtained for cephalosporin C and Antibiotic 842A under the conditions described above.

TABLE XVI Percent Inactivation After Incubation With Washed Bacterial Cells (Assayed on B. subtilis (MB-964) Plates) DEGRADATIVE 4 Hour Incubation ORGANISM Ceph C Antibiotic 842A Alcaligenes faecalis MB-9 99+ 0 A. viscosus MB-12 99+ 54.4 The ability of Antibiotic 842A and cephalosporin C to withstand the degradative effect of four other cultures was also determined. These cultures are: Escherichia coli 236, Proteus morganii 251, Proteus morganii 356 and Proteus mirabilis 241. Each is gram-negative and resistant to cephalosporin C. In conducting the assay individual mixtures of the organisms and one of the antibiotic were sampled after four hours incubation and assayed for residual antibiotic activity. The procedure is the same assay method as described above against Alcaligenes faecalis MB-9 and A. viscosus MB-12. The following table indicates the percent inactivation of cephalosporin C and 842A on B. subtilis (MB-964) via this method: TABLE XVII Culture Ceph C Antibiotic 842A Escherichia coli 236 >99 38 Proteus morganii 251 >99 80 Proteus morganii 356 >99 69 Proteus mirabilis 241 72 5 The foregoing data indicates that Antibiotic 842A is, apparently, more resistant than cephalosporin C to inactivation by A. faecalis , A. viscosus , Escherichia coli 236, Proteus morganii 251, Proteus morganii 236 and Proteus mirabilis 241.

The Antibiotic 842A which is obtained via the instant fermentation process is an amphoteric:compound with an apparent isoelectric point of about pH 3.5; it is unstable above pH 9.0 but relatively stable ht pH 1.5.

Since Antibiotic 810A and Antibiotic 842A and their salts e ffectively inhibit the growth of various species of Salmonella they can be used as disinfectants in various household and industrial applications . For example , 810A exhibi ts activity against Salmonella schottmuelleri and S . gallinarum and 842A exhibits activity against Salmonella schottmuelleri 3010 , S . gallinarum and S . typhosa and this property is indicative of their usefulness as sanitizing agents in household and industrial applications .

ISOLATION AND PURIFICATION Antibiotic 810A: Antibiotic 810A can be purified by adsorption on an ion exchange resin as , for example , on synthetic anion exchange resins derived from dextrose or acrylic copolymers or non-ionic cross-Inked polymers . The adsorbed antibiotic is eluted 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. Illustrative of the ion exchange resins and polymers which may be employed are , for example , the DEAE Sephadex A-25 , Amberlite IRA-68 and Amberlite XAD-2 mediums described below . If desired the eluate obtained according to the foregoing procedure can be further purified by a second and third adsorption and elution step. Concentrates of all the eluates are then obtained to afford the purified product. 810A Components ; Antibiotic 810A can be separated into its components , 7β- (D-5-amino-5-carboxyvaleramido) -3- (a-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy- 3-cephem-4-carboxylic acid and 7β- (D-5-amino-5-carboxyvaleramido) -3- (a-methoxy-p-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem- (1) Chromatography on a strongly hydrophylic anion exchange resin such as *DEAE Sephadex A-25, developed with an ammonium bromide- formic acidi^^^* system. Various concentration of this system may be employed but, in practice, a 0.5 M ammonium bromide - 0.1 M formic acid solution is preferred. (2) Chromatography on a weakly basic anion exchange resin such as **Amberlite IRA-68. This is a group separation where material in crude form is fed at a pH of about 3 to 3.5 and eluted first with an acid at a pH of about 2 and then with NaCl/HCl at a pH of about 1. (3) Chromatography on a non-ionic cross-linked poly- styrene polymer such as ***Amberlite XAD-2. Elution is effected with a suitable aqueous system but, in general, it is most advantageous to employ a mixture of water and a lower alkyl ketone. Typical of the eluants which may be employed are, for example, 10% methanol in water followed by 50% methanol in water. Alternatively, 20% acetone in water can be substituted for the 50% methanol in water solution. Λ* DEAE Sephadex A-25 is a synthetic anion ex- change resin derived from the polysaccharide, dextran in its chloride form, i.e., with chloride counter ions; Pharmacia Pine Chemicals, Inc., 800 Centenial Avenue, Piscataway, New Market, New Jersey 08854.

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

*** A non-ionic cross-linked polystyrene polymer sorbent; Rohm & Haas Co. , Philadelphia, Pennsylvania 19105.

The individual products obtained via the above methods may be purified by rechromatography . Thus, for example, 73- (D-5-amino-5-carboxyvaleramido) -3- (a-methoxy-p-sulfooxy-cinnamoyloxymethyl) -7-msthoxy-3-cephem-4-carboxylic acid (Ic) may be repurified by subjecting that product to the purifica-tion method described in Method 1, supra, followed by de-salting on Amberlite XAD-2 absorbent; and 7$ - (D-5- amino-5-carboxyvaleramido) -3- (α-methoxy-p-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid may be repurified by re-chromatography on a Sephadex A-25 anion exchange resin developed with 0.5 M ammonium bromide and 0.05 M acetic acid. Antibiotic 842A: The Antibiotic 842A can be purified by adsorption on an ion exchange resin as, for example, on resins composed of quaternary ammonium or sulfonic acid exchange media. The adsorbed antibiotic is eluted from the resin adsorbate with aqueous solutions or with an aqueous . alcoholic solution of a suitable salt such ammonium chloride, sodium chloride and the like. Suitable ion exchange resins which may be employed include, for example, the polystyrene nuclear sulfonic acid resins (45% or 53% water) or polystyrene trimethylbenzylammonium resins (43% water) which are taown as Dowex 50 and Dowex 1, respectively. If desired the eluate obtained according to the foregoing procedure can be further purified by a second and third adsorption and elution step. Concentrates of all the eluates are then obtained to afford the purified 842A product.

FORMULATIONS Antibiotic 810A and its individual components and Anti-b^iotic 842A may be used alone or in combination as the active in redient in an one of a variety of pharmaceutical preparations. These antibiotics and their corresponding salts may be employed in capsule form or as tablets, povdcrs or liquid solutions or as suspensions or elixirs. They be administered orally, intravenously or intramuscularly.

Suitable carriers which may be used in the composition include, for example, mannitol, sucrose, glucose or sterile liquids such as water, saline, glycols and oils of a petroleum, animal, vegetable or synthetic origin as, for example, peanut oil, mineral oil or sesame oil. Also, in addition to a carrier the instant compositions may also include other ingredients such as stabilizers, binders, antioxidants, preservatives, lubricators, suspending agents, viscosity agents, flavoring agents, and the like. In addition, there may also be included in the composition other active in-gredients to provide a broader spectrum of antibiotic activity .

The dosage to be administered depends to a large extent upon the condition of the subject being treated and the weight of the host. The parenteral route being preferred for generalized infections and the oral route for intestinal infections. In general, a daily dosage consists of from about 15 to about 175 mg. of active ingredient per kg. of body weight of the subject in one or more applications per day. A preferred daily dosage for Antibiotic 810A or its individual components lies in the range of from about 20 to 40 mg. of active ingredient per kg. of body weight. The preferred daily dosage for Antibiotic 842A is in the range of from about 40 to 80 mg. ,of active ingredient per kg. of body weight.

The instant composi ions may be administered in several unit dosage forms as, for example, in solid or liquid orally ingestible dosage form. The compositions, per unit docag^* > whether liquid or solid, will generally contain from about 15 mg. to about 700 mg. by weight of the active ingredient based upon the total of the compositions; however, in general, it is preferable to employ a dosage amount in the range of from about 80 mg. to 320 mg. In parenteral administration the unit dosage is usually the pure compound in a sterile water solution or in the form of a soluble powder intended for solution.

One typical unit dosage form consists in mixing 120 mg. of Antibiotic 810A or 120 mg. of one of its components or salt thereof, with 20 mg. of lactose and 5 mg. of magnesium stearate and placing the 145 mg. mixture into a No. 3 gelatin capsule. Similarly, by employing more of the active in-gredient and less lactose, other dosage forms can be put up in No. 3 gelatin capsules and should it be necessary to mix more than 145 mg. of ingredients together, larger capsules may be employed. In a similar manner other unit dosages such as compressed tablets and pills can also be prepared. The following examples are illustrative: Dry-filled Capsule Containing 120 rtg. of 70 - (D-5-amino-5-car~ boxy ale amido) -3- (a -methoxy-p-sulfooxycinnamoyloxymjthyl) -7-methoxy-3-cepheitt-4-carboxylic Acid (Ic) „ , Per Capsule 73 - (D-5-amino-5-carboxyvaleramido) - 3- (a-methoxy-p-sulfooxycinnamoyl- oxymethyl) -7 -methoxy-3-cephem- 4-carboxylic Acid (Ic) 120 mg.

Lactose 20 mg.

Magnesium Stearate 5 mg.

Capsule Size No. 3 145 mg.

The 7β - (D-5-amino-5-carboxyv.aleramido) -3- (a-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) is reduced to a No. 60 powder and then lactose and magnesium stearate are passed through a No. 60 bolting cloth onto the powder and the combined ingredients admixed for 10 minutes and then filled into No. 3 dry gelatin capsules. By substituting 40 mg. of Antibiotic 842A and 100 mg. of lactose for the 120 mg. of active ingredient and 20 mg. of lactose recited in the foregoing formulation, there is thus obtained a 145 mg. capsule which is also suitable for oral administration.

Tablet Containing 250 mg. of 7ρ- (D-5-amino-5-carboxyvaler-amido) -3- (a-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy^ 3-cephem-4-carboxylic Acid (Ic) · Per Tablet 7β- (D-5-amino-5-carboxyvaleramido) - 3- (a-methoxy-p-sulfooxycinnamoy1- oxymethyl) -7-methoxy-3-cephem- 4-carboxylic Acid (Ic) 250. mg.

Dicalcium Phosphate, U.S. P. 192. mg.

Magnesium Stearate 5. mg.

Lactose, U.S. P. 65. mg. 36281/2 \ The active component is blendod with the dicalciuin phosphate and lactose. The mixture is granulated with 3.5¾ cornstarch paste (6 mg.) and rough-screened. It is dried at 45°C. and screened again through a No . 16 screen. The magnesium stearate is added and the mixture is compressed into tablets approximately 0.5 inch in diameter.

Parenteral Solution Containing 500 mg . of 73- (D-5-amino-5- carboxyvaleramido) -3- (a-methoxy-p-sulfooxycinnamoyloxy-methyl) -7-methoxy-3-cephem-4-carboxylic Acid (Ic) Ampoule ; ..^. 7β- (D-5-amino-5-carboxyvaleramido) - 3- (a-methoxy-p-sulfooxycinnamoyloxy- methyl) -7-methoxy- 3-cephem-4- carboxylic Acid (Ic) 500 mg.

Ampoule: ' ^ Diluent: Sterile Water for Injection 2 cc. -84- 36281/2 The 70- (D-5-amino-5-carboxyvaleramido) -3- {α-methoxsp·»-^ p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) may be administered alone or in combination with other biologically active ingredients as, for example, with other antibacterial agents such as lincomycin, a penicillin, streptomycin, novobiocin, gentamicin, neomycin, colistin and kanamycln. -85- SYNTHETIC METHODS In addition to the above-described ferment tion products this invention includes derivatives in which the 3-carbamoyloxy moiety of 842A is replaced by a wide variety of substituents . In general, this replacement or substitu-tion is effected by simply treating 842A with a reagent capable of converting the 3-carbamoyloxy moiety to the de-sired R substituent (see Formula I, supra). However, in practice, prior to the replacement of the 3-carbamoyloxy radical, it is frequently necessary to 'protect' the free amino and carboxy groups by treating 842A with a nitrogen-blocking agent such as a N-lower alkoxycarbonylphthalimide and an esterifying agent so as to afford the corresponding 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (carbamoyl-oxymethyl) -7-methoxy-3-cephem-4-carboxylic acid diester (Compound II, infra) . Suitable esterifying agents include phenyldiazomethane or diphenyldiazomethane and the like. Also, other nitrogen-blocking agents which may be used in lieu of the N-lower alkoxycarbonylphthalimide include tert-iary-butyloxyazide or trihaloethoxycarbonyl halides such as trichloroethoxycarbonyl chloride and the like. The follow-ing equation wherein the nitrogen-blocking agent is an N-lower alkoxycarbonylphthalimide illustrates this reaction; however, it is to be understood that other nitrogen-blocking agents such as mentioned above may be substituted therefor in an otherwise similar reaction to afford the corresponding N-substituted derivative: wherein R1JR14C=N2 is diazomethane or an aryl substituted diazomethane such as phenyldiazo ethane or diphenyldiazo-methane and the like and R^3 and R^-4 are hydrogen or phenyl. The Compound (II) thus obtained is the starting material in the following synthetic methods.

The 3-hydroxymethyl analog of 842A is obtained by treat-ing Compound II, supra, with a nitrosyl halide such as nitrosyl chloride in a suitable solvent such as methylene chloride. The following equation illustrates this method of preparation: R13R1 H N0C1/CH2C12 l13R14HC wherein R1J and R are as defined above. The diester thus obtained, preferably the di-benzyl ester (Ilia, infra) , can then be converted to the corresponding free amino-acid (IV, infra) via catalytic hydrogenation and treatment with hydrazine hydrate in a basic solution. De-blocking by this method is most conveniently effected when the diester reactant is the dibenzyl ester (Ilia). The following equation illustrates this method of preparation: H2NH2 1 H -0 Base COOM IV and M is the cation derived from an alkali metal or alkaline earth metal.

The N-mono-substituted and Ν,Ν-di -substituted 3-carbamoyloxy analogs of 842A are conveniently obtained by treating the diester of 73- (D-5-phthaloylamino-5-carboxy-valeramido) -3- (hydroxymethyl) -7-methoxy-3-cephem-4-carboxylic acid, preferably in the form of its dibenz-hydryl ester (Illb, infra) , with a carbonyl halide such as carbonyl chloride (i.e., phosgene) or carbonyl bromide/ followed by the reaction of the dibenzhydryl ester of 73-(d-5-phthaloylamino-5-carboxyvaleramido) -3- (haloformyloxy-methyl) -7-methoxy-3-cephem-4-carboxylic acid (V, infra) thus obtained with the appropriate mono-lower alkylamine, di-lower alkylamine or heterocyclic amine: wherein COX2 is a carbonyl halide such as carbonyl chloride (i.e., phosgene) or carbonyl b^jmide and X is halo as, for example, chloro, bromo and the like, HNR^R1 is a primary or secondary amine selected from mono-lower alkylamines, di-lower alkylamines and heterocyclic amines and R^ and R-^ represent lower alkyl such as methyl, ethyl, n-propyl, n-butyl and the like and, taken together R^ ^ and may be combined with the nitrogen <^ ^ to which they are attached to afford a mononuclear heterocyclic amine selected from pyrrolidine, piperidine and mor pholine. The product (VI) thus obtained may then be converted to the corresponding free amino-acid by treatment with trifluoroacetic acid in xylene and then with hydrazine hydrate in a basic solution: wherein M and R and R are as defined above.

Another method for preparing the N-mono-substitutcd derivatives of 842 A consists in treating the compound i: infra, or a di-salt thereof, with an appropriate isocyanate. This reaction is most advantageously conducted in the presence of a strong organic base such as triethylamine and in the presence of an inert solvent such as dimethyl form-amide, methylene chloride, tetrahydrof uran or acetonitrile : R1 R13R14 wherein R 17 is lower alkyl, halo substituted lower alkyl such as chloromethyl , 2-chloroethyl or chloro-tertiary-butyl and the like, lower alkoxycarbonyl such as ethoxy-carbonyl and the like, mono-nuclear and bi-nuclear aryl such as phenyl, naphthyl and the like, mono-nuclear alkaryl-sulfonyl such as p-tolylsulfonyl and the like or benzhydryl. 17 Typical of the ioscyanate reactants (i.e., OC-NR ) which may be employed are, for example, methylisocyanate , ethyl-isocyanate, tertiary-butylisocyanate , chloromethyl isocyanate, {J-chloroethylisocyanate , carbethoxyisocyanate , phenyl-isocyanate and benzhydrylisocyanate. The products corresponding free amino-acid via the method previously described, i.e., by treatment with t ri fluoroacetic <^ d- in xylene followed by the reaction of the resulting intermediate with hydrazine hydrate in basic solution.

