IL43361A - 3-(tetrazolo(4,5-b)pyridiazin-6-ylthiomethyl)-3-cephem 4-carboxylic acids their preparation and pharmaceutical compositions containing them - Google Patents

Description

3-(Ietrazolo ¾f 5÷t^py^&a£i -6*y^ -3- cepli©ini*4-carb0¾i l±c aeid0,; their preparation and ptermace tical compositions containing them BBISTOL-MYERS 0. 41537 This invention relates to certain novel 3-thiolated-7-acylaminocephalosporanic acid derivatives which have "been found to be valuable as antibacterial agents, aa nutritional supplements in animal feeds, as agents for the treatment of mastitis in cattle and as therapeutic agents in poultry and animals, including man, in the treatment of infectious diseases caused by many Gram-positive and Gram-negative bacteria More particularly, this invention relates to 7- (o-aminomethyl-phenylacetamido)-3- (tetrazolo [ 4,5-blpyridazin-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(o-aminomethylphenylpropionamido)-3- (tetrazo lo [ 4,5-b ]pyr^azin-6-ylthiomethyl)-3-cephem-4-carboxyllc acid, the dimethanesulfonate derivatives of said acids, and nontoxic pharmaceutically acceptable salts and easily hydrolyzed esters thereof. Also included within the present invention is the novel intermediate 7-amino-3-(tetrazoloT4, 5-b]pyridazin-6-ylthiomethyl)-3-cephem-4-carboxylic acid which may be acylated as described herein to produce the novel active cephalosporin derivatives of this invention.

There is extensive literature relating to the cephalosporin antibiotics. The more important prior art in this field is given below 7-Phenylacetamidocephalosporanic acid also named N-phenylacetyl derivative of 7-ACA, cephaloram, PACA and apparently phenasporin is well-known from the scientific literature. Publications on the preparation and/or properties of this compound, with or without substituents in the benzene ring, and corresponding compounds in which the 3-acetoxymethyl group has been replaced by methyl, hydroxymethyl and/or pyridinium- methyl include the following: 1 2 Chauvette, R. R.> et al. "Chemistry of Cephalosporin Antibiotics II. 3 Preparation of a New Class of Antibiotics and the Relation 4 of Structure To Activity", Journal of the American Chemical 5 Society. 84, 3401-3402 (1962)·. 6 Chauvette, R. R. , et al. "Structure-Activity of Relationships 7 Among 7-Acylamidocephalosporanic Acids", Antimicrobial Agents 8 and Chemotherapy - 1Q62. 687-69 . 9 Cocker, J. D. et al., "Cephalosporanlc Acids. Part II. Dis- 10 placement of the Acetoxy-group by Nucleophiles", Journal 11 . of the Chemical Society. 5015~5031 (1965). 12 Cocker, J. D. et al., "Cephalosporanlc Acids. Part IV. 7-*3 Acylamidoceph-2-em-4-carboxyllc Acids", Journal of the 14 Chemical Society. 1142-1151 (I966). 15 Culp, H. W., et al., "Metabolism and Absorption of the 7-(Phenyl- 16 acetamido-l-Cli*)rCephalosporanic Acid", Antimicrobial 17 Agents and Chemotherapy - 1963, 243-246. 18 Jago, M„ "Antibacterial Activity of Some Derivatives of 7- *9 Aminocephalosporanic acid Against Staphylococcus Aureus and 20 Synergism Between these and Other Antibiotics", Brit. J. 21 Pharmacol..22, 22-23 (1964). 22 Ioder, B., et al., "The Cephalosporin C Nucleus (7-Amino- 23 cephalosporanic Acid) and some of its Derivatives", 24 Biochemical Journal.1 , 4o8-4l6 (1961). 25 Nishida, M. , et al., "Studies on Microbial Degradation of 26 Cephalosporin C Derivatives. II", The Journal of 27 Antibiotics, 2 . 375-378 (I968). 28 Nishida, M., et al., "Studies of Microbial Degradation of 29 Cephalosporin C Derivatives I.", The Journal of Antibiotics. 30 21, 165-169 (1968).

Spencer, J. L., et al., "Chemistry of Cephalosporin Antibiotics VTII. Synthesis and Structure-Activity Relationships of Cephaloridine Analogues", Antimicrobial Agents and Chemotherapy - 1Q66. 573"580 Stedman, R. J. et al., "7-Aminodesacetoxycephalosporanic Acid and its Derivatives", J. Med. Chem.. 7(1) . 117-119 ( 1964) . Sullivan, H. R., et al., "Metabolism of Oral Cephalothin and Related Cephalosporins in the Rat", Biochemical Journal. 102, 976-982 (1967) .

, Vymola, P., et al., "The Classification and Characteristics of Cephalosporin Antibiotics I. Systematic Study of the Quantitative Sensitivity of Some Pathogenic Microorganisms to Cephaloridine", Journal of Hygiene. Epidemiology.

Microbiology and Immunology. 10. I8O-I89 ( 1966) . · Many other 7-acyl derivatives of 7~aminocephalosporanic acid have been reported in the patent literature including 7- [4-(a-aminoalkyl)phenylacetamido]cephalosporanic acids (U.S. Patent 3,582,241), 7- [ (p-aminophenylthlo)acetamido]- cephaiosporanic acid (U.S. Patent 3,422, 100) , 7-halophenyl- thioacetamido)cephalosporanic acids (U.S. Patent 3,335, 136) and the nearly unlimited number of variations of such compounds encompassed by the generic formulae (and often not otherwise described) of such patents as Netherlands 69/02013 (Parmdoc 39, 172) . 7-(p-Aminophenylacetamido)-cephalosporanic acid is disclosed in U.S. Patent 3,^22, 103 as is the corresponding N-trJLtyl derivative; see also Japan 2712/67 (Parmdoc 25,4o6) .

U.S. Patent 3,219,662 includes claims to compounds of the structure R - CHg - CO - ACA in which R is phenyl, nitrophenyl (especially para-nitro), chlorophenyl, alkylphenyl and alkoxy- phenyl and the corresponding phenoxy and substituted compounds and for all of those the corresponding compounds in which the 3-acetoxymethyl group has been replaced by a 3-pyridiniummethyl group. A more extensive group of such compounds, including the series in which R is phenylthio and also the compounds in which R is benzyl [i.e., 7-0-phenylpropionamido)cephalosporanic acid], alkoxybenzyl, alkanoyloxybenzyl, aminobenzyl, etc. are disclosed, at least generically, for use as starting materials in Great Britain 1 ,012, 9*3 and 1, 153,421 (Parmdoc 23, 984) and see also Great Britain 1,001, 478 and U.S. 3,280, 118. Additional 7-phenylacetamidocephalosporanic acids having substituents on the benzene ring including hydroxy and amino are disclosed as starting materials in Great Britain 1,082, 943 and 1,082, 962.