The 3-acylated derivatives of ^his invention correspond- ing to Formula I wherein R is lower alkanoyloxy (except acetoxy) , aromatic-carbonyloxy, aralkanoyloxy or cycloalkane-carbonyloxy are conveniently obtained by treating the dibenzhydryl ester of 76- (D-5-phthal0ylamino-5-carboxyvalerarnido) -3- (hydroxy- methyl) -7-methoxy-3-cephem-4-carboxylic acid (Illb) with a suitable acyl halide. The following equation illustrates this method of preparation: wherein R1B is lower alkyl such as ethyl and the like, mono-nuclear and bi-nuclear aryl such as phenyl, a mcno nuclear nitrogen-containing heterocycle such as 4-pyridyl and the like, naphthyl and the like, mono-nuclear and bi- nuclear lower aralkyl such as benzyl, menaphthyl and the like cyclopentyl or cyclohexyl and the like and X is as defined above. Again, the product thus obtained may be to the corresponding free amino-acid by treatment with trifluoroacetic acid in xylene followed by the reaction of the resulting intermediate with hydrazine hydrate in a basic solution.

The 3-methyl analogs of 842A are conveniently obtained via the reduction of 842A or a salt thereof as, for example, by the catalytic hydrogenation of the corresponding alkali metal or alkaline earth metal salt. Catalysts which may be used in this process include, for example, any of the metals in Group VIII of the Periodic Table as, for example, palladium, platinum or nickel. The following equation il-lustrates this method of pareparation: HOOC H /Catalyst Those derivatives of 842A which are not prepared via the foregoing methods can be obtained by treating a 3-acyloxymethyl analog of 842A as, for example, the 3-lower alkanoyloxymethyl analog such as 70- (D-5-amino-5-carboxyvaleramido) -3- (acetoxy-methyl) -7-methoxy-3-cephem-4-carboxylic acid, with the dithiocarbamate , dithiocarboxylate , amine, pyridine, nuclear substituted pyridine, alkali-metal sulfinate, azide, poly-hydroxybenzene or N-lower alkylindole. The resulting products can then be purified by conventional means as, for example, by recrystallization from suitable solvents such as methanol and water or by fractionation through a suitable ion-exchange resin.

In addition to the 3-acyloxymethyl analog of 842A it has been found that 842A may be employed per se as a starting material in the reaction with thiourea and alkali metal azides, etc. Thus, for example, 842A may be treated with the appro-priate thiourea, N-substituted thiourea, N,N-disubstituted thiourea, alkali metal azide, thiazole, thiadiazole, halo-genating agent or alkali metal thiocyanate to afford the cor-responding 3-thiouroniummethyl , 3-azidomethyl , 3-thiazolyl-mereaptomethy1 , 3-thiadiazolylmercaptomethyl , halomethyl and 3-thiocyanatomethyl analogs of 842A. In principle, it is only necessary to combine the starting materials to effect the synthesis but, in practice, it is usually desirable to catalyze the reaction by the application heat as, for example, by heating at temperatures in the range of from about 45 °C. to 95°C. for a period of from about 2.5 days to several minutes. A preferred operating temperature lies in the range of from about 75-80°C. and good yields have also been obtained upon heating the reactants at 95°C. for about eight minutes. Also, in some instances, it is desirable to protect the free amino group in the 5-amino-5-carboxyvaleramido side chain of 842A by treating the latter with tertiary-butoxycarbonylazide or similar blocking group. The resulting 7f$- (D-5-N-tertiary-butoxycarbonylamino-5-carboxyvaleramido) -3- (car amoyloxy-methyl) -7-methoxy-3-cephem-4-carboxylie acid can then be or thiocyanate reactant so as to introduce the cesired sub-stituent into the 3-position of the 842A nucleus the risk of any untoward side reactions. The intermediate thus obtained can then be deblocked via treatment with a suitable reagent such as trifluoroacetic acid, to obtain the desired product.

The 3-azidomethyl derivative of 842A obtained via the foregoing method is converted to its corresponding 3-aminomethyl analog by reduction. Suitable means include, for example, molecular reduction and hydrogenation over a suitable metal catalyst such as the metals in Group vm of the Periodic System. Typical of the metals which may be employed include, for example, platinum, palladium and nickel and combinations thereof.

The molecular reduction of the 3-azidomethyl deriva-tive is most adva tageously conducted by adding zinc dust to an acidic solution of the 3-azidomethyl reactant. The resulting solution is then treated with hydrogen sulfide or an equivalent material to remove the soluble zinc from the solution in the form of a precipitate. The product thus obtained is the substantially pure 3-aminomethyl analog of 842A.

The products of this invention (I) form a wide variety of pharmacologically acceptable salts with inorganic and organic bases; these include, for example, metal salts such as are derived from alkali metal and alkaline earth metal hydroxides, carbonates and bicarbonates and salts derived from primary, secondary, and tertiary amines such as mono-alkylamines, dialky1amines , trialky1amines , lower alkanol-amines, di-lower alkanolamines , lower alkylenediamines , ,N-diaralky1 lower alkylenediamines , aralkylamines , amino - - substituted lower alkanols, amino-, polyamino- and guanidino-substituted lower alkanoic acids and nitrogen-^^ containing heterocyclic amines. Representative examples of these salts include salts derived from sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium hydroxide, calcium carbonate and the like and salts derived from such amines as trimethylamine, triethyl-amine, piperidine, morpholine, quinine, lysine, protamine, arginine , procaine, ethanolamine , morphine, benzylamine, ethylenediamine , Ν,Ν'-dibenzylethylenediamine, diethanol-amine, piperazine, dimethylaminoethanol , 2-amino-2-methyl-1-propanol, theophylline and N-methylglucamine and the like. The aforementioned salts can be mono-salts such as* the monosodium salt obtained, for example, by treating one equivalent of sodium hydroxide with one equivalent of product (I) , or mixed di-salts obtained by treating one equivalent of the mono-salt with one equivalent of a dif-ferent base. Alternatively, the said di-salts can be ob-tained by treating one equivalent of a base having a di-valent cation such as calcium hydroxide with one equivalent of the said product (I) . In addition, mixed salts and esters such as those obtained by treating the product (I) with one equivalent of sodium hydroxide and then with one equiva-lent of lactic acid are also contemplated.

The salts of this invention are pharmacologically ac-ceptable nontoxic derivatives which can be used as the active ingredient in suitable unit-dosage pharmaceutical forms. Also, they may be combined with other drugs to provide compositions having a broad spectrum of activity.

In addition, the instant salts and, also, the corresponding ester and amide derivatives, have utility as intermediates formula I, supra. And too, the said salts may be used prepare other pharmaceutically acceptable salts.

In addition to salts the instant products (I) may also be converted to their corresponding mono- and di-esters and mono- and diamides as, for example, the pivaloyl-oxymethyl or dibenzhydryl esters or alkyl, cycloalkyl, aryl or aralkyl esters as, for example, the methyl, ethyl, cyclohexyl, phenyl and benzyl esters or amides, diamides, N-lower alkyl amides, N,N-di-lower alkylamides, N-aralkyl-amides, Ν,Ν-diaralkylamides or heterocyclic amides such as the N-methyl and N-ethylamide, N,N-dimethylamide, Ν,Ν-diethylamide, N-benzylamide , Ν,Ν-dibenzylamide , piper-idide, pyrrolidide or morpholide and the like.

Methods for the preparation of the aforementioned esters and amide derivatives include the reaction of the carboxylic acid product (I) or corresponding acid halide with methanol, ethanol, cyclohexanol, phenol, benzyl-alcohol or dibenzhydrol. In a similar manner the amide derivatives may be obtained by treating the corresponding acid halide with ammonia or with the appropriate alkylamine, dialkylamine, aralkylamine or heterocyclic amine. These and other convenitonal methods for the preparation of the said esters and amides will be obvious to those skilled in the art.

The examples which follow illustrate the methods by which the products of this invention may be obtained. How-ever, the examples are illustrative only and it should be apparent to those having ordinary skill in the art ^pK that this invention includes other functionally equivalent products and methods for their preparation. Therefore, any modification of this synthesis which results in the formation of an identical product should be construed as constituting an analogous method. The claimed process is capable of wide variation and modification and, therefore, any minor departure therefrom or extension thereof is considered as being within the skill or the artisan and as falling within the scope of this invention.

EXAMPLE 1 Antibiotic 810A A lyophilized tube of Streptomyces griseus cultur (FA-2837) was opened asceptically. The contents were used to inoculate four slants of a nutrient medium having the following composition : Medium I: Difco Yeast Extract 10 . 0 g.

Glucose 10 . 0 g.

*Phosphate Buffer 2 .0 ml.

MgSOu · 7H20 0 . 05 g.

Distilled Water 1000 . 0 ml.

Difco Agar 25 . 0 g.

*Phosphate Buffer: KH2FCH 91 . 0 g.

N a2HF0 i> 95 . 0 g.

Distilled Water 1000 . 0 ml.

The slants were prepared by dispensing 14 ml. /22 x 75 mm. culture tube. The tube was then stoppered with cotton, heated at 120°C. for 15 minutes to effect sterilization and the medium allowed to solidify in a slanted position. The inoculated slants were incubated at 28°C. for one week and then stored at 4°C. until used. The culture on one of these slants was then used to inoculate baffled Erlenmeyer flasks (250 ml.) containing 50 ml. of Medium II , infra, by the addition of 5 ml. of sterile medium, scraping the s]ant sur-face to suspend the growth and asceptically pipetting 1 ml. into each of three seed flasks. Medium II has the following composition: Medium II: Beef Extract 3.0 g.

*N∑ Amine 10.0 g.

Dextrose 10.0 g.

NaCl 5.0 g.

Distilled Water 1000.0 ml. pH adjusted to 7.2 with KaOH *An enzymatic digested casein The seed flask was shaken on a 220 rpm. rotary shaker^^ with a two inch throw for three days. The seed flask cul- ture was then used to inoculate eleven two-liter baffled Erlenmeyer flasks each containing 350 ml. of Medium III using a 2-3% inoculum. Medium III has the following composi-tion: . Medium III: * .

Distilled Water 1000.0 ml. pH adjusted to 7.2 with NaOH *Trace Element Mix No. 2: FeSO^ · 7H20 1.0 g.

MnSC^ · H20 1.0 g.

CuCl2 · 2H20 25.0 mg.

CaCl2 100.0 mg.

H3BO3 56.0 mg.

( H_J6M07>2if * 4H20 19.0 mg.

ZnSO,. · 7H20 200.0 mg.

Distilled Water 1000.0 ml.

The flasks were then shaken on a 135-150 rpm. shaker with a two inch throw for four days at 28°C. At the end of the in-cubation period the contents of the eleven flasks were com-bined and assayed. The assay on the combined, centrifuged broth showed an inhibition zone of 22 mm. (1/2 inch discs) against Proteus vulgaris on a standard assay plate. This antibiotic was identified as 810A, that is, an antibiotic mixture comprising 7$- (D-5-amino-5-carboxyvaleramido) -3-(a-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid and 7g- (D-5-amino-5-carboxyvaleramido) -3-(ct-methoxy-p-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (la) .

EXAMPLE 2 Antibiotic 810A A lyophilized tube of Streptomyces griseus (MA-2837) was opened asceptically and the contents were used to inoculate the nutrient medium slants described as Medium I in Example 1. These slants were incubated at 28°C. for one week after which they were stored at 4°C. A portion of the culture on one of these slants was then used to inoculate a 250 ml. baffled Erlenmeyer seed flask containing 50 ml. of the medium described as Medium II in Example 1. This inoculated medium was incubated at 28°C. for two days on a 220 rpm. rotary shaker with a two inch throw. The inoculum was then washed asceptically by centifuging down the mycelia, pouring off the supernatant, resuspending in an equal volume of saline solution, re-centrifuging and again resuspending in a 0.9% sodium chloride solution. The washed mycelia was then used to inoculate (2% inoculum) two 250 ml. baffled Erlenmeyer flasks each containing 50ml. of a chemically defined production medium which has been sterilized at 120° C. for 15 minutes. The chemically defined production medium has the following composition: Production Medium; . pH (unadjusted) 7.1 The production flasks were then shaken at 220 rpm. on a shaker with a two inch throw for four days at 28°C.

Assays were run at three and four days. Samples were centrifuged and the supernatants assayed by the disc-Petii plate procedure. Using 1/2 inch discs these broths gave inhibition zones against roteus vulgaris (MB-838) of 21 mm. after three days and 26 mm. after four days. The product was identified as Antibiotic 810A.

EXAMPLE 3 Antibiotic 810A A lyophilized tube of Streptom ces griseus (MA-4125a) was opened asceptically and the contents transferred onto 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 to 1000.0 ml. pH 7.9-8.0 The slants thus obtained were then used to inoculate several Erlenmeyer flasks (250 ml.) each containing 50 ml. of Medium V, infra.

Medium V: Yeast Autolysate (Ardamine) 10.0 g.

Glucose 10.0 g.

*Phosphate Buffer 2.0 ml.

MgSOit * 7H20 0.05 g.

DistilLed Water 1000.0 ml. pH - adjust to 6.5 using NaOH *Phosphate Buffer Solution: ΚΗ2Ρ0_> 91.0 g. aaHPOi* 95.0 g.

Distilled Water 1000.0 ml. The seed flasks were shaken for one day at 220 rpm. at 28°C. The contents of the flasks were then used to inoculate 39 unbaffled Erlenmeyer flasks (250 ml.) containing 40 ml. of the Medium VI, infra, at 3.5 ml. of inoculum per flask.

Medium VI : Corn Steep Liquor (wet basis) 40.0 g.

Dextrose 20.0 g.

NaCl 2.5 g. gSO,, · 7H20 0.5 g. /liter Polyglycol 2000 0.25% by volume (add to each flask individually) Distilled Water 1000.0 ml. pH - adjust to 7.0 with NaOH The pi-oduction flasks were shaken on a rotary shaker wi. th a two- inch throw at 0 rpm. and at 24°C. for 40 bom after which time the flasks were pooled, an aliquot was ΐη!;· for assay and the remainder delivered for extraction stu'l' -s .

The sample for assay was acidified to pH 4.0 using hydro-chloric acid, filtered, diluted 1:4 in pH 5.0 phosphate buffer and placed onto Proteus vulgaris MB- 838 plater, using 1/2 inch discs. The zone of inhibition was 26.5 mm. The product was identified as Antibiotic 810A but primarily it consisted of 7ρ- (D-5-amino-5-carboxyvaleramido) -3- (a-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid with only trace amounts of 76- (D-5-amino-5-carboxyvaleramido) -3- (a-methoxy-p-hydroxycinnamoyloxymethyl)-7-meth0xy-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 250 ml. baffled Erlenmeyer flask.

Medium V: Yeast Autolysate (Ardamine) 10.0 g.

Glucose 10.0 g.

*Phosphate Buffer 2.0 ml, MgSOj, · 7H20 0.05 g.

Distilled II?0 100Ό.0 ml, pH adjusted to 6.5 with NaOH *P The flask was then shaken on a rotary shaker at 220 rpm. for one day at 28°C. Three ml. of this vegetative inoculum was used to inoculate a seed flask containing 50 ml. of ' the following synthetic medium (Medium VII) in a 250 ml. baffled Erlenmeyer flask.

Medium VII: Seed 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- K2 HPO i, 2.0 g. 2.0 g- CaCl2 * 2H20 0.4 g. 0.4 g- MnS<¾ * H20 0.1 g. 0.1 g.

FeS(\ · 7H20 0.1 g. 0.1 g.

ZnS04 · 7H20 0.05 g. 0.05 g- MgS<¾ · 7H20 1.0 g. 1,0 g.

Glycerol 20.0 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 The seed flask was again shaken at 220 rpm. for one day at 28°C. This synthetic seed was then used to inoculat several 250 ml. unbaffled Erlenmeyer flasks containing of Medium VII production, supra, at 1.5 ml. of inoculum per flask. The production flasks were shaken at 220 rpm. and 24 °C. and their contents were then pooled and assayed at four and five days age. In the assay the whole broth was acidified to pH 4.0 using hydrochloric acid and the broth was then filtered and diluted 1:4 in pH 5.0 phosphate using 0.5 inch discs. An inhibition zone of 25.5 mm. was obtained with this fermentation broth after four days in^^ cubation and the product thus obtained was identified as 810A.

EXAMPLE 5 Preparation of Antibiotic 810A and Separation into Components Step A: Fermentation Stage 1; The contents of a lyophilized tube of Strep- tom ces griseus (MA-2837) was suspended in two ml. of ' Medium I (described in Example 1) and the resulting inoculum was used to inoculate slants of the same medium. These slants were incubated at 28°C. for five days or until well-sporulated and then ten ml. of Medium VIII, infra, was added to the slants.

Medium VIII : Meat Extract 0.3% NaCl 0.5% NZ Amine 1 % Dextrose 1 % pH 7.0 The growth on each slant was scraped into suspension and the suspension was used as the inoculum in Stage 2, infra.

Stage 2: The suspension obtained in Stage 1 was used to inoculate a 250 ml. baffled Erlenmeyer flask containing 50 ml. of sterilized Medium VIII (described in Stage 1) . The inoculated flask was then placed on a 220 rpm. rotary shaker and incubated for 48 hours at 28°C.

Stage 3; The contents of an inoculum flask from Stage 2 was used to inoculate a two-liter baffled - was then placed on a 220 rpm. rotary shaker and incubated for 48 hours at 28°C.

Stage 4; An inoculum of 500 ml. of the resulting growth from Stage 3 was used to inoculate a 200 gal-Ion stainless steel fermentor containing 467 liters of a sterile Medium VIII (described in Stage 1) . The fermentation was allowed to proceed at a temperature of 28°C. with agitation (130 rpm.) while maintaining an air flow of 10 cfm for 65 hours. During the fcr-mentation an antifoam agent, Polyglycol 2000, was added in small quantities to prevent excessive foaming.

Stage 5 ; An inoculum of 100 gallons of the resulting growth from Stage 4 was used to inoculate a 1500 gal-Ion stainless steel fermentor containing 1200 gallons of Medium IX, infra.

Medium IX: Corn Steep Liquor 4% Dextrose 2% pH adjusted to 7.2 with NaOH The fermentation was allowed to proceed at a tempera-ture of 28°C. with agitation (120 rpm) . While main-taining an air flow of 55.3 cfm for 30-36 hours. Dur-ing the fermentation Polyglycol 2000 was added in small quantities to prevent excess foaming. The batch was harvested and activity was determined by disc-plate assay. Using 0.5 inch discs this broth gave an inhibition zone of 32.5 mm. against Proteus vulgaris MB-B38 when harvested at 31 hours age.

SJto _I3 s Isolation of Antibiotic Mixture 810A Filtered broth (1075 ga.1. ) frovu Step A, Stage 5, harvested after 36 hours and the pH adjusted from the range of 7-8 to .3.0 in the fermentor by the addition of phosphoric acid. The myceld were removed by passage through a plate-screen type filter press and discarded.

The filtered broth was then passed through a 100 gal. bed of Amberlite XAD-2 adsorbent resin at a flow rate of 10 gallons per minute. The spent broth was assayed and dis- carded and the resin bed was washed with two volumes of ' water. The antibiotic was eluted from the resin bed with a.60% solution of methanol and water at a flow rate of 5 gallons per minute. Forty fractions, each 5 gallons, were collected and assayed. Fractions 2 through 40 were combined and the methanol was removed by vacuum evaporation. The final concentrate (41.5 gal.) was adjusted to pH 3.5 by the addition of ammonium hydroxide and held frozen.

Samples were bio-assayed be the disc-plate method against Proteus vulgaris .

Filtered Broth: Assays run on 1060 gallons of filtered broth gave the following gone diameters.

Spent Broth and Wash: Ten fractions of 100 gallons each assayed zero without dilution. The water wash assayed zero.

Eluate Fractions: Assays were run on all fraction The zone diameters are tabulated below: Eluate Fractions Fraction Zone Size Fraction Zone Size 1 0 21 33 mm. 2 28 mm. 22 33 3 35 23 34 4 34 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 33 36 27 17 38 37 28 18 36 38 26 19 36 39 26 20 35 40 26 Eluate Composite and Eluate Concentrate: Assays were also run on 195 gallons of eluate composite and 43.5 gallons of Antibiotic 810A in the form of eluate concentrate.

Eluate Composite 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 Assay: Filtered Broth 119 kg. 195 Gallon Eluate Composite 7.23 kg. 41.5 Gallon Eluate Concentrate 7.20 kg.

Stop C: Adsorption on an Anion Exchange JXes n The concentrate from Step B (20.7 gal.) was dilutcdjp* 31 gallons with water and adsorbed on a 22.5 liter bed of weakly basic anion exchange . resin (Jvmberlite IRA-68 resin on the chloride cycle) at pPI 4.0 and a flow of 2 gallons per minute. This was followed by a 45 liter water wash whereafter the resin bad was eluted with a pH 7.5 solution of 1 M sodium nitrate and 0.1 M sodium acetate at a flow rate of 1.5 liters per minute. Ten five gallon eluate fractions were then collected and the pH adjusted to 4 · with hydrochloric acid as collected.

All fractions were bio-assayed by the disc-plate method against Proteus vulgaris as follows: —————————————————————— Feed Solution Eluate Fractions; Dilution 1:10 Dilution Zone Size Fraction Zone Size Fraction Zone Size 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 m . 4 26 mm. 9 21 mm . 5 26 mm . 10 17.5 mm.

The spent stream assayed 25 mm. without dilution and the water wash assayed 23 mm. without dilution.

Step D: Adsorption on Anion Exchange Resin Fractions 1 through 10 fron Step C were combined and fed to a 45 liter bed of Amberlite XAD-2 adsorbent at pH 3.0 and at a flow rate of 5 liters per minute. The resin bed was washed with 90 liters of water at the same rate.

The antibiotic was then eluted from the resin by a 25% solution of acetone and water at a flow rate of 5 liters per minute. Sixteen five gallon fractions were collected.

All fractions wore assayed by disc-plate method against Proteus vulgaris as follows: The feed (190 lite gave the following zone diameters: Feed Solution Dilution Zone Size 1:5 30 mm. 1:10 27.5 mm. 1:20 24.2 mm.

The zone diameters of the eluate fractions are tabulated below: The eluate fractions 2 through 16, supra, were combined and the acetone removed by vacuum evaporation to a final volume of 17.4 liters. The 17.4 liter concentrate was adjusted to pH 4.0 by ammonium hydroxide and freeze dried to yield 620 g. of Antibiotic 810A, i.e., a mixture consisting essentially of 7g- (D-5-amino-5-carboxyvaleramido) -3- (a- methoxy-p~sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem- 4-carboxylic acid and 7p- (D-5-amino-5-carboxyvaleramido) -3- (a-methoxy-p-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem- 4-carboxylic acid. This dry product had a bioassay potency ml for a 25 mm. zone. 7£- (D-5-amino-5-carboxyvaleramido) - 3- ( α-methoxy-p-hydroxycinnamoyloxymethy1 ) - 7 methoxy-3-cepheiu- 4-carboxylic Acid A one inch diameter chromatography column was packed to a bed height of 100 centimeters with DEAE Sephadex A-25 anion exchange resin in a system containing 0.5 M ammonium bromide and 0.05 M acetic acid. The mixture of Antibiotic 810A (10.0 g.) obtained in Step D was dissolved in 18 ml. of a solution of 0.5 M ammonium bromide and 0.05 M acetic acid and charged to the column. Eluting solution was pumped through the bed at a rate of 81 ml./hour and 10 ml. fractions of eluate were collected by machine. The eluate stream was monitored by a differential refractometer . The refractometer record showed mass peaks at tubes 19 , 36, 79, 109 and 206. Disc plate assays against Proteus vulgaris (MB-838) were run on every third fraction using 0.5 inch diameter discs buffered at pH 7.0. The zone dia-meters are tabulated below: (Fractions 1 through 66 assayed zero) .

Zone Zone Zone Fraction Fraction Fraction Diamete Diameter. Diameter 69 18 mm. 122 29 204 40 + 72 24 125 28 207 40 + 75 26 128 27 210 40 + 78 31 131 26 213 40 + 81 — 134 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 23 107 40 + 189 40 + ■ 243 19 110 40 + 192 40 + 246 17 113 40 195 40 + 249 0 116 38 198 40 + 252 0 119 33 201 40 + Fractions 80 through 133 were combined and fraction^ 170 through 230 were combined.

A repeat of the above run was made and fractions 82-130 were combined and fractions 180-234 were combined.

The fractions containing the first active component from the two above runs were combined and adsorbed on a 100 ml. bed of Amberlite XAD-2 resin. The bed was washed with one volume of water and then eluted with three volumes of a 90% solution of methanol and water. The methanol was removed by vacuum evaporation and the aqueous concentrate was freeze dried to afford 810 mg. of a product identified as 73- (D-5-amino-5-carboxyvaleramido) -3- (a-methoxy-p-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid. The bio-potency of this product determined by disc plate assay against Proteus vulgaris was 18 yg./ml. afford-ing a 25 mm. zone. Analysis by ultraviolet adsorption gave the following characterizing data: U.V. adsorption in 0.1 N HCL \max. 305 ^cm 524 U.V. adsorption in 0.1 N NaOH ¾max. 328 E*cm 564 Step F; 7&- (D-5-amino-5-carboxyvaleramido) -3- (a-methoxy-p-sulfooxycinnamoyloxymethyl) -7- methoxy-3-cephem-4-carboxy]jc Acid ■ The fractions from the two runs on Sephadex A-25 con-taining the second active component were combined and ad-sorbed on a 100 ml. bed of Amberlite XAD-2 resin. The bed was washed with one volume of water and then eluted with three volumes of a 90% solution of methanol and water. The rich eluates were combined and methanol was removed by vacuum evaporation. The aqueous concentrate was freeze dried and yielded 720 mg. of 7f$- (D-5-amino-5-carboxyvaler-amido) -3- (a-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3- e hem-4-carbox lic acid Ic . Anal sis b ultr i let absorption gave the following characterizing data U.V. adsorption in 0.1 N HCl max. 287 mm. E lcm.

U.V. adsorption in 0.1 N NaOH max. 280 mm. E* 432 c lcm.

EXAMPLE 6 Separation of 7f$- (D-5-amino-5-carboxyvaleramido) -3- ( oc-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic Acid from the Antibiotic Mixture 810A The antibiotic mixture 810A comprising 7β- (D-5-amino-5-carboxyvaleramido) -3- ( a-methoxy-p-sulfooxycinnamoyloxy-methyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) and 73-(D-5-amino-5-carboxyvaleramido) - 3- ( ot-methoxy-p-hydroxycin-namoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (20.0 g.) from Example 5, Step D, was dissolved in water (200 ml.) and the pH of the solution adjusted to 3.5. This solution was passed thru a 200 ml. bed of Amberlite IRA-68 anion exchange resin on the chloride cycle followed by the addition of 300 ml. of water wash. The bed was then eluted with 1 liter of 1% (v/v) formic acid in water. This was followed by the addition of two portions of dilute hydro-chloric acid pH 0.95. The fed solution, spent and wash, and eluates 1, 2 and 3 were analyzed by paper electrophoresis at pH 4.0 run one hour at 1000 volts D.C. The papergram was dried, exposed to ammonia vapor to neutralize acid and incubated on a nutrient agar plate seeded with Proteus vulgaris (MB-838) . Examination after 17 hours incubation at 37°C. showed two zones of inhibition in the feed material (in the direction of the anode) , only a single component (slower of the two) in the spent and formic acid eluates and a single component in the second hydrochloric acid eluate corresponding with the faster component in the feed. The following table indicates total-solid and bio-assay Total Biological Mass Vol me Units Feed 20 g. 200 ml. 60,000 units la and lb Spent St 11.15 g. 5Q0 ml. lb 7,500 units Wash Formi c 4.52 g. 1000 ml. 20,000 units lb Acid Eluate 1st Hydro500 ml. 1,000 units chloric Acid Eluate 2nd Hydro2.10 g, 500 ml. 10,000 units la chloric Acid Eluate These fractions were recovered by adsorption on Amberlite XAD-2 resin to separate out 70- (D-5-amino-5-carboxyvaleramido>-3- (a-methoxy-p-sulfooxycinnamoyloxymethyl) - 7-methoxy- 3-cephem-4-carboxylic acid (Ic) and this was eluted by a 50% solution of methanol and water to afford substantially pure product (Ic) .

EXAMPLE 7 Separation of 7&- (D-5-amino-5-carboxyvaleramido) -3- (a-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic Acid from Antibiotic 810A The Antibiotic 810A mixture of 7β- (D-5-amino-5-carboxy-valeramido) -3- (a-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (Ic) and 7f$- (D-5-amino-5-carboxyvaleramido) -3- (a-methoxy-p-hydroxycinnamoyloxy-methyl) -7-methoxy-3-cephem-4-carboxylic acid (5.0 g.) ob-tained according to Example 5, Step D, was dissolved in a 20% solution of acetone and water (20 ml.) and the pH adjusted to 4.0. This solution was fed to a 2 inch diameter x 100 cm. height bed of Amberlite XAD-2 adsorbent in 20% acetone and wate^r solution. A solution of 20% acetone and water was pumped through the bed at a rate of 880 ml./hour and 20 ml. fractions were collected automatically.

Disc plate assays against Proteus vulgaris (MB-838) were run on every third . fraction using 0.25 inch discs . ¾ The zone diameters are tabulated belov; • Zone „ .. Zone Zone Fraction Fraction Fraction Dieimeter Diameter Diamet* 1-41 0 131 0 221 18 44 12 mr . 134 0 224 18 47 22 137 0 227 18 50 25 140 8 230 17 53 29 143 11 233 17 56 31 146 13 236 15 59 35 149 13 239 15 62 30 152 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 13 185 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 13 200 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 zero Fractions 44 through 90 were combined, acetone was removei by vacuum evaporation and the aqueous concentrate was frei " dried to yield 3.3 g. of crude 7$- (D-5-amino-5-carboxyvaler- amido) -3- (o-methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy- 3-cephem-4-carbox lic acid (Ic) · Fractions 150 through 225 were combined and by similar treatment afforded 700 mg. of 7β- (D-5-amino-5-carboxy- valeramido) -3- (a-methoxy-p-hydroxycinnamoyloxymethyl) -7- methoxy-3-cephem-4-carboxylic acid.

A repeat of the above run afforded 3.1 g. of crude 7β- (D-5-aminO"5-carboxyvaleramido) -3- (a-inethoxy-p-sulfooxycinnamoyloxyrnethyl) -7-methoxy-3-cephem-4-carbox lic acid (Ic) and 400 mg . of 7β- (D-5-arnino-5-carboxyvaleramido) -3- (a-niethoxy-p-hydroxycinnareoyloxymethyl) -7-methoxy-3-cephera-4-carboxylic acid.

The two quantities of 7β- (D~5-amino-5-carboxyvaler-amido) -3- (ct-methoxy~p-sulfooxycinnamoyloxyrnethyl) -7-methcxy-3-cephem-4-carboxylic acid (Ic) obtained according to the foregoing method were combined and the 6.4 g. of material was charged to a 2 inch diameter x 100 cm. height bed of Amberlite XAD-2 adsorbent in a 5% solution of methanol and v/ater. A 5% methanol and water solution was pumped through the bed at a flow rate of 880 ml. /hour and 20 ml. fractions were collected automatically. Two hundred and eighty-seven fractions were collected and every fourth fraction was assayed by the disc-plate method against Proteus vulgaris (MB-838) using 0.25 inch discs. The assay results are tabu-lated below. Fractions 1 through 50 were not assayed.

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 7 147 16 87 9 151 15 91 13 155 13 95 19 159 11 99 21 163 9 103 22 167 0 107 22 171 0 111 21 287 zero Fractions 95 through 159 were combined, methanol vacuum evaporated, and the aqueous concentrate freeze dried to cephem-4-carboxylic acid (Ic) . The ultra violet spectra ·* r of 76- (D-5-amino-5-carboxyvaleramido) -3- ( a-methoxy-p-sul fooxycinnamoyloxymethy1) -7-methoxy-3-cephem-4-carboxylic acid (Ic) gave the following adsorption data: U.V. adsorprtion in O.l^ HC1 max. 285 lcm. 160 U.V. adsorpction in 0.1 N NaOH max. 277 El.cm. 166 When assayed with 0.5 inch diameter discs by the disc-plate method against Proteus vulgaris the 73- (D-5-amino-5-carboxyvaleramido) -3- ( a-methoxy-p-sul fooxycinnamoyloxymethy1) -7-methoxy-3-cephem-4-carboxylic acid (Ic) sample gave a 25 mm. zone at 88 meg. /ml. and 73- (D-5-amino-5-carboxyvaleramido) -3- (a-methoxy-p-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid gave a 25 mm. zone at 167 meg. /ml.

Examples 8-13 are concerned with the preparation and purification of Antibiotic 842A whifch is 'ari intermediate for the synthesis of some of the compounds of the present invention.

EXAMPLE 8 , " Antibiotic 842A^ Step A; Shake Flask Production A lyophilized tube of Streptbmyces lactamdurans culture ( A-2908) was opened asceptically. The tube was then used to inoculate a 250 ml. baffled Erlenmeyer flask containing 50 ml. of nutrient Medium V by breaking the tube in sterile gauze and transferring the pellet asceptically into the flask. The Medium V has the following composition: Medium V: Yeast Autolysate (Ardamine) 10.0 g.

Glucose 10.0 g.

♦Phosphate Buffer 2.0 ml.

MgSOij * 7H20 0.05 g.

Distilled Water 1000.0 ml. pH 6.5 ♦ This seed flask was shaken at 28°C. on a 220 rpm. > rotary shaker with a two inch throw for three days. Five ml. aliquots (10% inoculum) of this growth were then transferred, using sterile pipettes, to four second-stage seed flasks of the same size and containing the same medium as described above and these flasks were then shaken in the manner in-dicated above. The second-stage seed flasks were then pooled asceptically into one flask and used to inoculate 11 two-liter baffled Erlenmeyer flasks, each containing 350 ml. of Medium IX with 2-3% inoculum using sterile pipettes. Medium IX has the following composition: Medium IX: Amber Yeast #300 10.0 g.