U.S. Patent 3,341, 531 describes the 7~(o-, m- and p- carboxamidomethylphenylacetamido)cephalosporanic acids and their betaines. A variety of ' 7'-(.halor, dihalo- nitro- and halonitro-phenylacetamido)cephalospbrahic acids are named as starting materials for reaction with certain nucleophiles in U.S. Patent 3,^31,259 (Parmdoc 27,715) . Additional 7-phen lacetamido)cephalosporanic acids having various substituents on the benzene ring are disclosed in Japan 2712/67 (Parmdoc 25, 4o6) , Japan 26105/69 (Parmdoc 40,86o) , Great Britain 1, 178, 471 (Parmdoc 27,715, see Netherlands 67/ΟΟ906) and Japan 25785/69 (Parmdoc 40,847) .

Replacement of the 3-acetoxy group of a cephalosporin by various heterocyclic thiols has been disclosed a) in South Africa 70/2290 [see also Netherlands 70/05519 (Farmdoc 80, 188R)] where the sidechains were, for example, 7-a- aminophenylacetamido and typical heterocyclic thiols were 1 2-methyl-l,3, 4-thiadiazole-5-thiol and l-methyl-l,2 ,3 , 4-tetra.*>le 2 5-thiol, and 3 b) in U.S. 3,516, 997 where the sidechalns at the 7-posltion 4 had structures such as R5-(alk)m-CO-NH- and R5-S-(alk)m-CO-NH- 5 m which R3 was one of many aromatic heterocycles and the numerou 6" heterocyclic thiols at the 3-position included, for example, 7 l-methyl-tetrazole-5-thiol and 2-methyl-l,3 , 4-thiadiazole-5-thiol 8 and 9 c) in U.S. Patent 3 ,563 , 983 . 10 Various cephalosporins, including cephalosporin C on 11 occasion, have been reacted with nucleophilic, aromatic 12 mercaptans to produce compounds having the structure 13 Xg In U.S. Patent 3,278,531 Ar is phenyl or certain substituted 20 phenyls or certain aromatic heterocyclic rings named, for example 21 in column 5 . Similar nucleophiles, e.g. 2-mercaptopyrimidines, 22 are disclosed in U.S. 3, 261, 832 and Great Britain 1, 101,422 23 and U.S. 3, 479,350 and U.S. 3,502, 665 , all issued to Glaxo. 24 A parallel disclosure is found in Great Britain 1, 109, 525 to Ciba 25 e.g. in definition "h" for R^. Additional nucleophiles of this 26 type were disclosed by Fujisawa in Belgium 714 ,518 (Farmdoc 2? 35 ,307; Netherlands 68/06129 and. South Africa 2695/68) , in Canada 28 818,501 (Farmdoc 38,845), in Great Britain 1, 187 , 323 (Farmdoc 29 31, 936; Netherlands 67/14888) and especially in U.S. 3,516, 997 30 (Farmdoc 34 ,328; Netherlands 68/05179) which includes the Ί compound named cefazolin, whic has a tetrazolylacetyl sidechain on the 7-amino group and a 5-methyl-thiadiazolylthiomethyl group 3 at the 3-position and is described at some length in the il scientific literature, e.g. in Antimicrobial Agents and Chemotherap 5 1969, American Society for Microbiology, Bethesda, Maryland at 6 pages 236-243 and in J. Antibiotics (Japan) 23(3). 131-148 ( 1970) . 7 More recently, replacement of the 3~acetoxy group of a 8 cephalosporin by various heterocyclic thiols has been disclosed 9 in U.S. 3,563, 983 and in Netherlands 70/05519 (Parmdoc 80, l88R) 0 where the sidechains were, for example, 7-a-aminophenylacetamido 1 and typical heterocyclic thiols were 2-methyl-l,3, 4-thiadiazole- 2 5-thiol and l-methyl-l,2,3, 4-tetrazole-5-thiol; the latter corre- 3 sponds to U.S. patent 3,641, 021 Issued February 8, 1972 on an 1 application filed April 18, 1969. Additional similar disclosures ^ are found in U.S. patent 3* 63, 983, Belgium 771, 189 (Parmdoc 16 12, 8l7T) , Japan 72/05550 (Parmdoc 12,921T) and Japan 72/0551 17 (Farmdoc 12,922T). 18 Various cephalosporins having the structure CH2 - S - alkyl 26 In which acyl represents various sidechains including a-amino- 2γ phenylacetyl have been described in some of the above and by 28 Qlaxo in Belgium 734 ,532 (Farmdoc 4l, 6l9) and in Belgium 734,53 29 (Farmdoc 4l,620) and in U.S. 3,668,203. 30 1 Cephalosporins having the structure 2 S 0 II II where X includes - S - C - and - S - C - are disclosed in some 0 of the above and in U.S. 3,239,515; 3,239,516; 3,243,435; 11 3,258,461; 3,431,259 and 3, 46, 803. 12 Related publications in the scientific literature include 13.

J. Med. Chem. £, 174-181 (1965) and J. Chem. Soc. (London) I 1595-J.605 (1965), 5015- 031 (1965) and 1959-1963 (1967). 15 6-Mercaptotetrazolo[4,5-b]pyridazine was described by 16 B. Stanovnik et al., J. Org. Chem. 3 ( ^ . 1138-1141 (1970) as 17 their compound 8 (R=H) under the name 6-mercaptotetrazolo[ 1,5-b] 18 pyridazine . . 19 The literature contains various disclosures of methane-20 sulfonates of antibiotics. Thus Belgium 742,728 (Farmdoc 21 4l466R) discloses the mono-methanesuIfonate of ampicillin. 22 U.S. patents 3,268,508 and 3,295,246 disclose the conversion 23 of one or more of the four free amino groups of kanamycin to the 24 methanesulfonate derivative. Sodium bacitracin methanesulfonate 25 26 is disclosed in U.S. 3,205,137 and for neomycin see J. Antibiotics (Japan) 12, 114-115 (1959) . The injectable antibiotic 28 colistimethate (marketed by Varner-Chilcott as "Coly- ycin") 29 is the sodium salt of the methanesul Γonate of colistin; Merck 30 Index cites Koyama et al., Japan 48p (1957) . Colistimethate is reviewed in detail on pages 515-^20 of the U.S. Dispensatory 26th Edition, J.B. Lippincott Company, Phildelphia, Penna . It is described as the pentasodium salt of the penta (methane- sulfonic acid) derivative of Colistin A at page's 621-622 of U.S.P. XVII.· U.S. Patent 2 , 599, 950 in Example VIII discloses "Polymyxin - formaldehyde-sodium bisulfite compound". See also li. . 874 ,028, 902, 992 and 896, 77^ and Japan 15948/61.

This invention comprises the amphoteric compounds of the formula w which exist primarily as the zwitterion, and their nontoxic, pharmaceutically acceptable salts including the dimethanesulfonat sodium salt, and easily hydrolyzed esters.

The nontoxic pharmaceutically acceptable salts referred to 2 ^ above include the nontoxic carboxylic acid salts, e.g. nontoxic ^ metallic salts such as sodium, potassium, calcium and aluminum, t 5 ammonium salt and substituted ammonium salts, e.g. salts of such 6 nontoxic amines as trialkylamines including triethylamine, procai 7 dibenzylamine, N-benzyl-beta-phenethylamine, 1-ephenamine, 8 N N'-dibenzylethylenediamine, dehydroabietylamine, Ν,Ν'-bis- 9 dehydroabietylethylenediamine, N-(lower) -alkylpiperidine, e.g. 0 N-ethylpiperidine, and other amines which have been used to form salts with benzylpenicillin; and the nontoxic, acid addition salts thereof (i.e., the amine salts) including the mineral acid addition salts such as the hydrochloride, hydro-bromide, hydroiodide, sulfate, sulfamate and phosphate and the organic acid addition salts such as the maleate, acetate, citrate, oxalate, succinate, benzoate, tartrate, fumarate, malate, mandelate, ascorbate and the like.

Particularly preferred salts of the amphoteric compounds of this invention are the dimethanesulfonate sodium salts.

These salts being water-soluble are especially useful in the preparation o injectible formulations.

Also included in this invention are the compounds (used as either intermediates or metabolic precursors) in which the amino group is "blocked" by substituents such as t-butoxycarbonyl, carbobenzyloxy, formyl, o-nitrophenyl-sulfenyl, β,β,β-trichloroethoxycarbonyl, 4-oxo-2-pentenyl-2, 1-carboethoxy—l-propenyl-2-and the like. Particularly included in such blocking groups are the ketones (especially acetone) and aldehydes (especially formaldehyde and acetaldehyde) disclosed, for example, in U.S. Patents 3, 198, 804 and 3,3 7, 851 and the β-ketoesters and β-diketones disclosed, for example, in U.S. Patent 3,325 , 479 and the β-ketoamides disclosed in Japan 71/24714.

There is also provided, according to the present invention, the process for the preparation of the compounds having the formula wherein n is 1 or 2 and the nontoxic pharmaceutically acceptable, salts and easily hydrolyzed esters thereof which process comprises reacting a compound, of the formula or a salt or easily hydrolyzed ester thereof with an acylating derivative of an acid having the formula wherein B represents an amino-protecting group and n is as above to produce a compound having the formula CHgNHB (CH¾-nC-NH-CH — \ wherein B and n are as above, or a salt or easily hydrolyzed ester thereof, and subsequently removing the amino-protecting group B to produce the desired compound of formula I or a nontoxic pharmaceutically acceptable salt or easily hydrolyzed ester thereof.

The amphoteric compounds of the present invention are prepared by coupling with a particular 3-thiolated-7-amino-cephalosporanic acid designated II, that is, 7-amino-3- (tetrazolo-Γ 4, 5-b]pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid , or a salt or easily hydrolyzed ester thereof (including, but not limited to, those of U.S. patent 3, 284, 451 and U.K. 1 ,229, 453 and any of the silyl esters described in U.S. patent 3,249, 622 for use with 7-aminopenicillanic acid and used in Great Britain 1, 073 , 530 and particularly the pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl and β,β,β-trichloroethyl esters), an acid of formula III or its functional equivalent as an acylating agent for a primary amino group. After ••oupling , the amino-blocking group B is removed to give the desired product. In formula III, B represents a blocking group of the type used either in peptide syntheses or in any of the numerous syntheses of amplcillin or cephaloglycin or cephalexin frow 2-phenylglycine. Particularly valuable blocking groups are a proton, as in a compound of the formula 1 or a β-diketone or β-ketoester as n Great Britain 1,123,333 and 2 U.S. 3,325 , ^79 and U.S. 3, 316,247, e.g., methyl acetoacetate, 3 or a β-ketoamide as in Japan 71/24714 (Farmdoc 7, 32IS) in 4 which case the acid containing the blocked amino group is 5 preferably converted to a mixed anhydride, as with ethyl 6 chloroformate, before reaction with compound II or a salt 7 thereof to form the desired product I after cleavage 8 of the blocking group. 9 Further to the discussion above of blocking groups used 10 on the free amino group of the sidechain acid during its coupling 1 with compound II, the blocking group is then removed to form the 12 products of the present invention, e.g., the t-butoxy-carbonyl 13 group may be removed by treatment with formic acid or tri-li+ fluoroacetic acid, the carbobenzyloxy group may be removed by ^ catalytic hydrogenation, the 2-hydroxy-l-naphthcarbonyl group may l6 ' be removed b acid hydrolysis, the trichloroethoxycarbonyl group * may be removed by treatment with zinc dust in glacial acetic 18 acid and the l-carboethoxy-l-propenyl-2 group is preferably re- 19 moved by treatment with formic acid. Obviously other functionally 20 equivalent blocking groups for an amino group can be used and 21 such groups are considered within the scope of this invention. 22 Thus, with respect to the acid of formula III to be used to couple with compound II, functional equivalents include the corre- 24 sponding acid anhydrides, including mixed anhydrides and particular- 25 ly the mixed anhydrides prepared from stronger acids such as the 26 lower aliphatic monoesters of carbonic acid, or alkyl and aryl sul- 27 fohic acids and of more hindered acids such as diphenylacetic acid. 28 In addition, an acid azide or an active ester or thioester (e.g., 29 with p-nitrophenyl, 2 , -dinitrophenol, thiophenol, thioacetic acid) 30 may be used or the free acid itself may be coupled with compound II after first reacting said free acid with Ν,Ν1 -dimethylchloro- formiminium chloride [c . Great Britain 1,008,170 and Novak 3 and Weichet, Experientla XXI. 6, 360 (19^5)] or by the use of 4 enzymes or of an Ν,Ν' -carbonyldiimldazole or an Ν,Ν' -carbonyl- 5 ditriazole [cf . South African patent specification 63/2684] or a 6 carbodiimide reagent [especially Ν,Ν' -dicyclohexylcarbodiimide, 7 Ν,Ν'-diisopropylcarbodlimide or N-cyclohexyl-N ' - (2-morpholino- 8 ethyl)carbodiimide; cf. Sheehan and Hess, J. Amer . Chem. Soc .. 9 21, IO67 (1955)], or of alkylylamine reagent [cf. R. Buijle and 0 H. G. Viehe, Angew. Chem. International Edition 3. 582,(1964)] 1 or of an isoxasolium salt reagent [cf. R. B. Woodward, R. A. 2 Olofson and H. Mayer, Amer. Chem. Soc.. 83,, 1010 (1961)], 3 or of a ketenimine reagent [cf. C. L. Stevens and M. E. Mond, 4 J. Amer. Chem. Soc. 80. (4θβ5)]. 5 Another equivalent of the acid chloride is a corresponding azolide, i.e., an amide of the corresponding acid whose amide nitrogen is a member of an quasiaromatic five membered ring Xg containing at least two nitrogen atoms, i.e., imidazole, 20 pyrazole, the triazoles, benzimidazole, benzotriazole and 21 their substituted derivatives. As an example of the general 22 method for the preparation of an azolide, Ν,Ν1 -carbonyl- 23 diimidazole is reacted with a carboxylic acid in equimolar 2 proportions at room temperature in tetrahydrofuran, chloroform, 25 dlmethylformamide or a similar inert solvent to form the 26 carboxylic acid imidazolide in practically quantitative yield 27 with liberation of carbon dioxide and one mole of imidazole. 2^ Dicarboxylic acids yield dimidazolide . The by-product, 2 imidazole, precipitates and may be separated and the imidazolide J isolated, but this is not essential., The methods for carrying out these reactions to produce a cephalosporin and the methods used o isolate the cephalosporin so produced are well known in- the art.