Distiller's Solubles 20.0 g.

Dextrose 10.0 g.

Distilled Water 1000.0 ml. pH 7.0 The production flasks were then shaken at 28°C. on a 145 rpm. shaker with a two inch throw for four days. 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 Antibiotic 842A, i.e. , the product 78- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (lb) , in the broth was determined by agar diffusion assays performed with 0.5 inch filter paper discs soaked in the broth and set on the sur-face of assay plates containing 10 ml. of nutrient agar (Difco) plus 0.2% yeast extract (Difco) medium seeded with the bacterial inoculum. The zones of inhibition were measured in mm. after overnight incubation at 28°C. The assays of broth harvested after fermentation for four days showed an inhibition zone of 31.5 mm. diameter on plates h brio rcolans Β 2 Step B: Adsorption On An Anion Exchange Resin The filtered broth was adjusted to pH 7.0 with di hydrochloric acid and 2900 ml. was adsorbed on 100 ml. of a strongly basic anion exchange resin having a styrene-divinyl ben zene matrix (Dowex 1 x 2 chloride cycle resin) at 10 ml/minute. The spent was collected in 500 ml. fractions. The resin column was washed with water and eluted with 3% NH.Cl in 90% methanol. The eluate was collected in 100 ml. 4 fractions .

Disc plate assays against Vibrio percolans (MB-1272) were run on all fractions; the zone diameters are tabula-ted below.

Filtered Broth Spent Fraction Eluate Fraction Dilution Zone Size Fraction Zone Size Fraction Zone 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 assays indicate about 60% of the activity is in the spent and about 18% is in the eluates. Furthermore, they indicate that the resin capacity is only two fractions or 10 column volumes of broth. Eluate fractions 1 through 4 were combined and concentrated to remove methanol. Spent fractions 3 through 6 were combined to give 1960 ml. of solution. An 1860 ml. portion of the solution was adjusted from pH 7.2 to 8.0 with dilute sodium hydroxide and adsorbed on 100 ml. of a strongly basic anion exchange resin having a styrene-divinylbenzene matrix (Dowex 1 2 chloride cycle resin) at 14 ml/minute. The spent was collected in four equal fractions and assays indicate that 5%of the activity The column was washed with water and eluted with 5% aqueous sodium chloride. The eluate was collected in 50 ml. frac-tions and assayed. The assays indicated that 90% of the activity was present in cuts 3 through 16 so these were combined.

Step C; Adsorption On A Cation Exchange Resin A 50 ml. portion of a concentrate as prepared in Step B was diluted to 500 ml., adjusted from pH 8.8 to pH 2.0 with dilute hydrochloric acid and adsorbed on 25 ml. of a strongly acidic cation exchange resin of the sulfonate type having a styrene-divinylbenzene matrix (Dowex 50 x 2 hydro-gen cycle resin) at 2.5 ml/minute. 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.). Assays of the spent fraction and eluate indicated 9% of the activity in the spent and 90% in the eluate. The eluate -was identified as the pyridinium salt of Antibiotic 842A.

The 842A product is amphoteric with an apparent iso-electric point at about pH 3.5. The product is unstable above pH 7 but stable at pH 1.5. The eluate thus obtained was adjusted to pH 8.0 with dilute sodium hydroxide and con-centrated under vacuum to remove pyridine. The product thus obtained was identified as the monosodium salt of Anti-biotic 842A. The molecular weight is 468 based on the empirical formula.

Analysis for 6H2 ^SOg a: 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 HI vi^ro studies this product, i.e., Antibiotic inhibits the growth of the following gram-negative bacte Escherichia coli, Proteus vulgaris, Alcaligenes faecalis Brucella bronchiseptica , Salmonella gallinarum Vibrio percolans and Xanthomonas vesicatoria. Also the product inhibits the growth of the following gram-positive bacteria: Staphylococcus aureus, Sarcina lutea and Bacillus subtilis.

In in_ vi o studies in mice Antibiotic 842A also exhibits the following activities. Administration was by subcutaneous injection, At the completion of the test period, usually seven days after administration, the amount of product re-quired to protect 50% of the mice (ED^Q) from this otherwise fatal injection was calculated: ED50 bY Subcutaneous Route X Two Doses Proteus vulgaris 51 yg.

Proteus mirabilis 276 yg. ¾>roteus morganii 3202 276 yg.

Salmonella schottmuelleri 103 yg.

Klebsiella pneumoniae AD 125 yg.

Klebsiella pneumoniae B 125 yg.

Paracolobactrum arizoniae 125 yg.

Escherichia coli 200 yg.

Aerobacter aerogenes 49 yg.

Pasteurella multocida 57 yg.

Salmonella typhosa 34 yg.

Diplococcus pneumoniae E400 566 yg.

*Cephaloridine and Cephalothin failed to protect at 4000 mg. x 2 doses In addition to the aforementioned in vivo trials of the product^ a clinical isolate of Proteus morganii 356 which is resistant to cephalosporins and capable of degrading cephalosporin C, was employed in a mouse protection test performed in the same manner as reported above. The ED5Q for these tests is as follows: ED50 ky Subcutaneous Route X 2 Doses Infection Antibiotic (average of Two Trials) EXAMPLE 9 Antibiotic 842A Shake Flask Production; The inoculum was prepared as described in Example 8. Two second-stage seed flasks were pooled and the broth used to inoculate (at 1 ml. /flask) 62 Erlenmeyer flasks (250 ml.) each containing 50 ml. of Medium X. The Medium X has the following composition: Medium X: Staley's 4S-Soybean Meal 30.0 g.

Distiller's Solubles 7.5 g.

Cerelose 20.0 g.

NaCl 2.5 g.

CaC03 (after pH to 7.0) 10.0 g.

Distilled Water 1000.0 ml.

The flasks were shaken at 28°C. on a 220 rpm. shaker with a two inch throw for five days. At the end of the incubation period the contents of 60 such flasks were combined and a sample was centrifuged to remove the mycelium. The presence of Antibiotic 842A was determined by fol-lowing the procedure described in Example 1 via agar dif-fusion assays performed on 0.5 inch filter paper assay discs. After incubation for four days, assay of broth gave an inhibition zone of 33 mm. versus Vibrio percolans (MB-1272) .

EXAMPLE 10 Antibiotic 842A Fermentation: Stage 1: A lyophilized tube of Streptomyces lactam- durans culture (MA-2908) was used to inoculate 50 ml. of sterile Medium V in a baffled 200 ml. Erlenmeyer flask.

Medium V: Yeast Autolysate (Ardamine) 10.0 g. Glucose 10.0 g. *Phosphate Buffer 2.0 ml. MgS<¾ - 7H20 0.05 g. Distilled Water 1000.0 ml. pH - adjust to 6.5 using NaOH *Phosphate Buffer: KH2 P04 91.0 g . a2HPO-> 95.0 g.

Distilled Water 1000.0 ml. The inoculated flask was placed on a 220 rpm. rotary shaker with a two inch throw and incubated for 72 hours at 28°C.

Stage 2: An inoculum of 10.0 ml. of the resulting vegetative growth was then used to inoculate a two-liter baffled Erlenmeyer flask containing 50 ml. of the sterilized Medium V described above. The inocul-ated flask was then placed on a 220 rpm. rotary shaker and incubated for 48 hours at 28°C.

Stage 3; The contents of the inoculum flask was then used to inoculate a 50 gallon stainless fermentor con-taining 160 liters of the same Medium V described above. The inoculated medium was incubated at 28°C. for 48 hours with agitation while maintaining an air-flow of 3 cfm through the fermenting broth. During the fermentation period, small amounts of Polyglycol 2000 were added to control foaming.

Stage 4: An inoculum of 43 liters of the resulting growth was then used to inoculate a 200 gallon stain-less steel fermentor containing 467 liters of a sterile Medium XII having the following composition: Medium XI * .

Amber Yeast #300 10.0 g.

Distiller's Solubles 20.0 g.

Distilled Water 1000.0 ml. pH 7.0 The fermentation was allowed to proceed at a temperature of 28°C. with agitation while maintaining an airflow of 10 cfm for 72 hours. During the fer- mentation an antifoam agent, Polyglycol 2000 was added in small quantities to prevent excessive foaming. The batch was harvested .and activity was determined by disc plate assay. The fermentation broth was then filtered through diatomaceous earth at a pH of 7.8 and the product thus obtained was identified as 8¾2A by following the procedure described in Example 1. Disc-plate assays of a 1:10 dilution gave an inhibition zone of 21.5 mm. ,v. Vibrio percolans (MB-1272).

EXAMPLE 11 Purification; Monosodium Salt of 7ρ- (D-5-Amino-5-Carboxy-valeramido) -3- (Carbamoyloxymethyl) -7-Methoxy-3-Cephem-4-Carbox lic Acid Adsorption on Carbon; Four fermentation batches harvested according to Example 8, were each adsorbed on 100 ml. of a strongly basic anion exchange resin having a styrenedivinylbenzene matrix (Dowex 1 x 2 chloride cycle resin) and eluted with 1% aqueous sodium chloride. The eluate was collected in 50 ml. fractions and assayed. Eluate fractions from all four batches were adjusted to pH 5 with dilute hydrochloric acid and combined to give 4300 ml. of solution. 4200 ml. of this solution was stirred with 42 g. of carbon (Darco G-60) for 1/2 hour. The carbon was collected by filtiation and washed with water. The trate and wash were void of activity. The carbon cake was eluted twice with a one liter portion of 60% aqueous acetone by stirring the mixture for 1/2 hour and filtering each time. The eluates were concentrated under vacuum to 108 ml. and 100 ml., respectively. Assays indicated that the first eluate contained 76% of the activity, 18 times as potent as the starting material and that the second contained 17% of the activity, 14 times as potent as the starting material. The two concentrates were combined and concentrated further to 61 ml. and adjusted from pH 4 to pH 5 with dilute sodium hydroxide. This concentrate contained 40 mg/ml. of dry solids and gave a 25 mm. zone against MB-1272 at a dilution of 1:100 (400 mcg./ml.).

The product was identified as the monosodium salt of 76- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (la) .

EXAMPLE 12 Purification; Monosodium Salt of 7ρ- (D-5-Amino-5-Carboxy-valeramido) -3- (Carbamoyloxymethyl) -7-Methoxy-3-Cephem-4-Carboxylic Acid Adsorption on Gel: A 22 ml. portion of the eluate obtained according to Example 8, Step B, was adjusted to pH 7.0 with dilute sodium hydroxide and chromatographed on a column containing 3.88 ml. of BioGel P-2. The column was developed with water, the effluent monitored with a dif-ferential refractometer and 5 ml. fractions collected automatically and bioassayed. The bioactivity appeared in fractions 47 through 63 while sodium chloride appeared in fractions 62 through 72. Fractions 50 tirough 60 were pooled, reassayed and concentrated to dryness yielding 10.8 mg. of residue identified as the monosodium salt of 7g-(D-5- amino-5-carboxyvaleramido) -3- ( carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (la) . On assay with Vibrio percolans this product gave a 25 mm. zone at 8 meg. /ml.

EXAMPLE 13 7β- (D-5-Amino-5-Carboxyvaleramido) -3- (Carbamoyloxymethyl) -7-Methoxy- 3-Cephem-4-Carboxyli c Acid Modified Fermentation Process: Step A: Slants A lyophilized tube of Streptomyces lactamdurans cul-ture (MA-2908) was opened asceptically and the organism transferred to a medium of the following composition: Medium XII: 1% Blackstrap Molasses 1% National Brewer's Yeast 2.5% Difco agar pH 7.0 Water to volume The slants are incubated for seven days at 28°C. When stored in the cold, the slants are stable for more than 13 weeks .

Step B: Seed Stages: Two Stage System First Seed: The first seed is inoculated directly from the slant of Step A to 40 ml. of 1% Primary Dried Yeast N.F. , pH 7.0 (obtained from the Yeast Product Corporation) in a 250 ml. baffled Erlenmeyer flask. The flasks were then shaken on a 220 rpm. rotary shaker with a 2 inch throw at 28°C. for a period of from two to three days.

Second Seed: A 2,5% inoculum from the first seed stage was added to a flask containing a 2% Fleischmann S-150 yeast autolysate, pH 7.0. The growth in this stage is character-istically light and the incubation, performed as in the first Step C; Production Medium The production medium contains per liter of distilled water: 30 g. distiller's solubles; 7.5 g. Primary Dried Yeast N.F. and 0.25% v/v Mobilpar-S defoamer. The medium is adjusted to pH 7.0 with a small amount of concentrated NaOH solution, dispensed into Erlenmeyer flasks and auto-claved for 15 or 20 minutes at 121 °C. After cooling the" medium received a 2.5% inoculum of the seed obtained in Step B. The time of incubation can vary from about 50 hours to 100 hours but an incubation period of about 72 hours is preferred. The volume of media in each flask can vary from 30 to 50 ml. but 40 ml. was used routinely. The level of inoculum can vary from 1% to 5%; but, in practice, a 2.5% level is generally employed.

Step D; Assay When the fermentation was complete, the cells were re-moved by centrifugation and the broth was diluted with phos-phate buffer, pH 7.0. The concentration of 7β- (D-5-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid in the fermentation broth was determined by the standard biological-disc assay method.

The assay organism employed was Vibrio percolans (ATCC 8461) . Filter paper discs are emersed into the diluted broths and placed on the surface of agar-containing Petri dishes that had been inoculated with the assay organism Vibrio percolans (ATCC 8461) . Also placed on these Petri dishes are discs that had been dipped previously in standard solutions containing known concentrations of 842A. The discs were incubated overnight at 28°C. and the diameters of the zones of inhibition recorded. The concentration of 842A and the fermented broth is calculated by interpola-tion from the standard curve which relates zone diameter with the known concentrations of standard 842A solutions. By this procedure it was calculated that Streptomyces lactamdurans MB-2908 produced 78.6 yg./ml. of 842A in the modified fermentation process.

EXAMPLE 14 Disodium. Salt of 7g- (D-5-Amino-5-Carboxyvaleramido) - 3- (N ,N- Dimethylcarbamoyloxymethyl) -7-Metho-y-3-Cephem-4-€arbo>ylic Acid Step A: 7β- (D-5-Phthaloylamino-5-Carboxyvaleramido) - 73- (D-5-Amino-5-carboxyvaleramido) -3- (carbamoyloxy-methyl) -7-methoxy-3-cephem-4-carboxylic acid (2.005 g., 4.27 m. moles) is dissolved in a solution of 10% aqueous dipotassium hydrogen phosphate (26 ml.). The mixture is then filtered and the resulting solid washed with 10% aqueous dipotassium hydrogen phosphate (2 ml.).

To the filtrate is added 10 ml. of acetone. The solu-tion becomes slightly cloudly and has a pH of 8.45. To this solution was added 50% aqueous tripotassium phosphate to adjust the pH to 9.18. N-Ethoxycarbonylphthalimide (1.492 g., 6.69 m. moles) in acetone (5.0 ml.) is then added over a five minute period. The pH is adjusted to 9.1 by adding increments of 50% aqueous tripotassium phosphate over the next 1.5 hours. The mixture is then evaporated to remove acetone and the pH adjusted to 2 to 2.5 with 2.5 N hydro-chloric acid. The mixture is extracted with four washes of ethyl acetate (25 ml.) and the combined pale yellow extracts are washed with 25 ml. of water, dried with anhy-drous sodium sulfate overnight and evaporated to afford 2.356 g. of a yellow foam. Nuclear magnetic resonance and electrophoresis confirm that the product thus obtained is 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (carbamoyloxy-methyl) -7-methoxy-3-cephem-4-carboxylie acid.

Step B: Dibenzhydryl Ester of 7g- (D-5-Phthaloylamino- 5-Carboxyvaleramido) -3- (Carbamoyloxymethyl) - 7-Methoxy-3-Cephem-4-Carboxylic Acid A solution of 7β- (D-5-phthaloylamino-5-carboxyvaler- of freshly prepared diphenyldiazomethane. After standing overnight at room temperature the solution is evaporated .3^" to afford 1.053 g. of residue identified as the dibenzhydryl ester of 76.- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (carbamoylxoymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Step C: Dibenzhydryl Ester of 70- (D-5-Phthaloylamino- 5-Carboxyvaleramido) -3- (Hydroxymethyl) -7- Methoxy-3-Cephem-4-Carboxylic Acid The dibenzhydryl ester of 73- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (0.5 g.) obtained in Step B is dissolved in purified dioxane (5 ml.) containing 1.5 equivalents of pyridine. A solution of nitrosylchloride (1.3 equivalents) in methylene chloride is added and the solution stirred in an ice bath for one hour. The product thus obtained is a solution of the dibenzhydryl ester of 7β- (D-5-phthaloyl-amino-5-carboxyvaleramido) -3- (hydroxymethyl) -7-methoxy-3-cephem-4-carboxylic acid which is used directly in the following step.