Mention was made above of the use of enzymes to couple the free acid with its blocked amino group with compound II.

Included in the scope of such processes are the use of an ester, e.g. the methyl ester, of that free acid with enzymes provided by various microorganisms, e.g. those described by T. Takahashi et al., J. Amer. Chem. Soc, SilllL 4035- 037 (1972) and by T. Nara et al., J. Antibiotics (Japan) (^>) . 521-325 (1971).

After completion of the coupling and deblocking reactions described above, the zwitterion products having the formula may, if desired, be converted to the dimethanesulfonate sodium salts thereof having the formula COONa Conversion of the zwitterion cephalosporanic acid to the dimethanesulfonate derivatives may be carried out by methods known in the art. A preferred method comprises reacting the 1 zwitterion. with sodium formaldehyde bisulfite (or a source 2 thereof such as for example sodium bisulfite and formalin) 3 and water in the presence of a strong sodium base, e.g. 4 sodium 2-ethylhexanoate. The above mixture is warmed above 5 room temperature, e.g. above 40° C. and preferably in the 6 range of kO-h 0 C, for a brief period of time until there 7 is produced a solution of the desired product. The 8 product is recovered as a solid by precipitation,' e.g. by 9 the addition of a water-soluble, substantially anhydrous 0 solvent which is preferably an alcohol such as ethanol or 1 ieopropanol. Alternatively, the zwitterion products may 2 be converted by methods known per se to nontoxic, pharmaceuticall 3 acceptable salts or easily hydrolyzed esters thereof.

^ It will be obvious to those skilled in .the art that the 1C> compounds of formula I may alternatively be prepared by 1°" first acylating 7-aminocephalosporanic acid or a salt thereof with the desired 7-sidechain acid or functional equivalent ΑΟ thereof followed by displacement of the 3-acetoxy group with 9 the 6-mercaptotetrazolo- r , ^-b^yridazine to give the desired 20 product. The above alternate process is intended to be 21 included within the scope of the present invention. 22 There is also provided, according to the present invention, the process of preparing the novel compound of the formula 24 25 1 or a salt or easily hydro lyzed ester thereof; which process 2 comprises reacting 7-aminocephalosporanic acid or a salt 3 thereof with the thiol of the formula 8 9 or a salt thereof, preferably a sodium or potassium salt, 10 and, if desired, converting by methods known per se the 11 so-produced 7-amino-3- (tetrazolof" , 5-blpyridazin-6-ylthio- 12 methyl) -3-cephem-4-carboxylic acid to a salt or easily 13 hydrolyzed ester thereof. l^ The displacement of an ester group, e.g. the 3-acetoxy group of 7-aminocephalosporanic acid, with a thiol group ^ is a well-known reaction and may be" accomplished in aqueous ^ solution in the presence of a mild base such as sodium 18 bicarbonate. The displacement is preferably carried out 19 at a temperature within the range of about 50° to 100° C. 20 The 7-amino-3- (tetrazoloi"4, 5-blpyridazin-6-ylthiomethyl)- 21 3-cephem-'4-carboxylic acid intermediate is useful in preparing 22 active 7-acylamino cephalosporins such as those of formula I. 23 In the treatment of bacterial infections in man, the 24 2^ compounds of this invention are administered parenterall , 2g in accordance with conventional procedures for antibiotic 2γ administration, in an amount of from about 5 to 200 mg./kg./day 28 and preferably about 5 to 20 mg./kg./day in divided dosage, 29 e.g. three to four times a day. They are administered in 30 dosage units containing, for example, 125, 2 0 or 500 mg. \ of active ingredient with suitable, physiologically acceptable carriers, or excepients. The dosage units are in the form of liquid preparations such as solutions or suspensions.

STARTING MATERIALS Methyl o-bromomethylphenylacetate A mixture of methyl o-methylphenylacetate (82.0 g., 0.50 mole), N-bromosuccinimlde (89.0 g., 0.50 mole), benzoyl peroxide (1.0 g.) and carbon tetrachloride (800 ml.) was heated under reflux for 2 h. while Irradiated with a 750 watt light source. The succinimide was removed by filtration, the solvent removed from the filtrate and the residue distilled in vacuo to give 90.1 g. ( $) of product, b.p. 95-105° (0.4 mm) j n.m.r. (CCl^): singlets at ^ 2.85 (4H), 5.5Ο (2H), 6.31 (2H) and 6.38 (3H) . o-Azldomethylphenylacetic acid A mixture of methyl .o-bromomethylphenylacetate (90.1 g., O.37I mole), sodium azide (26.0 g., 0.40 mole) and 10$ aqueous acetone (750 ml.) was stirred at room temperature for 3 h. The solvent was removed under reduced pressure and the residue treated with ether (300 ml.) and water (100 ml.). The crude methyl .0- \ and concentrating the ether solution, was dissolved in 150 ml. of methanol. This solution was cooled in ice and treated with 150 ml. 3 ϋ methanolic sodium hydroxide. The mixture was left at room temperature for 1 h., then concentrated to dryness and the residue dissolved in water. The aqueous solution was acidified, the product collected by filtration, dried and recrystallized from ethyl acetate-n-hexane to give 9.5 g. (70$) of the acid, m.p. 116-118°; n.m.r. (CDCl^): sharp singlets at T2.75 (4H), 5 .62 (2H) and 6.28 (2H); » SΓΠ3jΧi01 2100 and 1700 cm"1.

Anal. Calcd. for C, 56. 3 H, 4.75; N, 21.98.

Found: C, 56.37; H, 4.65; N, 21.74 o-Amlnomethylphenylacetlc acid A mixture of ο,-azidomethylphenylacetic acid (9.6 g., O.O50 mole), 10$ Pd on charcoal (2.5 g.), methanol ( 150 ml.) and I N hydrochloric acid (50 ml.) was hydrogenated at 30 p.s.i. for 3.5 h. The mixture was filtered, concentrated under reduced pressure to a volume of approximately 30 ml. and extracted with ether. Prom the ether extract 1-2 g. of impure starting material was recovered. The aqueous solution was adjusted to pH 5 .0 with dilute ammonium hydroxide and cooled in ice. The white solid precipitate was collected by filtration, washed successively with ice-water, methanol and ether, and dried in vacuo over Ρ2°ρ: Yield 5 .4 g. ( 5$) , m.p. 179-181° (decomp.); n.m.r. (CP^CO^): -Γ 2.54 (s, 4H) 5.48 (q, 2H) and 6.00 (s, 2H) . o-tert-Butoxycarbonylamlnomethylphenylacetic acid Triethylamine (14.4 g., 0.143 mole) was added to an ice-cooled suspension of ο,-aminomethylphenylacetic acid ( 10.3 g., Ο.Ο624 mole) in 100 ml. of water followed by the addition of a solution of tert-butoxycarbonyl azide ( 11.4 g., 0.080 mole) in for l6 h., then most of the THF was removed under reduced pressure. The aqueous solution was washed with ether, layered with 125 ml. of ethyl acetate and with ice-cooling brought to pH 3.5 with dilute hydrochloric acid. The ethyl acetate solution was dried, concentrated and the solid residue recrystallized from ethyl acetate-jn-hexane (1:1) to give 14.4 g. (67$) of white needles m.p. 114-116°.

Anal. Calcd. for Cl2|HigN04: C, 63.39; H, 7.22; N, 5.28. Found: C, 63.44; H, 7.21; N, 5.42. 2.4-Dinltrophenyl o-tert . -butoxycarbonyla inophenylacetate NiN'-Dicyclohexylcarbodiimide (1.0 g., 0.0050 mole) v;as added to an ice-cooled solution of o-tert-butoxvcarbonvlamlnomethvl- phenylacetic acid (1.33 g., 0.0050 mole) and 2,4-dinitrophenol (Ο.92 g., O.OO5O mole) in 12 ml. of anhydrous tetrahydrofuran.

The reaction mixture was kept at room temperature for one hour, then the precipitated Ν,Ν'-dicyclohexylurea was removed by filtration. The solvent was removed from the filtrate to give the activated ester as a viscous yellow oil. ■ o-tert .-Butoxycarbonylaminomethylphenylacetlc acid can be prepared in quantitative yield from ^ert^-butoxycarbonyl azide and the amino acid by using triethyla ine as the base.

The BOC-amino acid reacts with thlonyl chloride in the presence of triethylamine (methylene chloride as solvent) or pyridine (benzene as solvent) to give the BOC-amino acyl chloride which can bo directly coupled with the compound of the formula wherein R has the meaning set out above in methylene chloride solution in the presence of triethylamine . The protecting group can subsequently be removed by treatment with cold trifluoro-acetic acid .

Exactly 200 g. of 7-aminocephalosporanic acid (7-ACA) was suspended in 500 ml. of acetone and a solution of 240 g. of £-toluenesulfonic acid in 500 ml. of acetone was added in one charge. After stirring for five minutes, at room temperature, the mixture was filtered through diatomaceous earth ("Super Cel") and the bed washed with 150 ml. of acetone (the insoluble matter weighed about 50 g.). Then 80 ml. of water was added to the filtrate and, while stirring, the p-toluene-sulfonate salt crystallized out after scratching on the inside of the flask with a glass rod. The suspension was stirred in an ice-salt bath for thirty minutes and filtered cold. It was washed with 2 x 200 ml. of cold acetone (0° C.j and air dried; yield 250 g. of salt. This x>-toluene sulfonate salt of 7-ACA was stirred in 2 liters of methanol and the insoluble matter filtered through "Super Cel". The filtrate was placed in a five liter 3 neck flask and 2 liters of water were added. Then the pH was adjusted to k by the addition of concentrated ammonium hydroxide with cooling and the/suspension stirred for one hour at 0° C. The product was collected by filtration and washed with 2.x 100 ml. HgO (0° C.) and 3 x 1 liter acetone (room temperature) . After air drying, the yield of 7-ACA was 1^5 g. o-Aminomethylphenylacetic acid is also provided by Beckmann rearrangement of 2-indanone oxime followed by hydrolysis of the resulting lactam according to the following I. II. III.