Step D; Dibenzhydryl Ester of 7B- (D-5-Phthaloylamino- 5-Carboxyvaleramido) -3- (Chlorocarbonyloxy- methyl) -7-Methoxy-3-Cephem-4-Carboxylic Acid An excess of phosgene is bubbled into a stirred solution of the 7β- (D-5-phthaloylamino-5-carboxyvaleramido)-3- (hydroxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained in Step C and the mixture is allowed to stand at room temperature overnight. The excess phosgene is removed by bubbling dried nitrogen through the solution for several hours. The product thus obtained is identified as the di-benzhydryl ester of 78- (D-5-phthaloylamino-5-carboxyvaler-amido) -3- (chlorocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Step E; Dibenzhydryl Ester of 7β- (D-5-Phthaloylamino-5- ~ Carboxyvaleramido) -3- (N,N-Dimethylcarbamoyloxy- methyl) -7-Methoxy-3-Cephem-4-Carboxylic Acid . The dibenzhydryl ester of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (chlorocarbonyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid obtained according to Step D is cooled in an ice bath and 2.5 equivalents of dimethylamine is added. The mixture is stirred at room temperature for one hour and the excess amine hydrochloride filtered off.

The product thus obtained is identified as the dibenzhydryl ester of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (N,N-dimethyIcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Step F: Disodium Salt of 7β- (D-5-Phthaloylamino-5-Carboxy- valeramido) -3- (N, -DimethyIcarbamoyloxymethyl) -7- Methoxy-3-Cephem-4-Carboxylic Acid The dibenzhydryl ester of 7β~ (D-5-phthaloylamino-5-carboxyvaleramido) -3- (Ν,Ν-dimethyIcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained in Step E is dissolved in anisole (3.5 ml.) and treated with trifluoro-acetic acid (10 ml.) at room temperature for 10 minutes.

The trifluoroacetic acid and anisole are then removed under reduced pressure while maintaining the temperature 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 hour at room temperature and the organic phase is separated and washed with water. The combined aqueous phase is then washed twice with methylene chloride and lyophxlized to afford the disodium salt of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (N,N-dimethyIcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic Step G; Disodium Salt of 7β- (D-5-Amino-5-Carboxy- valeramido) -3- (N ,N-Dimethylcarbaraoyloxynethyl)- .7- ethoxy-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. One equivalent of hydrazine hydrate is then added and the mixture is allowed to stand at room temperature overnight. The aqueous solution is extracted with ethyl acetate before lyophilization to afford the disodium salt of 73- (D-5-amino-5-carboxyvaleramido) -3- ( ,N-dimethylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carbojylic acid.

EXAMPLE 15 Disodium Salt of 7β- (I 5-AirarD-5-CarboxyvaleramMo) -3- (Piper-idinocarbonyloxymethyl)-7-Mettoxy-3-Cepem-4-Carb<^lic Acid Step A: Dibenzhydryl Ester of 7β- (D-5-Phthaloyl- amino-5-Carboxyvaleramido) -3- (Piperidino- carbonyloxymethyl) -7-Methoxy-3-Cephem-4- Carboxylic Acid Upon substituting an equivalent amount of piperidine for the dimethylamine of Example 14, Step E, and following the procedure described therein there is thus obtained the dibenzhydryl ester of 7β- (D-5-phthaloylamino-5-carboxy-valeramido) -3- (piperidinocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Step B; Disodium Salt of 7β- (D-5-Amino-'5-Carboxy- valeramido) -3- (Pi eridino carbonyloxymethyl) - 7-Methoxy-3-Cephem-4-Carboxylic Acid Upon substituting the dibenzhydryl ester of 7β-(ϋ-5-phthaloylamino-5-carboxyvaleramido) -3- (piperidinocarbonyl-oxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained according to Step A for the dibenzhydryl ester of 7β-(ϋ-5-phthaloylamino-5-carboxyvaleramido) -3- (Ν,Ν-dimethylcarbamoyl- Example l4, Step F, and following the de-esterification method described therein there is thus obtained the di-sodium salt of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (piperidinocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid which, when treated with hydrazine hydrate according to the method described in Example 14 , Step G, affords the disodium salt of 7*3- (D-5-amino-5-carboxyvaler-amido) -3- (piperidinocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

EXAMPLE 16 Disodium Salt of 7g-(D^-AiTdno-5-Caitoxyvaleramido)-3-(Pyrroli-din^carbonyloxymethyl) -7-Methoxy-3-Cephem-4 -Carboxylic Acid Step A; Dibenzhydryl Ester of 7β- (D-5-Phthaloyl-o -amino-5-Carboxyvaleramido) -3- (Pyrrolidinylt- carbonyloxymethyl) -7-Methoxy-3-Cephem-4- Carboxylic Acid Upon substituting an equivalent amount of pyrrolidine for the dimethylamine of Example 14, Step E, and following the procedure described therein there is thus obtained the dibenzhydryl ester of 7β- (D-5-phthaloylamino-5-carboxy- o valeramido) -3-¾>yrrolidinylcarbonyioxymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Step B: Disodium Salt of 76-^5-Amino-5-Carboxyvaler- amido) -3- (Pyrrolidin¾-icarbonyloxymethyl).-7- Methoxy-3-Cephem-4-Carboxylic Acid Upon substituting the dibenzhydryl ester of 7β-(ϋ- o 5-phthaloylamino-5-carboxyvaleramido) -3- (pyrrolidinyicarbony-loxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained according to Step A for the dibenzhydryl ester of 7β-(ϋ-5-phthaloylamino-5-carboxyvaleramido) -3- (N,N-dimethylcarbamoy-loxymethyl) -7-methoxy-3-cephem-4-carboxylic acid recited in Example 14 , Step F, and following the de-esterification method described therein there is thus obtained the di-sodiura^f salt of 7β - (D-5-phthaloylamino-5-carboxyvaleramido) -3- 0 (pyrrolidinyicarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid which, when treated with hydrazine hydrate according to the method described in Example 14, Step G, affords the disodium salt of 7β- (D-5-amino-5-carboxyv ler- o amido) -3- (pyrrolidinylcarbonyloxymethyl) -7-methoxy-3-cephem- 4-carboxylic acid.

EXAMPLE 17 Disodium Salt of 7B- (D-5-Amino-5-Carboxyvaleramido) -3- (Mor-pholinocarbonyloxymethyl) -7-Methoxy-3-cephem-4-Carboxylic Acid Step A: Dibenzhydryl Ester of 7β- (D-5-Phthaloylamino- 5-Carboxyvaleramido) -3- (Morpholinocarbonyl- oxymethy1) -7-Methoxy-3-Cephem-4-Carboxylic Acid Upon substituting an equivalent amount of morpholine for the dimethylamine of Example 14, Step E, and following the procedure described therein there is thus obtained the dibenzhydryl ester of 7β- (D-5-phthaloylamino-5-carboxyvaler-amido) -3- (morpholinocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Step B: Disodium Salt of 7β- (D-5-Amino-5-Carboxy- valera ido) -3- (Morpholin o carbonyloxymethyl) - 7-Methoxy-3-Cephem-4-Carboxylie Acid Upon substituting the dibenzhydryl ester of 7g-(D-5-phthaloylamino-5-carboxyvaleramido) -3- (morpholinocarbonyl-oxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained according to Step A for the dibenzhydryl ester of 76-(D-5-phthaloylamino-5-carboxyvaleramido) -3- (N ,N-dimethylcar-ba o loxymethyl) -7-methoxy-3-cephem-4-carboxylic acid recited in Example 14, Step F, and following the de-ester-ification method described therein there is thus obtained the disodium salt of 7 g- (D-5-phthaloylamino-5-carboxy-valeramido) -3- (morpholinocarbonyloxymethyl) -7-methoxy-3-ir cephem-4-carboxy.lic acid which, when treated with hydrazine hydrate according to the method described in Example 14, Step G, affords the disodium salt of 73- (D-5-amino-5-carboxyvaleramido) -3- (morpholinocarbonyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

EXAMPLE 18 (Illustrative Example) Disodium Salt of 73- (D-5-Amino-5-Carboxyvaleramido) -3- (Acetoxy-methyl) -7-Methoxy÷-3-Cephem-4-Carboxylic Acid Step A: Dibenzhydryl Ester of 73- (D-5-Phthaloylamino- 5-Carboxyvaleramido) -3- (Acetoxymethyl) -7- ethoxy-3-Cephem-4-Carboxylic Acid An excess of acetyl chloride is added with stirring to the dibenzhydryl ester of 7ρ- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (hydroxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained in Example 14, Step C. The mixture is allowed to stand for several hours at room temperature. The mixture is then concentrated in vacuo to remove excess acetyl chloride and solvent. The residue thus obtained is the dibenzhydryl ester of 73- (D-5-phthaloylamino-5-carboxy-valeramido) -3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

The dibenzhydryl ester of 73- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid can also be obtained by substituting ketene for acetyl chloride and otherwise following the procedure described in this Step A.

Step B: Disodium Salt of 73- (D-5-Phthaloylamino-5- Carboxyvaleramido) -3- (Acetoxymethyl) -7-Methoxy- 3-Cephem-4-Carboxylic Acid The dibenzhydryl ester of 7β- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3- cephem-4- carboxylic acid obtained in Step A is dissolved in dioxane^ and the mixture is subjected to the de-esterification method described in Example 14, Step F. There is thus obtained a residue comprising the disodium salt of 7ρ- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3~cephem-4-carboxylic acid.

Step C: Disodium Salt of 7 - (D-5-Amino-5-Carboxyvaler- a ido) -3- (Acetoxymethyl) -7-Methoxy-3-Cephem- 4-Carboxylic Acid The disodium salt of 7β- (D-5-phthaloylamino-5-carboxy-valeramido) -3- (acetoxymethyl) -7-methoxy- 3-cephem-4-carboxylic acid obtained in Step B is dissolved in methylene chloride and the solution extracted with 5% sodium bicarbonate solu-tion. The resulting mixture is then acidified to pH 2 with sulfuric acid and extracted with ethyl acetate. Upon drying the extract is concentrated in vacuo and the residue is dissolved in water. One equivalent of hydrazine hydrate is then added and the mixture is allowed to stand at room temperature overnight. The aqueous soltuion is extracted with ethyl acetate before lyophilization to afford the disodium salt of 7fi- (D-5-amino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy- 3-cephem- 4-carboxylic acid.

In a manner similar to that described in Example 18 all of the 3-acyloxy substituted derivatives of this in-vention may be obtained. Thus, for example, by substitut-ing the appropriate acyl halide for the acetyl chloride of Example 18, Step A and following the procedure described in Steps A, B and C of that example all of the corresponding 3-alkanoyloxy , 3-aromatic-carbonyloxy , 3-aralkanoyloxy and 3-cyclo-alkane-carbonyloxy substituted derivatives of this invention may be obtained. The following equation and Table XVIII illustrate this method and the products ob- COONa EXAMPLE 28 ; Disodium Salt of 7β- (D-5-Amino-5-Carboxyvaleramido) -3- ( ^ 1 Methylcarbamoyloxymethyl) - 7-Methoxy- 3;-Cephein-4-C arboxyUc Acid Step A: Dibenzhydryl Ester of 7β- (D-5-Phthaloyl- amino-5-Carboxyvaleramido) -3- (N-.Methyl- carbamoyloxymethy1) -7-Methoxy-3-Cephem-4- Carboxylic Acid The dibenzhydryl ester of 7f5- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (hydroxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (250 mg.) is suspended in dimethylform-amide (5 ml.). The reaction mixture is placed under nitro-gen and agitated by ultrasonic waves. Triethylamine (0.20 ml.) and methylisocyanate (0.625 ml.) are then added and after dissolving the mixture is allowed to stand for 0.5 hour.

Ethyl ether is added to the mixture and after centri-fuging, the ether is decanted. An additional quantity of ethyl ether is added to the oily residue and solidification of the product is facilitated by scratching. The resulting solid is recrystallized from a hot mixture of methanol and isopropanol (180 mg.) in two crops and the combination of these two crops are recrystallized again to afford a product identified as the dibenzhydryl ester of 7β- (D-5-phthaloyl-amino-5-carboxyvaieramido) -3- (N-methylcarbamoyloxymethyl) -7-methoxy-3-cephem-4- carboxylic acid.

Step B: Disodium Salt of 7$- (D-5-Phthaloylamino-5- Carboxyvaleramido) -3- (N-Methylcarbamoyloxy- methyl) -7-Methoxy- 3-Cephemr4-Carboxylic Acid The dibenzhydryl ester of 7&- (D-5-phthaloylamino-5-car-boxyvaleramido) -3- (N-methylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained in Step A is dissolved in anisole (3.5 ml.) and treated with trifluoroacetic acid (10 ml.) at room temperature for 10 minutes. The trifluoro-acetic acid and anisole are then removed under reduced pres-sure while maintaining the temperature below 40eC, and the residue is taken up in 25 ml. of chloroform and treated with 20 ml. of water, containing 0.120 g. of sodium bicar-bonate. The mixture is stirred for 0.5 hour at room temp-ature and the organic phase is separated and washed with water. The combined aqueous phase is then washed twice with methylene chloride and lyophilized to afford the di-sodium salt of 7B- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (N-methylcarbamoyloxymethyl) - 7-methoxy-3- cephem- -carboxy-lie acid.

Step C: Disodium Salt of 7g- (D-5-Amino-5-Carboxyvaler- amido) -3- (N- ethylcarbamoyloxymethyl) -7- Methoxy-3-Cephem-4-Carboxylic Acid The disodium salt of 7β- (D-5-phthaloylamino-5-carboxy-valeramido) -3- (N-methylcarbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid obtained in Step B is dissolved in water. One equivalen of hydrazine hydrate is then added and the mixture is allowed to stand at room temperature overnight. The aqueous solution is extracted with ethyl acetate before lyophilization to afford the disodium salt of 73- (D-5-a-nino-5-carboxyvaleramido) -3- (N-methylcarbamoyloxy-methyl) -7-methoxy-3-cephem-4-carboxylic acid.

By following the procedure described in Example '28 all of the 3- (N-mono-substituted carbampyloxymethyl) - derivatives of this invention may be obtained. Thus, by substituting the appropriate isocyanate for the methylisocyanate of Example 2 S , Step A, and following the procedure described in Steps A, B and C of that Example all of the correspond-ing 3-carbamoyloxymethyl substituted derivatives of this invention may be synthesized. The following equation il-lustrates the reaction of Example 28, StepsA, B and C and, together vith Table DJ infra, illustrate the starting EXAMPLE 38 76- (D-5-Aitiino-5-Carboxyvaleramido) -3- (Pyridiniummethy1) -7-Methoxy-3-Cephem-4-Carboxylic Acid A solution of 7β- (D-5-amino-5-carboxyvaleramido) -3-(acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (1.0 g.) is brought to pH 2.5. Pyridine (8 ml.) is added and the solution is heated at 60°C. for two hours. The reaction mixture is then lyophilized and the residue is dissolved in water and passed through a polystyrene trimethylbenzyl-ammonium anion exchange resin (43% H2O) . The resulting mixture of 76- (D-5-amino-5-carboxyvaleramido) -3- (pyridinium-methyl) -7-methoxy-3-cephem-4-carboxylic acid is diluted with water and selected fractions are lyophilized to afford sub-stantially pure 76- (D-5-amino-5-carboxyvaleramido) -3- (pyri-diniummethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Upon substituting an equivalent amount of trimethyl-amine and triethylamine for pyridine in the foregoing process and otherwise following the procedure described therein, there is thus obtained 76- (D-5-amino-5-carboxy-valeramido) -3- (trimethylammoniummethyl) -7-methyl-3-cephem- 4-carboxylic acid and 76- (D-5-amino-5-carboxyvaleramido) - 3- (triethylammoniummethyl) -7-methoxy-3-cephem-4-carboxylic acid.

All of the 3-pyridiniummethyl derivatives of this invention maybe obtained by substituting the appropriate mononuclear substituted pyridine for the pyridine reactant recited in the foregoing example. The following equation and accompanying Table illustrate this method of preparation and the products obtained thereby: TABLE XX EXAMPLE 65 S- [7g (D-5-Amino-5-Carboxyvaleramido) -4-Carboxy-7-Methoxy-3-Cephem- 3- ylmethyl] Isothiourea A solution of 7B- (D-5-amino-5-carb6xyvaleramido) - 3-(aceto methyl) -7-methoxy-3-cephem-4-carboxylic acid (1.0 g.) and thiourea (1.0 g.) in 25 ml. of water is main-tained at 37°C. for five days. Acetone (200 ml.) is added and the mixture is chilled in an ice-bath. The re-suiting product is then filtered and fractionated through a polystyrene trimethylbenzylammonium anion exchange resin (43% H20) . Selected fractions are lyophilized and the crude product is then recrystallized from a mixture of methanol and water to afford substantially pure S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl]-isothiourea.