J. Org. Chem. 9_, 380-391 ( 19 ) and 28, 2797-2804 ( 1963) .

Materials Vol.. ml. Moles 2-Indanone oxime 6.78 Phosphorus pentachloride 7. 13 Chloroform 5660O 10$ Sodium Hydroxide Solution 680 "Darko KB" activated charcoal Procedure 1. Dissolve 100 g. of 2-indanone oxime in 3l600 ml. of chloroform at 20-25° C. 2. Cool the solution to -300 C. [On cooling the 2-lndanone oxime solution to -30° C, some of the oxime crystallizes ~ 3. Add 1482 g. of phosphorus pentachloride to the vigorously stirred suspension in portions. Control the temperature of the reaction at -28° to -32° C. by the rate of addition of the solid phosphorus pentachloride. [The best results were obtained on running the reaction at -30° C. It can also be successfully run at -10 to -5° C. or perhaps even higher but it appears that more tar and side products are produced which then complicate isolating the lactam.] 4. Stir the reaction at^ -30° C. for 10 minutes after completing the addition and then warm it to 25° C. over 3/4 hr. During this period the solids dissolve and then a new solid reprecip- 5. Stir the reaction at 25° C. for 3 additional hours and then with thorough mixing add it to 3l600 ml. of water at O-50 C. [The reaction can be followed by TLC (thin layer chromatography). In the system of 8 parts benzene and 2 parts acetic acid the lactam has an R = .36 and the oxime has as Rf » .64. The spots are developed by .05$ potassium permanganate spray. The oxime spot may not completely disappear but it should become quite faint.] Subsequent washing operations were conducted at 20-25° C. 6. Separate the layers and wash the chloroform phase with I58OO ml. of water. 7. Combine the water fractions and extract them with 158OO ml. of chloroform. 8. Combine the chloroform fractions, layer with 158OO ml. of water and with good mixing titrate the mixture to/v pH 7 with 10$ sodium hydroxide solution. This may take about 68Ο ml. of sodium hydroxide solution and the titration is slow. [This basic wash is important in removing the tar producing side products. The titration may take 1 to 2 hours.] 9. Separate the layers and wash the chloroform with I58OO ml. of water. 0. Combine the water fractions, wash with 9200 ml. of chloroform and combine the chloroform fractions. 1. Carbon treat the chloroform solution with 1000 g. of activated charcoal ("Darko KB") atn>25° for 15-30 min. 2. Filter the slurry through diatomaceous earth ("Dicalite") wash the cake with chloroform and concentrate the filtrate at reduced pressure to leave o-aminomethylphenylacetic acid lactam as a dry solid. 5. The yield of crude lactam is nearly 100$. It is a yellow crystalline solid, [if the tar making materials have not been removed by the washes, this product will come out dark. It can be recrystallized from hot water after first adjusting the water slurry to pH 7.0 or from toluene-heptane J Materials Vol.. ml. Moles Crude o-aminomethylphenyl- ^1000 ^ 6 .78 acetic acid lactam from 1000 g. of 2-indanone oxime Cone. Hydrochloric Acid 80.00 "Dario KB" activated charcoal 100 Methylene Chloride 6000 Methyl isobutyl ketone (MIBK) As needed 6 N Ammonium Hydroxide As needed Procedure 1. Add 8OOO ml. of concentrated hydrochloric acid to the approximately 1000 g. of crude lactam obtained from oxime rearrangement . 2. Stir the mixture and heat it cautiously to reflux for 3 hours. [On heating this reaction an excessive amount of foam is formed as excess HC1 leaves. This foam can fill the whole apparatus. It can be reduced by silicon antifoam agent. After the initial foam stage is passed, the reaction can be refluxed without difficulty.] 3 . Cool the dark slurry to about 40-50° C. and add 100 g. of activated charcoal ("Darko KB") and continue stirring. 4. Carbon treat for 15-20 min., filter the slurry through a "Dicalite" cake and wash the cake with about 4000 ml. of hot water. 5 . Extract the clear yellow filtrate with 6000 ml. of methylene chloride ( i/2 volume) and separate the methylene chloride. Save the CHgClg layer for checking possible recovery of unchanged lactam. 6. Concentrate the water phase at reduced pressure to give solid o-aminomethylphenylacetic acid hydrochloride. 7. Add MIBK to the wet solids and continue the reduced pressure distillation and MIBK addition until all the water has been removed from the solids. 8. Continue the reduced pressure distillation until all the ΜΓΒΚ has been removed from the solids. [The MIBK distillation not only azeotropes the water but also takes excess HCl with it.] 9. Redissolve the solids in 5900 ml. of water and add 650 ml. of MIBK. 0. While stirring at 20-25° C. adjust the pH of the solution to 5.O with 6 ammonium hydroxide. The o-aminomethylphenylacetic acid zwitterion starts to crystallize at about pH 3.5 . 1. Stir the zwitterion slurry and cool it to 0-5° C. for 1 hr. 2. Filter the slurry, wash the cake carefully with -1000 ml. of ice cold water, then 2000 ml. of MIBK and then 5000 ml. of ice cold acetone. The combined filtrate and washes should be checked for lactam content. 3. Suck the cake dry and then dry it in an air circulating oven at ° C. The yield is 6 0- 30 g.j 60-6 $ based on oxime. 4. (1 The reactions and processing may be followed by TLC using the solvent system acetone, 1. benzene, 1.0 acetic acid, 1.5 water; developed by KMnO^; Rf lactam r 0.88, R^. amino acid z Ο.69. \ 1 o-Aminomethylphenylacetic acid is also provided by 2 Schmidt rearrangement of 2-indanone followed by hydrolysis 3 of the resulting lactam according to the following equation; 4 8 I. II. III. 9 Organic 10 Syntheses 4l, 53 (196l). 11 2-Indanone (I) was prepared from indene according to a 12 procedure described in Organic Syntheses (ibid). When a 15 technical grade reagent of indene is used, the yield of 2-].4 indanone will decrease to around 4 $ (see note 2 of Organic 15 Syntheses) . 2-Indanone thus prepared was used as soon as 16 possible because of a description in note 7 of Organic Syntheses 17 that 2-indanone is unstable to air at room temperature. 18 Hydrolysis of the ^-lactam (II) was carried out success¬ 19 fully using hydrochloric acid to give the amino acid (III) in 0 7 yield. Barium hydroxide was also used for the hydrolysis 1 but the yield was 32^. o-Amlnomethylphenylacetic acid ^-lactam (ilV 4 To a cooled suspension of 12.3 g. (0.093 mole) of freshly 5 prepared 2-indanone (I) and 13.0 g. (0.2 mole) of sodium azide 6 in 500 ml. of chloroform was carefully added dropwise 50 ml. 7 of concentrated sulfuric acid at such a rate as to maintain 8 the temperature below 4o° C. under stirring. After the addition 9 was completed and the heat evolution ceased, the mixture was then poured into OO g. of crushed ice. The chloroform layer was separated and the aqueous layer was extracted with three 300 ml. portions of chloroform. The chloroform layer was combined with the chloroform extracts, dried with anhydrous sodium sulfate and concentrated under reduced pressure to afford crystalline precipitate, which was collected by filtration and recrystallized from 250 ml. of n-hexane-benzene ( 1 : 1) to give 12.0 g. (88$) of the desired product (II). Colorless prisms, m.p. 152-153° C.

IR (KBr): .

NMR (CDCl^): J (ppm from TMS) 3 .50 (2H, t, 1, 5 Hz), 4.40 (2H, br-s, converted to a triplet (J = 1.5 Hz) by an addition of I>20) , 7.06 ( 1÷H, s), 7 . ^5 (1H, br-s, disappeared by an addition of DgO) .

Anal. Calcd. for CgHgN0: C, 73 . ^5 ; H, 6 . 16; N, 9.52 .