Upon substituting an equivalent amount of N-methyl-thiourea, N-ethylthiourea, N-propylthiourea, ,N-dimethyl-thiourea, Ν,Ν-diethylthiourea and Ν,Ν-dipropylthiourea for the thiourea recited in the foregoing process and otherwise following the procedure described therein, there is thus obtained N-methyl-S- [7g- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] isothiourea, N-ethyl-S- [7g- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] isothiourea, N-propyl-S- [7g-(D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] -isothiourea, N,N-dimethyl-S- [7β- (D-5-amino-5-carboxyvaler-amido) -4-carboxy-7-methoxy-3-cephem-3-ylntethyl] isothiourea, N,N-diethyl-S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy- 3-cephem-3-ylmethyl] isothiourea and ,N-dipropy1-S- [7β- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy- 3-cephem-3-ylmethyl] isothiourea.

EXAMPLE 66 73- (D-5-Amino-5-Carboxyvaleramido) -3- (Ethylthiomethyl) -7-Methoxy-3-Cephem-4-Carboxylic Acid A mixture of 73- (D-5-amino-5-carboxyvaleramido) - 3-(acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (0.654 g.) and ethanethiol (0.37 ml.) in a mixture of one part acetone and one part water (10 ml.) is stirred at room temperature and a 10% sodium hydroxide solution (2.0 ml.) is added with stirring. The mixture is then heated in a sealed tube for 100 hours and the resulting mixture is con-centrated in vacuo . The residue is dissolved in water and fractionated through a polystyrene trimethylbenz/lammonium anion exchange resin (43% H20) . Selected fractions are combined and lyophilized to afford 73- (D-5-amino-5-carboxy-valeramido) -3- (ethylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Upon substituting 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-3 ,4-thiadiazole-5-thiol for the ethanethiol recited in the foregoing process and otherwise following the procedure described therein, there is thus obtained 7g- (D-5-amino-5-carboxyvaleramido) -3- (meth lthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 73- (D-5-amino-5-carboxy-valeramidoKHpropylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 73- (D-5-amino-5-carboxyvaleramido) -3- (2-pyridylthio-methyl) -7-methoxy-3-cephem-4-carboxylic acid, 73- (D-5-amino-5-carboxyvaleramido) -3- (3-pyridylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 73- (D-5-amino-5-carboxyvaleramido) -3- (4-pyridylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 73- (D-5-amino-5-carboxyvaleramido) -3- (2-benzothiazolylthio- carboxyvaleramido) -3- (4-methylpyrimidin-2-ylthiomethyl) -j^ 7-methoxy-3-cephem-4-carboxylic acid and 73- (D-5-amino-5-carboxyvaleramido) -3- (2-methyl-3, -thiadiazol-5-ylthiomethyl) 7-methoxy-3-cephem-4-carboxylic acid.

EXAMPLE 67 S- [7&- (D-5-Amino-5-Carboxyvaleramido) -4-Carboxy-7-Methoxy-3-Cephem-3-y lmethyl] ,N-Dimethyldithiocarbamate A solution of 7β- (D-5-amino-5-carboxyvaleramido) - 3-(acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (6.82 g. and sodium N ,N-di me byldith i.ocarbamate (2.86 g.) in 60 ml. of water is heated to 50°C. for 24 hours. The product is lyophilized and then fractionated through a polystyrene trimethylbenzylammonium anion exchange resin (43% H20) .

Selected fractions are then lyophilized to afford S-[7Q-(D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyU-N,N-dimethyldithiocarbamate.

Upon substituting an equivalent amount of the following reactants: sodium N-methyldithiocarbamate , sodium N,N-diethyl dithiocarbamate , sodium ,N-di-n-propyldithiocarbamate , sodium N-methyHi-C2-dLmethylaminoethy1) d thiocarbamate , sodium N-ethyl-N- (2-diethylaminoethyl) dithiocarbamate , sodium N- (2-di-n-propylaminoethy1) dithiocarbamate , sodium N-methyl-N- (2-morpholinoethyl) dithiocarbamate, sodium N-methyl-N- (3-diethylaminopropyl) dithiocarbamate, sodium N-phenyl-N- (2-methylaminoethy1) dithiocarbamate , sodium N,N-tetramethylenedithiocarbamate , sodium Ν,Ν-pentamethylene-dithiocarbamate, sodium N,N-bis- (2-hydroxyethyl) dithiocar-bamate and the sodium salt of 4-methyl-piperazinodithiocar-boxylate for the sodium dimethyldithiocarbamate recited in the foregoing process, omitting the sodium bicarbonate, but otherwise following the procedure described therein, there is thus obtained S- [76 - (D-5-amino-5-carboxyvaleramido)^^ 4-carboxy-7--nethoxy-3-cephem-4-ylmethylKJ-methyldithio-carbamate, S- [76 - (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl>N,N-diethyldithio-carbamate, S- [76- (D-5-arnino- 5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl]- ,N-di-n-propyldithiocar-bamate, S- [76- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl}-N-methyl-N- (2-dimethylamino-ethyl) dithiocarbamate , S- [76- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] -N-ethyl-N- (2-diethylaminoethy1) dithiocarbamate , S- [76- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] -N- (2-di-n-propylaminoethyl) dithiocarbamate, S- [76- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] -N-met yl-N- (2-morpholinoethy1) dithiocarbamate, S-[76-(D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethy-- -methyl-N- (3-diethylaminopropyl) dithiocarbamate , S- [76- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl]-N-phenyl-N- (2-methylaminoethyl) dithio-carbamate, S- [76- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] - ,N-tetramethylenedithiocar-bamate, S- [76- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] -N,N-pentamethylenedithiocar-bamate, S- [76- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl]-N,N-bis- (2-hydroxyethyl) dithio-carbamate and S- [76- (D-5-amino-5-carboxyvaleramido) -4-carboxy-7-methoxy-3-cephem-3-ylmethyl] -4-methyl-piperazinodithio-carboxylate.

EXAMPLE 68 7g- (t)-5-Amino-5-Carboxyvaleramido) -3- (Benzoylthiomethy 1) -7-Methoxy-3-Cephem-4-Carboxylic Acid A mixture of 7 g- (D-5-aini no-5-carboxy valeramido) -3-(acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (0.654 g.) , sodium bicarbonate (0.504 g.) and thiobenzoic acid (0.414 g.) in 5.0 ml. of water is heated at 50°C. overnight under a nitrogen atmosphere. The product is pre-cipitated by the addition of acetone and crystallized from a mixture of alcohol and water to afford 7 ø- (D-5-amino-5-carboxyvaleramido) -3- (benzoylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Upon substituting an equivalent amount of potassium ethyl xanthate, potassium n-propyl xanthate, potassium isopropyl xanthate', potassium n-butyl xanthate, potassium n-hexyl xanthate, potassium cyclopentyl xanthate and potassium cyclohexyl xanthate for the thiobenzoic acid recited in the foregoing process and otherwise following the procedure described therein, there is thus obtained 7$- (D-5-amino-5-carboxy valeramido) -3- (ethoxythiocarbonylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 7g- (D- 5- amino- 5- carboxy -valeramido) -3- (n-propoxythiocarbonylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 7β- (D-5-amino-5-carboxyvaler-amido) -3- (isopropoxythiocarbonylthiomethyl) -7-methoxy-3-cephem- 4- carboxy lie acid, 7β- (D-5-amino-5-carboxyvaleramido) -3- (n-butoxythiocarbonylthiomethyl) -7-methoxy-3-cephem-4-carboxylic acid, 7β- (D-5-amino-5-carboxyvaleramido) -3- (n-hexyloxythiocarbonylthiomethyl) -7-methoxy-3-cephem-4-car-boxy lie acid, 7 - (D- 5- ami no- 5- carboxy aleramido) -3- (cyclo-pentyloxythiocarbonylthiomethyl) -7-methoxy-3-cephem-4-car-boxylic acid and 7B~ (D-5-amino-5-carboxyvaleramido) -3- EXAMPLE 69 73- (D-5-Amino-5-Carboxyvaleramido) -3- ( oluene-p_-Sulfonyl-methyl) -7-Methoxy-3-Cephem-4-Carboxylic Acid A mixture of 7g- (D-5-amino-5-carboxyvaleramido) -3-(acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (0.654 g.) and sodium toluene-p_-sulfinate (1.0 g.) in 5.0 ml. of water is heated at 50 °C. for 24 hours. The mixture is concentrated in vacuo and crystallized from a mixture of methanol and water to afford 73 - (D-5-amino-5-carboxyvaleramido) -3- ( toluene- p_-sulfonylmethyl) -7-met.hoxy-3-cephem-4-carboxylic acid.

EXAMPLE 70 7β- (D-5-Amino-5-Carboxyvaleramido) -3- (Azidomethyl) -7-Methoxy-3-Cephem-4-Carboxylic Acid A mixture of 76 - (D-5-amino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylie acid (2.0 g.) and sodium azide (1.0 g.) are dissolved in 10 ml. water and heated at 50°C. overnight. The mixture is then lyophilized to afford crude 7β- (D-5-amino-5-carboxyvaleramido) -3- (azido-methyl) -7-methoxy-3-cephem-4-carboxylie acid.

Alternatively, in lieu of treating 7β- (D-5-amino-5-carboxyvaleramido) -3- (acetoxymethyl) -7-methoxy-3-cephem-4-carboxylic acid with sodium azide, it is possible to sub-stitute 842A per se therefor in an otherwise analogous process to afford an identical product. The following example illustrates this method of preparation: 7 β- (D-5-Amino-5-carboxyvaleramido) -3-fcarbamoyloxy-methyl) -7-methoxy-3-cephem-4-carboxylic acid (100 mg.) in a 0.5 M phosphate buffer solution (5 ml., obtained by adding 3.5 g. of sodium dihydrogen phosphate and 3.4 g. of disodium phosphate in 100 ml. of water followed by the addition of heated in the presence of sodium azide (20 mg.) at 95°(¾^^ for eight minutes. Preparative thin layer chromatography gave 20 mg. of 7β- (D-5-amino-5-carboxyvaleramido) -3- ( zido-methyl) -7-methoxy-3- ephem-4-carboxylic acid as indicated by infra-red and nuclear magnetic resonance identification. Treatment of this material with trifluoroacetic acid (1.0 ml.) at 0°C. for 5 minutes followed by quenching in a large volume of ether and evaporation of the solvent afforded 15 mg. of 7$- (D-5-amino-5-carboxyvaleramido) -3-(azidomethyl) -7-methoxy-3-cephem-4-carboxylic acid in the form of a white powder. This material possessed bioactivity against several strains of bacteria. This product was characterized by the following data: Thin Layer Chromatography: conducted on cellulose plates in a 75% aqueous acetonitrile solvent; the Rf value for this product was 0.7; the Rf for the disodium salt of 842A was 0.3.

Ultra-Violet : the λ maximum was 263 millimicrons (i.e., my); the Molecular Extinction Coefficient was approximately 3400 (theoretical: 8000); Infra-red in nujol was 1780cm~^ indicating the presence of a β-lactam ring; also, the product gave a positive ninhydrin test indicating the presence of an a-aminoadipoyl side chain.

Bioassay : The disc zone sizes in the following table are expressed in mm. for ΙΟΟγ/ml of antibiotic.

Escherichia coli W-MB-60 Pseudomonas EXAMPLE 71 76- (D-5-Amino-5-Carboxyvaleramido) -3- (Aminomethyl) -7-Methoxy-3-Cephem-4-Carboxylic Acid The crude sample of 73- (D-5-amino-5-carboxyvaleramido) -3- (azidomethyl) -7-methoxy-3-cephem-4-carboxylic acid ob-tained in Example 70 is hydrogenated over platinum oxide in an aqueous methanol solution containing acetic acid. The catalyst is filtered and the solution concentrated. A solu-tion of the residue is then fractionated through a poly-styrene trimethylbenzylammonium anion exchange resin (43% H20) and selected fractions are combined and lyophilized to afford 7g- (D-5-amino-5-carboxyvaleramido) -3- (aminomethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Acylation of the 73- (D-5-amino-5-carboxyvaleramido) -3-(aminomethyl) -7-methoxy-3-cephem-4-carboxylie acid with acetyl halide, propionyl halide and 2-phenylacetyl halide yields the corresponding N-acylated derivatives: 73-(D-5-amino-5-carboxyvaleramido) -3- (acetamidomethy1) -7-methoxy-3-cephem-4-carboxylie acid, 76- (D-5-amino-5-carboxyvaler-amido) -3- (propionamidomethyl) -7-methoxy-3-cephem-4-carboxylic acid and 73- (D-5-amino-5-carboxyvaleramido) -3- (2-phenyl-acetamidomethyl) .

Alternatively, in lieu of the catalytic process re-cited above 73- (D-5-amino-5-carboxyvaleramido) -3- (azido-methyl) -7-methoxy-3-cephem-4-carboxylie acid may also be reduced to the corresponding amine via molecular hydrogena-tion. The following example illustrates this method of preparation. 73- (D-5-Amino-5-carboxyvaleramido) -3- (azidomethyl) -7-raethoxy-3-cephem-4-carboxylic acid was dissolved in a e a id nd 10% water. The mixture was 10 minutes the mixture was filtered and treated with hydrogen sulfide to remove soluble zinc. The resulting mixture was then diluted with a large volume of water and lyophilized to afford substantially pure 73- (D-5-amino-5-carboxyvaleramido) -3- (aminomethyl)-7-methoxy-3-cephem-4-carboxylic acid. This product was characterized by the following data: Thin Layer Chromatography; conducted on cellulose plates in a 75% aqueous acetonitrile solvent; the Rf value for this product was 0.2; the Rf for the disodium salt of 842A was 0.3.

Ultra-Violet; the λ maximum was 263 my; the Molecular Extinction Coefficient was approximately 3500 (theoretical: 8000); Infra-red in nujol was 1780cm-"" indicating the presence of a 3-lactara ring; also, the product gave a positive nin- hydrin test indicating the presence of an γ-aminoadipoyl side chain.

Bioassay ; The disc zone sizes in the following table are expressed in mm. for equal concentrations of antibiotic.

Vibrio percolans MB-1272 18 20 (5y/ml) Salmonella gallinarum MB-1287 27 21 (ΙΟΟγ/ml.) Pseudomonas stutzeri MB-1231 21 21 (ΙΟΟγ/ml.) 14281 EXAMPLE 72 76- (D-5-Amino-5-Carboxyvaleramido) -3- (2 , 4-Dihydroxybenzyl-7-Methoxy-3-Cephem-4-Carboxylic Acid A mixture of 73- (D-5-amino-5-carboxyvaleramido) -3-(acetoxymethyl) -7-methoxy-3-cephem-4-carboxylie acid (0.654 g.), resorcinol (1.1 g.) and water (10 ml.) are heated at 50°C. for two days. The reaction mixture is then lyophilized to afford crude 76- (D-5-amino-5-carboxy-valeramido) -3- (2 , 4-di-hydroxybenzyl) -7-methoxy-3-cephem-4-carboxylic acid.

EXAMPLE 73 73- (D-5-Amino-5-Carboxyvaleramido) -3- EXAMPLE 74 Disodium Salt of 73- (D-5-Amino-5-Carboxyvaleramido) -3-(Hydroxymethyl) -7-Methoxy-3-Cephem-4-Carboxylic Acid Step A; Dibenzyl Ester of 7β- (D-5-Phthaloylamino-5- Carboxyvaleramido) -3- (Carbamoyloxymethyl) - 7-Methoxy-3-Cephem- 4-Carboxylic Acid A solution of 73- (D-5-phthaloylamino-5-carboxyvaler-amido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-car-boxylic acid (0.653 mg.) (Example 14, Step A) in methanol is treated with a slight excess of freshly prepared phenyl- 14281 the solution is evaporated to afford 1.053 g. of residue identified as the dibenzyl ester of 73- (D-5-phthaloyl-amino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid.

Step B; Dibenzyl Ester of 7β- (D-5-Phthaloylamino-5- Carboxyvaleramido) -3- (Hydroxymethyl) -7- Methoxy-3-Cephem-4-Carboxylic Acid The dibenzyl ester of 7fJ- (D-5-phthalo lamino-5-car-boxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy- 3-cephem-4-carboxylic acid (0.5 g.) obtained in Step A is dissolved in purified dioxane (5 ml.) containing 1.5 equivalents of pyridine. A solution of nitrosylchloride (1.3 equivalents) in methylene chloride is added and the solution stirred in an ice bath for one hour. The product thus obtained is a solution of the dibenzyl ester of 76- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (hydroxymethyl) -7-methoxy- 3-cephem-4-carboxylic acid which is used directly in the following step.