Pound: C. 73 .73J H, 6 .08; N, 9.23 . o-Aminomethylphenylacetic acid rf- lactam (II) Ten milliliters of sulfuric acid was added to a stirred suspension of 1.32 g. (0 .01 mole) of freshly prepared 2- indanone (I) in 50 ml. of chloroform and to the mixture was added portionwise 1.30 g. (0.02 mole) of sodium azide at room temperature under stirring. After stirring for half an hour, the reaction mixture was poured into 200 ml. of ice-water and extracted with two 100 ml. portions of chloroform. A large amount of insoluble material which appeared during the extraction procedure was filtered off. The chloroform extracts were combined, dried on anhydrous sodium sulfate, treated with active carbon and filtered. Evaporation of the filtrate followed by recrystallization « - - ~ 1 o-Aminomethylphenylacetlc acid fill) 2 A mixture of 7.37 g. (0.05 mole) o II and 50 ml. of 5 concentrated hydrochloric acid was refluxed for 3 hours and the reaction mixture was treated with 1.0 g. of active carbon 5 and filtered. The filtrate was concentrated to dryness under 6 reduced pressure to give 8.5 g. as the crystalline residue. 7 Recrystallization from 400 ml. of aqueous acetone (acetone: water 8 = 10:1) gave 8.0 g. (79#) of hydrochloride of the desired amino 9 acid (III). Colorless plates, m.p. 164-166° C. 11 IR (KBr): yTmnaaxv(cm"1) 1695, l6l0, 1585, 1230, ll80. 12 NMR (DMSO-dg): ti(ppm from TMS) 3.69 (2H, s), 3.94 1 "3 (2H, s), 4.10-4.7 (3H, broad), 7.0-7.5 (4H, m), 7.6 - 8.7 14 C, 53. 0; H, 6.00; 6.79; CI, 17.94. 19 o-Aminomethylphenylacetlc acid (III) 20 A mixture of 40 mg. (2.3 m.moles) of II and 730 mg. 21 (2.3 m. moles) of barium hydroxide octahydrate in 20 ml. of water 22 was heated in a sealed tube at 150° C. for one hour. The 2¾ reaction mixture was cooled to room temperature and 300 mg. 2^ (2.5 m. moles) of ammonium carbonate was added to precipitate „c barium carbonate which was filtered and washed well with 50 ml. of water. The aqueous filtrate was combined with the washings and evaoorated to dryness. The residue was crystallized from ,Q 5 ml. of 50$ aqueous alcohol to give 163 mg. (30 ) of colorless plates which was identical with the free amino acid obtained (KBr): ymax (cm"1) 3100-2200, I660-I5OO, l400, I38O, 770, 755, 720.

MR (CF^COOH): (^(ppm from T S) 3. 6 (2H, br-s), 4.4 (2H, br-s), 6.3-8.0 (3H, br), 7.38 (4H, s) .

Anal. Calcd. for C^^O^. C, 65.44; H, 6.7I; N, 8.48.

Pound: C, 65.24; H, 6.4γ; N, 8.31. o-Amlnomethylphenylacetic Acid -lactam (II) .

In a 2 liter three-necked round-bottom flask, fitted with a reflux condenser, drying tubing, overhead stirrer, thermometer, and a 100 ml. dropping funnel, are placed 24.6 g. (O.186 mole) of 2-indanone and 26 g. (0.4 mole) of sodium azide in 1 liter of chloroform. The suspension is stirred and to it added dropwise 100 ml. of concentrated sulfuric acid at such a rate to maintain the temperature between 33~37° C. (Note l) . After the addition is complete the reaction mixture is stirred at room temperature for an additional 2 hours, and then poured into 800 g. of crushed ice. The chloroform layer is separated and the aqueous phase is extracted 3 times with 300 ml. portions of chloroform. The aqueous phase is treated with sodium nitrite (Note 2) .

The chloroform extracts are combined, dried over magnesium sulfate (anhydrous) and evaporated to dryness. The solid is redissolved in 150 ml. of hot water and treated with 2.0 g. of charcoal ("Darko KB") while hot. The solution is filtered and the charcoal is washed 3 times with 25 ml. portion of hot water.

The solution is cooled, and the solid is collected by filtration. It is washed with 10-20 ml. of ice-cold water, and dried in a vacuum desiccator over Ρ2°5· ^e PRODUCT melts at 144-145° C; 18.3 g. (yield 69$).

Notes l. Caution; During the sulfuric acid addition, hydrazoic acid is evolved. Hydrazoic acid boils at 38-39° C., therefore, the temperature must be maintained below 40° C. The entire experiment should be carried out in a well ventilated hood. 2 . Residual hydrazoic acid in the aqueous phase is decomposed by adding slowly sodium nitrite until a positive test is obtained with iodine paper and/or ferric chloride solution. o-Aminomethylphenylacetlc acid hydrochloride (III) In a 500 ml. round-bottom flask, fitted with a reflux condenser, and a magnetic stirrer, is placed 10. 1 g. ( 0.075 moles) of II and 100 ml. of concentrated hydrochloric acid.

The mixture is then refluxed for 3 hours. While hot, the reaction mixture is treated with 2 .0 g. of charcoal ("Darko KB") for 5 minutes, and filtered. The filtrate is concentrated to dryness at 50-6-° /15mm and finally at high vacuum over ρ2° (Note l) . The solid is recrystallized from a preformed mixture of acetone: water, 15 : 1 (Note 2) . The hydrochloride is dried in a vacuum desiccator over P2°5 « The yield of the pure product, melting at 188- 190° , is 11.4 g. (78$) .

Anal. Calcd. for CgH12N0gCl: C, 53 .73; H, 5 . 97; N, 6 .96 ; CI, 17 .66 .

Found: C, 53 .56; H, 6 .02 ; N, 6 .89; CI, 17 .76 .