Step C; Disodium Salt of 73- (D-5-Phthaloylamino-5- Carboxyvaleramido) -3- (Hydroxymethyl) -7-Methoxy- 3-Cephem-4-Carboxylic Acid A solution of 10% palladium (0.5 g.) on carbon is added, together with 0.5 ml. of glacial acetic acid, to the dibenzyl ester of 73- (D-5-phthaloylamino-5-carboxyvaler-amido) -3- (hydroxymethyl) -7-methoxy- 3-cephem-4-carboxylic acid obtained in Step B. The mixture is hydrogenated at 40 lb. per square inch with agitation for two hours; the catalyst is removed by filtration through diatomaceous earth and the solvent removed by concentration in_ vacuo. The residue is dissolved in methylene chloride and the solution extracted with a 5% sodium bicarbonate solution. The re-suiting mixture is then acidified to pH 2 with sulfuric 14281 extract is concentrated in vacuo to afford a residue identified as the disodium salt of 76- (D-5-phthaloylamino-5-carboxyvaleramido) -3- (hydroxymethyl) -7-methoxy- 3- cephem-4-carboxylic acid.

Step D; Disodium Salt of 76- (D-5-Amino-5-Carboxy- valeramido) -3- (Hydroxymethyl) -7-Methoxy- 3-Cephem-4-Carboxylic Acid The disodium salt of 76- (D-5-phthaloylamino-5-carboxy-valeramido) -3- (hydroxymethyl) -7-methoxy- 3-cephem-4-carboxylic acid obtained in Step F is dissolved in water. One equiva-lent of hydrazine hydrate is then added and the mixture is allowed to stand at room temperature overnight. The aqueous solution is extracted with ethyl acetate before lyophiliza-tion to afford the disodium salt of 76- (D-5-amino-5-carboxy-valeramido) -3- (hydroxymethyl)-7-itethoxy-3-cepem-4-carboxylic acid.

EXAMPLE 75 Purification; Monosodium Salt of 76- (D-5-Amino-5-Carboxy-valeramido) -3- (Carbamoyloxymethyl) -7-Methoxy-3-Cephem-4-Carboxylic Acid The monosodium salt of 78- (D-5-amino-5-carboxyvaler-amido) -3- (carbamoyloxymethyl) -7-methoxy- 3-cephem-4-carboxylic acid (1.0 g.) prepared according to Example 12 was dissolved in 20 ml. of 1% aqueous n-butanol and chromatographed on a column containing 2,530 ml. of Sephadex G-10, a modified dextran gel in bead form. The column was developed with 1% aqueous n-butanol at 10 ml/minute and 10.5 ml. fractions were collected automatically. The effluent was monitored with a recording refractometer and the fractions were bio-assayed. The bioactivity appeared in fractions 99 through 122 and these were pooled and concentrated to dryness to yield 670 mg. of product containing primarily the monosodium - - - - - - - - The column was subsequently calibrated by chromato-graphy on a mixture of Blue Dextran 2000 and sodium chloride under identical conditions. Blue Dextran 2000 was detected in fractions 85 through 93 and sodium chloride was detected in fractions 140 through 155, thus indicating that the bioactive product can be separated from impurities of this nature.

EXAMPLE 76 7β- (D-5-Amino-5-Carboxyvaleramido) - 3-Methy1- 7-Methoxy- 3-Cephem-4-Carboxylic Acid A 10% palladium on charcoal catalyst was suspended in water (80 ml.) and treated with hydrogen. The catalyst was then filtered and suspended again in water (50 ml.) and to this mixture (2.67 g.) was added the sodium salt of 842A (1.0 g.) in water (10 ml.). The resulting mixture was shaken for twenty-two hours at room temperature.

The catalyst was removed by filtration and washed once with water (50 ml.) . The combined wash and filtrate was then concentrated to dryness to afford a 52.8% yield of 7&- (D-5-amino-5-carboxyvaleramido) -3-methyl-7-methoxy-3-cephem-4-carboxylic acid (528 mg.) .

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

Silica-gel plates were used with the upper phase consisting of 4 parts n-butyl alcohol, one part acetic acid and 4 parts water. The hydrogenolysis product exhibited two spots with a higher rate of flow (Rf) than the starting material. The spots were detected by ninhydrin and ultra-violet fluorescence. The following table lists the observed dif- - -5- 7 - (D-5-amin Measurement Sodium Salt of 842A 7-methoxy-3- Bioactivity 5 ug/ml gave a 25 mm zone Inactive at against Vibrio percolans (MB-1272 Thin Layer Rf 0.1 ninhydrin (positive) Rf 0.25 nin Chromatography ultra violet (positive) ultra viole 15 ug/spot RF 0.35 nin ultra viole Ultra Violet λ maximum 266 nm E 142 λ maximum 2 lcm EXAMPLE 77 L S- [ 7g - (D-5-Amino-5-Carboxyvaleramido) -4-Carboxy-7-Methoxy-3-Cephem-3-yl-Methyl] Thiouronium Antibiotic 842A (100 mg.) was heated for eight minutes with thiourea (26 mg.) at 95°C. in a 0.5 M phosphate buffer solution (5 ml.; obtained by adding 3.5 g. of sodium dihydrogen phosphate and 3.4 g. of disodium phosphate in 100 ml. of water followed by the addition of sufficient hydrochloric acid to bring the pH to 5) . Electro phoresis of the solution at pH 7 showed the thiouronium compound as a non-mobile entity whereas 842A had an Rf of about 0.2. The mixture was purified by absorption on a polystyrene nuclear sulfonic acid cation exchange resin on the hydrogen cycle (Dowex 50) to remove the phosphate buffer Elution was carried out using a 0.1N pyridine solution. The pH was adjusted to 8 with IN sodium hydroxide and evaporated under vacuum to remove residual pyridine. Lyophilization gave a tan powder identified as S- [73- (D-5-amino-5-carboxy-valeramido) - 4-carboxy-7-methoxy-3-cephem-3-yl-methyl] thio-uronium. This product was characterized by the following data: Thin Layer Chromatography: conducted on cellulose plates in a 25% aqueous acetonitrile solvent; the Rf value for this product was 0.15; the Rf for the disodium salt of 842A was 0.3.

Ultra-Violet: the λ maximum was 263 mu; the Molecular Extinction Coefficient was approximately 5700 (theoretical: 8000) ; Infra-red in nujol was 1780cm-1 indicating the presence of a β-lactam ring; also, the product gave a positive ninhydrin test indicating the presence of an a-aminoadipoy 1 Electrophoresis ; the introduction of a positive into the product was confirmed by electrophoresis at 1000 volts using 0.05 M phosphate buffer at H7; the Rf for the product was 0.01; the Rf for the disodium salt of 842A was 0.4.

Bioassay : The disc zone sizes in the following table are expressed in mm. for equal concentrations of antibiotic.

Disodium Salt S-j7β-(p-5-amino-5-carboxy- Organism of 842A valeramxdo) -S-carboxy^^ethoxy- ilOOv/ml) -3-cephem-3-yl-methyl]- y/ ' thiouronium (ΙΟΟγ/ml) Vibrio percolans B-1272 >35 27 Salmonella gallinarum MB-1287 27 23 Pseudomonas stutzeri MB-1231 22 22 Proteus vulgaris MB- 838 27 17 EXAMPLE 78 70- (D-5-Amino-5-Carboxyvaleramido) -3- (4-Methylthiazol-2-ylmercaptomethyl) -7-Methoxy-3-Cephem-4-Carboxylic Acid Step A: 70- (D-5-tI-Tertiary-Butoxycarbonylamino-5- Carboxyvaleramido) -3- (Carbamoyloxymethyl) - 7-Methoxy-3-Cephem-4-Carboxylic Acid The disodium salt of 842A (1.0 g.) was dissolved in 5% di-basic potassium phosphate (31.6 ml.) and acetone (20.8 ml.) was added with stirring. The pH of the solution was then adjusted to 9.5 to 9.6 with sodium hydroxide and tertiary-butoxycarbonylazide (1.0 ml.) was added with stirring. Stirring was continued for 4-6 hours at room temperature while maintaining the pH between 9.0 and 9.6 via the addition of sodium hydroxide. The reaction mixture was then stirred overnight, by which time the pH had The resulting mixture was then extracted with one-1* half volume of ethyl acetate and the extract was discarded. One-half volume of ethyl acetate was added and the aqueous layer was adjusted to pH 2.5 with concentrated hydrochloric acid in an ice bath to maintain the temperature below 5°C. After separation of the ethyl acetate the aqueous solution was extracted twice more with one-half volumes of ethyl acetate at pH 2.5. Based on ultra-violet measurements 50% of the starting ultra-violet absorbent was in the extracts and 22% in the spent aqueous phase. The extracts were combined and concentrated to dryness to yield 645 mg. of 7$- (D-5-N-tertiary-butoxycarbonylamino-5-carboxyvaleramido) -3- (carbamoyloxymethyl) -7-methoxy- 3-cephem-4-carboxylic acid having one-tenth the biopotency of the starting material.

Thin-layer chromatograms were run using Analtech silica gel G.F. plates and the upper phase of a 4:1:4 n-butanol, acetic acid, water system. The product had an Rf of 0.54 by ultra-violet detection and was ninhydrin negative.

Starting material had an Rf of 0.1 and was ultra-violet and ninhydrin positive.

Removal of the tertiary-butoxycarbonyl protecting group from a 10 mg. sample of the 7β- (D-5-N-tertiary-butoxycarbonylamino-5-carboxyvaleramid) -3- ( carbamoyloxy-methyl) -7-methoxy-3-cephem-4-carboxylie was accomplished by dissolving the material in trifluoroacetic acid (0.2 ml.) and allowing the solution to stand at room temperature for five minutes. The solution was then concentrated to dryness at room temperature. Thin-3ayer chromatography as previously described gave a spot for this product with an Rf of 0.1 - n iti e.

M Step B; 76 - (D-5-Airdno-5-Carboxyvaleramido) -3- ( 4-Methy1thiazo1-2-yli^ifiapJtome hy1) - 7- Methoxy-3-Cephem-4-Carboxylic Acid 73- (D-5-N-Tertiary-butoxycarbonylamino-5-carboxyvaler-amido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (100 mg.) in 5 ml. of a pH 7 buffer (a 0.5 M solution of a mixture of 3.5 g. sodium dihydrogen phosphate 3.4 g. of disodium phosphate in 100 ml. of water) containing 2-mercapto-4-methylthiazole (50 mg.) is heated at 95°C. for eight minutes. There is thus obtained 73- (D-5-N-tertiary-butoxycarbonylamino-5-carboxyvaleramido) -3- (4-methyl- ylthiotnetbyl acid which, upon treatment with trifluoroacetic acid (0.2 ml.), generates 73- (D-5-amino-5-carboxyvaleramido) -3- (4-methyl- y1thiome-thy3L thiazol-2-yi«e-?e«p*ef«e-fchy4) -7-methoxy-3-cephem-4-carboxylic acid. This product was characterized by the following data: Thin Layer Chromatography; conducted on cellulose plates in a 25% aqueous acetonitrile solvent; the Rf value for this product was 0.6; the Rf for the disodium salt of 842A was 0.3.

Ultra-Violet: the λ maximum was 269 m ; the Molecular Extinction Coefficient was approximately 2000 (theoretical: 8000) ; Infra-red in nujol was 1780cm-^ indicating the presence of a 3-lactam ring; also, the product gave a positive ninhydrin test indicating the presence of an a-aminoadipoyl side chain.

Bioassa : The disc zone sizes in the following table are expressed in mm. for equal concentrations Qf antibiotic.

Pseudomonas stutzeri MB-1231 22 13.5 Escherichia coli W-MB-60 18 14 Vibrio percolans MB-1272 23 20 EXAMPLE 79 By substituting 2-mercapto-l , 3-4-thiadiazole for the 2-mercapto-4-methylthiazole of Example 7¾, Step B, and other-wise following the procedure described therein there is thus obtained 7β- (D-5-amino-5-carboxyvaleramido) -3- (1, 3, 4-thiadi- -7-methoxy-3-cephem-4-carboxylic acid.

EXAMPLE 80 73- (D-5-Amino-5-Carboxyvaleramido) -3- (Thiocyanatomethyl) -7-Methoxy-3-Cephem-4-Carboxylic Acid Antibiotic 842A (100 mg.) was added to a 0.5 M buffer solution (5 ml.) consisting of 3.5 g. sodium dihydrogen phosphate and 3.4 g. disodium phosphate in 100 ml. of water and the pH of the mixture was brought to pH 5 by the addition of hydrochloric acid. Sodium thiocyanate (20 mg.) was added and the mixture was heated at 95°C. for eight minutes.

There is thus obtained 7β- (D-5-amino-5-carboxyvaleramido) -3- ( thiocyanatomethyl) -7-methoxy-3-cephem-4-carboxylic acid.

EXAMPLE 81 7g- (D-5-Amino-5-Carboxy valeramido) -3- (Oiloromethyl) -7- "^™ Methoxy-3-Cephem-4-Carboxylic Acid Step A: Dibenzhydryl Ester of 70- (D-5-N-Tertiary- Butoxycarbonylamino-5-Carboxyvaleramido) - 3- ( Carbamoy loxyme thy 1 ) - 7-Me thoxy- 3-Cephem- 4-Carboxylic Acid To a solution of 7g- (D-5-N-tertiary-butoxycarbonyl-amino-5-carboxyvaleramido) -3- (carbamo loxy methyl) -7-me thoxy -3-cephem-4-carboxylic acid (15.0 g.) in ethyl acetate (500 ml.) is added diphenyldiazomethane (5.5 g.) in 70 ml. of ether. The mixture is warmed to 40°C. with stirring and then treated after 30 minutes with additional diphenyldiazo-methane (5.5 g.) in ether (70 ml.) . After three hours the solvent is removed in vacuo and replaced by a mixture of methanol (500 ml.) and water (20 ml.) . The methanol -water solution is extracted four times with hexane and then evaporated in vacuo . The residue is dissolved in ethyl acetate, dried over sodium sulfate and evaporated iri vacuo to afford the dibenzhydryl ester of 7β- (D-5-N-tertiary-butoxycarbonylamino-5-carboxy valeramido) -3- ( carbamoy loxy-methyl) -7-methoxy-3-cephem-4-carboxylic acid.

Step B; Dibenzhydryl Ester of 7&- (D-5-N-Tertiary- Butoxycarbonylamino-5-Carboxy valeramido) -3- ( Chloromethyl) -7-Me thoxy- 3-Cephem-4-Carboxy lie Acid _____ The dibenzhydryl ester of 7&- (D-5-N-tertiary-butoxy-carbonylamino-5-carboxy valeramido) -3- (carbamoyloxymethyl) -7-methoxy-3-cephem-4-carbox lic acid (1 m. mole) is dis-solved in methylene chloride (5 ml.) and the mixture is cooled to 0°C. Collidine (1 m. mole) is added followed by the dropwise addition of a solution of phosphorous penta-chloride (0.6 m. mole) in methylene chloride (5 ml.) . The mixture is then stirred for an hour in an ice bath at 0°C. 81 of sodium chloride. The mixture is evaporated to dryness and then isolated by chromatography on a cooled silica column using chloroform as the eluant. The product thus obtained is the dibenzhydryl ester of 7β- (D-5-N-tertiary-butoxycarbonylamino-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-4-Carboxylic Acid A solution of dibenzhydryl ester of 7β- (D-5-N-tertiary-butoxycarbonylamino-5-carboxyvaleramido) -3- (chloromethyl) -7-methoxy-3-cephem-4-carboxylic acid (1 m. mole) in anisole (13 ml. is poured into 6J5 ml. of cold (0°C.) trifluoroacetic acid with stirring. After 5 minutes the solution is poured, with stirring, into an ether solution (1800 ml.) maintained at 0°C. The solid precipitate which results is then collected and dried to afford 76- (D-5-amino-5-carboxy-valeramido) -3- (chloromethyl) -7-methoxy-3-cephem-4-carboxylic acid trifluoroacetate .

The novel compounds of this invention have been described as having the 5-amino-5-carboxyvaleramido radical in the beta configuration with respect to the cephem nucleus. While this is based upon information currently available and is believed to be correct, applicants do not wish to be bound by this designation of spatial con-figuration in the event later information proves this to be incorrect.

The following organisms referred to in this specifica-tion are on deposit in the Culture Collection of the American Type Culture Collection where they are available under the following ATCC designations: 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 Also, in this specification several of the materials employed are referred to by trade name. These have the following composition and are available from the following suppliers: Amber Yeast No. 300: a fraction of autolyzed brewers yeast Amber Laboratories, Juneau, Wisconsin.

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

Polyglycol 2000: a defoamer; polypropylene glycol polymer having an average molecular weight of 2000; Dow Chemical Company, Midland, Michigan.

Biogel P-2: a gel filtration medium; a spherical poly- acrylamide . cross-linked with methylene bis-aerylandde ; B - L b t ies R chm nd a f rnia.

Dowex 50: a polystyrene nuclear sulfonic acid cation exchange resin; Dow Chemical Company, Midland, Michigan.

Analtech G.F. Plates: silica gel with Calcium sulfate binder and a fluorescent indicator 250 micrometers in thickness; Analtech Inc., 100 South Justison Street, Wilmington, Delaware, 19801.

Claims (9)

1. 36281/2 CLAIMS : ·' 1. 7β~ (D--5'~amino-5-carboxyvaleraniido) -7--methoxy-3'~cepheJ'- 4-carboxylic acids of the formula: R wherein R is hydrogen, halo, hydroxy, lower alkanoyloxy ^except ac aryl-carbonyloxy , pyridyl-carbonylox , aralkanoyloxy , cycloalkanecarbonyloxy , a-methoxy-p-sulfooxy cinna oyloxy , a-methoxy-p-hydroxycinnamoyloxy ; a carbamoyloxy radical of 1 2 l the formula: -OOCNR R wherein R and R are hydrogen, but at lea one of them is / lower alkyl, halo-lower alkyl, lower alkoxycarbony1 , aryl, alkarylsulfonyl , benzhydryl or, taken together with the nitrogen atom to which they are attached, an heterocycle selected from pyrrolidinyl , piperidino and morpholino; a 3 3 thio radical of the formula: -SR wherein R is lower alkyl or an heterocycle selected from pyridyl, alkyl substituted thiazolyl, 1, 3, 4-thiadiazol-2-yl , alkyl substituted 1,3,4- thiadiazol-2-yl , 2-benzothiazoly1 and 4-lower alkyl pyri»-Mmi«din-2-yl ; a pyridinium radical of the formula: wherein X1 is hydrogen, halogen, trifluoro ethyl , cyano, carboxy, carbamoyl, N-lo er alkyl carbamoyl, N,N-di- lower alkyl carbamoyl, carboxymethy1 , lower alkanoyl, lower alkyl, hydroxymethyl or sulfo; thiouronium; an amino- thiocarbonylthio radical of the formula: S - -S NCNR7R80 wherein R 7 and R8 are hydrogen, lower alkyl, hydroxy-lower 36281/2 alkyl, N-ary 1-N-lower alk laminoalky1 or, taken together^ with the nitrogen atom to which they are attached, an heter-ocycle selected from morpholino, piperidino, pyrrolidinyl or a piperazino radical of the formula: 9 wherein R is lower alkyl; aroylthio; an oxy-thiocarbony1 thio radical of the formula: S « 10 -SCOR wherein R"*"^ is lower alkyl or lower cycloalkyl; alkarylsul-fonyl; azido; amino or an amido radical of the formula: -NHR11 wherein R11 is lower alkanoyl or aralkanoyl; polyhydroxy-phenyl; N- .lower alky1-indol- 3-yl or thiocy anato; and the nontoxic, pharmacologically acceptable salts,' esters and amides( monoalkylamides , di- alkylamides , aralkylamides and nitrogen-containing heterocyclic amide derivatives thereof.

2. Compounds of Claim 1 wherein R is lower alkanoyloxy except acetoxy, aryl-carbonyloxy , 4-pyridylcarbonyloxy , aralkanoyloxy , cyclopentanecarbonyloxy , cyclohexanecarbony-loxy, ot-methoxy-p-sulfooxycinnamoyloxy or a-methoxy-p-hydroxy cinnamoyloxy . 4

3. Compounds of Claim ^wherein R is a carbamoyl- 1 ■? . 1 2 oxy radical of the formula: -OOCNR-'-R^ wherein R and R are the same or different radicals selected from hydrogen, lower alkyl, halo-lower alkyl, lower alkoxy carbonyl , phenyl, p-tolysulfonyl, benzhydryl, but cannot both be hydrogen, or 1 2 R and R taken together with the nitrogen atom to which they are attached form an heterocycle selected from i 36281/2

4. The compound of Claim 1 wherein R is a radical ^ the formula: wherein X-1 is hydrogen, rifluorojnethyl , cyano, carboxy, carbamoyl, N-lower alk l carbamoyl, Ν,Ν-di-lower ' alkyl carbamoyl, carboxy ethy 1 ,- lower alkanoyl, lower alkyl, hydroxyme thyl or sulfo.

5. The compound of Claim 1 wherein R is a radical of the formula: S wherein R7 and R8 are hydrogen, lower alkyl, hydroxy-lower alkyl, di-lower alkylamino lower alkyl, N-phenyl-N-lower alkylamino-alkyl or taken together with the nitrogen atom to which they are attached, an heterocycle selected from orpholino, piperidino, pyrrolidinyl or a piperazino radical of the formula: wherein R9 is lower alkyl. / compound according to Claim of the formula wherein R is hydroxy, lower alkanoyloxy except acetoxy, or N,N-di-lower alkyl carbamoyloxy; and the salts, esters and amide derivatives thereof. -171- 36281/2 . 7. The compound of Claim 6 wherein R is N#N-dimethy lcarbamoyloxy . 8. ' The compound of Claim 6 wherein R is hydroxy. 9. 7An antibiotic mixture obtained by growing- in an aqueous nutrient medium under aerobic conditions a new strain of Streptomy ces griseus , S treptomy cos viridochromo-genes , Streptomy ces f imbriatus , Streptomy ces hals tedii Streptomy ces rochei , Streptomy ces cinnamonensis or S trepto-myces chartreusis,. and comprising 7β- (D-5-amino-5-carboxy valeramido) -3- (ct-methoxy-p-sulfooxycinnamoyloxy-methyl) -7-methoxy-3-cephem-4-carboxylic acid and 7(3- (D-5-amino-5-carboxyvaleramido) -3- (a-methoxy- p- hydroxy cinnamoy-loxymethyl) ~7-methoxy-3-cephem-4-carboxylic acid. 10. Compounds according to Claim 1 of the formula: -toxic, pharmacologically acceptable salts, esters and amide derivatives thereof. - 172 _ 36281/2 11· A product according to Claim 10 wherein R is. sulfooxy and the alkali metal and alkaline earth metal salts thereof. 12 12. A product according to Claim 10 wherein R is hydroxy and the alkali metal and alkaline earth metal salts thereof. 13. The sodium salt of the compound of Claim 10. 14. Compounds of formula I in Claim 1, substantially as described herein with reference to the Examples. 15. A pharmaceutical composition comprising from 15 to 700 mg. of a compound of the ■ formula I in Claim 1 or a non-toxic, pharmacologically acceptable salt, ester and amide, mono-alkylamide , di-alkylamide , aralkylamide and nitrogen-containing heterocyclic amide derivative thereof, and a pharmaceutically acceptable carrier. .1

6. A pharmaceutical composition according to Claim/5 comprising from about 15 mg. to 700 mg. of 7β- (D-5-amino- 5-carboxyvaleramido) -7-methoxy-3-cephem-4-carboxylic acid substituted in the 3-position by hydroxymethyl , lower oxymethyl alkanoyloxymethyl except acetyl/ or an N,N-di-lower alkyl- carbamoyloxymethyl; and the salts , esters and amide derivatives thereof; and a pharmaceutically acceptable carrier. -173- 36281/4 1

7. A pharmaceutical composition according to Claim 15 comprising from about 15 mg. to 700 mg. of 7β- (D-5-amino-5-carboxyvaleramido) -3- ( -methoxy-p-sulfooxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboyxlic acid and 7(3-(D-5-amino~5-carboxyvaleramido)-3- (a-methoxy-p-hydroxycinnamoyloxy~methyl) -7 -met-hoxy-3-cephem 4-carboxylic acid or a mixture thereof or a salt, ester and amide derivative thereof; and a pharmaceutically acceptable carrier. 1

8. A pharmaceutical composition according to Claim 15 comprising 73- (D-5-5amino-5-carboxyvaleramido)~3- (a-methoxy-p-sulfooxycinna^jmoyloxymethyl) -7-iuethoxy-3~eephem-4-carboxylic acid or a salt thereof at a concentration of at least 2 to 4 micrograms per ml. as determined by disc-plate assay with Proteus vulgaris; and a pharmaceutically acceptable carrier. 1

9. Process for producing a compound of formula I in claim 1 which comprises preparing the intermediate 3-carbamoyloxy-methyl-7B- (D-5-amino-5-carboxyvaleramido) -7-methoxy-3-cephem-4-carboxylic acid by growing in an aqueous nutrient medium under aerobic conditions a new actinomycete capable of producing the desired product, wherein the actinomycete is Streptomyces lactamdurans, and converting the obtained product by conventional methods to the corresponding ϊ$Π¾½2 derivative. - 174 - 36281/2 20. The method of Claim 19 wherein the aqueous nutrient medium contains between about 1% and 6% by weight of carbohydrate and between about 0.2% and 6% by weight of available nitrog n. 21. The method of Claim 19 wherein the fermentation is conducted at. a temperature in the range of from about 20°C. to 37°C. 22. The method of Claim 19 wherein the pH of the aqueous nutrient medium is in the range of from about 6.0 to 8.0. 23. A method for the preparation of a mixture of. 73- (D-5-amino-5-carboxyvaleramido) -3- ( a-me thoxy-p-sulfooxy-cinnamoyloxymethy 1) -7-methoxy-3-cephem-4-carboxylic acid and 73- (D-5-amino-5-carboxyvaleramido) -3- ( a-methoxy-p-hydroxy cinnamoy loxymethyl) -7-me hoxy-3-cephem-4-carboxy lie acid, which comprises growing in an aqueous nutrient medium under aerobic conditions an actinomycete capable of producing the desired product, wherein the actinomycete is a new strain of Streptomyces griseus, Streptomyces viridochromogenes , Streptomyces fimbriatus , S treptomy ces hals tedii , Streptomyces rochei , S reptomyces cinnamonen-sis or Streptomyces chartreusis . -175- 36281/2, .^-^ 24. The method of Claim 23 wherein the actinom cete is a new strain of Streptom ces griseus . 25. The method of Claim 23 wherein the aqueous nutrient medium contains between about 1% and 6% by weight of carbohydrate and between about 0.2% and 6% by weight of available nitrogen. 26. The method of Claim 23 wherein the fermentation is conducted at a temperature in the range of from about 20°C. to 37°C. 27. The method of Claim 23 wherein the pH of the aqueous nutrient medium is in the range of from about 5.5 to 8.0. -176- 14281 A method for the preparation of a salt of 73- BF (Dr5-amino-5-carboxyvaleramido) -3- (hydroxymethy1) ~7-methoxy-3-cephem-4-carboxy lie acid; which comprises treating a dinster of 73 - (D-5-amino-5-carbox valeramido) -3- ( carbamoy loxymethyl) -7-methoxy-3-cephem-4-carboxylic acid in which the 5-amino group has been blocked, with a nitrosyl halide to afford the corresponding diester of 73-(D-5-amino-5-carboxyvaleramido) -3- (hydroxymethyl) -7-methoxy- 3-cephem-4-carboxylic acid which, when subjected to catalytic hydrogenation and treatment with a de-blocking agent, affords the desired product. 3-7". A method for the preparation of a salt of a compound of the formula: H wherein are lower alkyl or, taken together with the nitrogen atom to which they are attached, a mononuclear heterocycle selected from pyrrolidine, piperidine and morpholine; which comprises treating a diester of 73 -(D- 5-amino- 5-carboxy aleramido) - 3- (hydroxymethyl) - 7-methoxy-3-cephem- 4-carboxylie acid in which the 5-amino-group has been blocked, with a carbonyl halide and then with an appropriate primary or secondary amine to afford the corresponding diester of 73 - (D- 5-amino- 5-carboxyvaleramido) - 3 - (car-bamoyloxymethyl) - 7-methoxy- 3-cephem- 4-carboxylic acid which, upon de-esterification and treatment with a de-blocking agent, affords the desired product. 36281/2 30. A method for the preparation of a salt of a - compound of the formula: wherein R is lower alkyl, halo-lower alkyl, lower-alko carbonyl, aryl, alkarylsulfonyl or benzhydryl; which comprises treating a di-salt o diester of 7&-(D-5-amino-5-carboxyvaleramido) -3- (hydroxymethy1) -7-methoxy-3-cephem-4-carboxylic acid in which the 5-aminb group ha been blocked, with the appropriate isocyanate, followed de-esterification of the resulting diester intermediate and treatment with a de-blocking agent to afford the desired product. 31. A method for the preparation of a salt of a compound of the formula: 18 wherein R is lower alkyl , aryl, " pyridyl, aralkyl or cycloalkyl, which comprises treating a diester of 78- (D-5-amino-5-carboxy aleramido) -3- (h drox meth l) -7-methoxy-3-cephem-4-carboxylic acid in which the 5-amino-group has been blocked, with the appropriate acylhalide, followed by de-esterification of the resulting diester intermediate and treatment with a de-blocking agent to afford the desired product. 36281/2 32. A method for the preparation of a compound of the formula: in which R is a thio radical of the formula: -SR 3 wherein R is lower alkyl or an heterocycle selected from pyridyl, alkyl substituted thiazolyl, 1 , 3 , 4- thiadi azol-2-y1 , alkyl substituted 1,3,4-thiadiazol-2-yl , 2-benzothiazolyl and 4-lower alkyl pyrid-imidin-2-yl; an aminothiocarbonylthio radical of the formula 7 8 wherein R and R have the same meanings as in Claim 1 or a piperazino radical of the formula: wherein R is lower alkyl; aroylthio; an oxy-thiocarbonylthio radical of the formula: wherein R10 is lower alkyl or lower cycloalkyl; alkarylsul-fonyl; azido; polyhydroxyphenyl; N-lower alkyl-indol-3-yl; or pyridinium radical of the formula: 36281/2 . 1 . : J wherein X is hydrogen, halogen, trifluoromethyl , cyano, carboxy, carbamoyl, N-lower alkyl carbamoyl, N,N-di-lower alkyl carbamoyl, carboxymethyl , lower alkanoyl, lower alkyl, hydroxymethyl or sulfo; which comprises treating 7β- (D-5-amino-5-carbox valeramido) -3- (acyloxy-methyl) -7-methoxy-3-cephem-4-carboxylic acid with the appropriate thiourea, N- substituted thiourea, N,N-di-substituted thiourea, thiol, dithiocarba ate , thiobenzoic acid, dithiocarboxylate , alkali metal sulfinate, azide, polyhydroxybenzene, N-lower alkylindole, tri-lower alkyl-amine or pyridine. 33. The method of Claim 31 wherein the starting material is a 7β- (D-5-amino-5-carboxyvaleramido) -3- (lower alkanoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid . 34. The method of Claim 31 wherein the starting material is a 7β- (D-5-amino-5-carboxyvaleramido) -3- (acetoxy-methyl) -7-methoxy-3-cephem-4-carboxylic acid. 35. The method of Claim 31 wherein the 7β-(ρ-5-amino-5-carboxyvaleramido) -3- (azidomethyl) -7-methoxy-3-cephem-4-carboxylic acid1 thus obtained is reduced to afford the corresponding 7β- (D-5-amino-5-carboxyvaleramido) -3- (aminomethyl) -7-methoxy-3-cephe 4-carboxylic acid. -180- 1 8 Τ 26 Λ4. A method for the preparation of a compound of the formula : COOH wherein R 11 is lower alkanoyl or aralkanoyl, which comprises treating a 7β- (D-5-amino-5-carboxyvaleramido) -3- (amino-methy1) -7-methoxy-3-cephem-4-carboxylic acid with the appropriate acyl halide. 27 .4-5T A method for the preparation of 7β- (D-5-amino-5-carboxyvaleramido) -3-methyl-7-methoxy-3-cephem-4-carboxy-lic acid, which comprises the reniction of 7β- (D-5-ajtiino-5-carboxyvaleramido) -3- (carbamoyloxy ethy1} -7-methoxy-3-cephem-4-carboxylic acid or a salt thereof. 36281/2 38. A method for the preparation of a compound the formula: 21 wherein R is thiouronium, azido, thiazolylthio , thiadiazolylthio , 3-thiocyanato which comprises treating 73- (D-5-amino-5~carboxyvaleramido) - 3- ( carbamoyloxymethyl) -7-methoxy- 3- cephem-4- carboxylie acid with the appropriate thiourea, alkali metal azide, mercaptothiazole , mercaptothi adiazole or alkali metal thiocyanate. 39. A method for the preparation of 7β- (D-5-amino-5- carboxyvale amido) -3- (aminomethyl) - 7-methoxy-3- cephem- 4- carboxylic acid, which comprises the reduction of 7&-(D-5- amino-5- carboxyvaleramido) -3- (azidomethyl) -7-methoxy-3 cephem-4-carboxylie acid. 40. Methods for the preparation of compounds of formula I in Claim 1 substantially as described herein with reference to the Examples.