Notes - 1. For a successful recrystallization absolutely dry material is needed. 2 . The ratio of acetone to water may vary, depending on the Preparation of o-amlnomethylphenylacetlc acid from indene I 4 2-Indanone (2) To a stirred mixture of 370 ml. of 80 formic acid, and 70 ml. of 30$ hydrogen peroxide was added dropwise 58 g. (0.5 mole) of Indene (!) at 35~^0° C. over a period of one hour. An additional 40 ml. of formic acid was used to rinse the last of the indene from the dropping funnel into reaction flask. The reaction mixture was stirred at room temperature for 15 hours. The mixture was concentrated to remove the formic acid under reduced pressure (20-30· nim) below 40° C. The residue was dissolved in 1000 ml. of 10$ sulfuric acid and that solution was refluxed for 20 minutes and then steam-distilled. The distillation was carried out at the rate of about 2 L per hour until 7..L of distillate had been collected. The distillate was extracted with six 400-ml. poritions of chloroform. The combined extracts were dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated to dryness to give 1 o-Amlnomethylphenylacetic acid -lactam (3_) 2 To a chilled suspension of 46 g. (0.35 mole) of freshly prepared 3 2-indanone (2) and 27 g. (0.42 mole) of sodium azide in 75 ml. of 4 chloroform was added dropwise 75 ml. of concentrated sulfuric 5 acid at such a rate as to maintain the temperature at 30-40° c . 6 under stirring. After the addition was complete and the evolution 7 of heat ceased, the stirring was continued for an additional 3 8 hours. The reaction mixture was poured into about one Kg. of 9 crushed ice. The chloroform layer was separated and the aqueous 0 layer was extracted with three 200-ml. portions of chloroform. 11 The chloroform layer was combined with the chloroform extracts, 12 dried over anhydrous sodium sulfate and evaporated to dryness 3 to give 44.5 g. (87$) of_3_as pale yellow prisms melting at 1H 146-149° C. 5 o-Amlnomethylphenylacetic acid hydrochloride (4.) 16 A mixture of 43.4 g. (0..30 mole) of o-aminomethylphenylacetic ΐγ acid -lactam (3) in l40 ml. of 6 N hydrochloric acid was refluxed 8 for 4 hours. The reaction mixture was treated with one g. of 1 charcoal and evaporated to dryness. The residual oil was 20 triturated with 500 ml. of acetone to give g. (75 ) of _4_as 21 colorless plates melting at l63-l64° C. 22 23 24 25 26 27 28 29 Preparation of o-aminomethylphenylacetlc acid from o-nitrotoluene 3) KCu(CN)_ 6 o-Nltrophenylacetlc acid (6) A mixture of 135 ml. (ca. 1 mole) of ethyl oxalate and 118 ml. (ca. 1 mole) of o-nitrotoluene (^) was added dropwise' under cooling to a stirred solution of 23 g. ( 1 g. atom) of sodium in 300 ml. of absolute ethanol and the mixture was stirred for 1 .5 hours at room temperature and then refluxed for 1.5 hours. The mixture was cooled to 6o° and about 500 ml. of water was added. The mixture was steam-distilled to remove the unreacted nitrotoluene . The residue was treated with 5 g. of active carbon and filtered. The clear filtrate was adjusted to pH 8-9 with dil. sodium hydroxide. A solution of 6$ hydrogen peroxide was added at 30-40° C. until a small sample of the reaction mixture when made alkaline with sodium hydroxide no longer gave the dark color characteristic of alkaline pyruvic acids. The reaction mixture was treated with r acidified with cone, hydrochloric acid and extracted with four - 25Ο ml. portions of ethyl acetate. The combined extracts were washed with water (3 x 100 ml.) and a saturated sodium chloride solution and dried over anhydrous sodium sulfate. The solvent was removed and the residue was crystallized from ethanol-water (200 ml. - 600 ml.) to give 81 .2 g. ( 45#) of o-nitrophenylacetic acid (6) melting at l4o-l4l° C. ^1710 cm"1. vNQ 1525 , 1 βθ cm"1. 2 o-Cyanophenylacetic acid (7_) A solution of 43 .4 g. (0.24 mole) of o-nitrophenylacetic acid (6) and 9.6 g. (0.24 mole) of sodium hydroxide in 200 ml. of water was hydrogenated at room temperature under 50 psi of · hydrogen pressure with one g. of 10 palladium-charcoal. The theoretical amount of hydrogen was absorbed during 4 hours. The catalyst was removed by filtration. To the filtrate was added a solution of 16.6 g. (0.24 mole) of sodium nitr te in 50 ml. of water under cooling at 0° C. The mixture was added dropwise to 80 ml. of cone, hydrochloric acid at 0-3° C. under stirring.

After the addition was completed, a chilled solution of 16.6 g. (0.12 mole) of potassium carbonate in 100 ml. of water was added to the reaction mixture with stirring. The diazonium salt solution was added with vigorous stirring at' 0-5° C. to a solution of potassium cuprocyanide which was prepared from 40 g. (0 .62 mole) of potassium cyanide and 28 g. (0. J1 mole) of cuprous cyanide in 130 ml. of water. The mixture was stirred for 30 minutes at 0° C. for one hour at 15-20° C.?for one hour at 50° C. and finally stood at room temperature overnight . The reaction mixture was diluted with one L of water and filtered. The filtrate was acidified with cone, h drochloric acid and extracted with V. back-extracted with four 200 ml. portions of sodium bicarbonate solution. The combined alkaline extracts were treated with a small amount of charcoal, acidified with cone, hydrochloric acid and extracted with three 300 ml. portions of ethyl acetate. The combined extracts were washed with 200 ml. of water and 200 ml. of a saturated sodium chloride solution, dried with anhydrous sodium sulfate and concentrated to give 36 g. of the crude product 7_. Recrystallization from benzene gave 29.6 g. {71 ) of tan needles melting at 121-122° C. ^c=N2220cm" 1.

^CFO 1690 ^ After the addition was completed the heating was continued for g 4 hours and then allowed to stand overnight in a refrigerator γ to give 285 g. ( 8 $) of 9. as massive pillars, m.p. > 290° C. 8 .6-Dichloropyrldazine (lO) g A mixture of 150 g. ( 1 .33 moles) of and 250 g. of phosphorus oxychloride was refluxed for 3 hours under ^ protection from moisture. The excess of phosphorus oxychloride ^2 was removed under reduced pressure and the dark residue was yn poured into one Kg. of crushed ice. The resulting precipitate ^ was collected by filtration. The second crop of the product ^ was obtained from the mother liquor by the extraction with five 300 ml. portions of chloroform followed by treating with 1 g. of charcoal and evaporating the solvent. The first and second crops were combined, dissolved in 500 ml. of chloroform ^ and treated again with one g. of charcoal and concentrated to 20 give 165 g. ( 83 ) of 10 as fine needles melting at 60-6l° C. 21 (in a sealed tube). 22 3-Chloro-6-hydrazlnopyridazlne (ll) , A mixture of 40 g. (0 .27 mole) of 3 * 6-dichloropyridazine ( 10 2ij and 40 ml. of 80# hydrazine hydrate in 80 ml., of ethanol was -c refluxed for one hour. The reaction mixture was evaporated to 2g dryness and the residue was recrystallized from benzene to give 27 39 g. ( 100#) of 11 melting at 114-115° C. 28 29 6-Chlorotetrazolo[4.5-b.pyrldazine (12) To a solution of 25.7 g. (0.174 mole) of 11 In 100 ml. of Vj>% acetic acid, was added dropwise a solution of 13.8 g. (0.2 mole)-of sodium nitrite in 50 ml. of water with vigorous stirring at 5-10* C. Stirring was continued for one hour at the same temperature. The precipitate which separated was filtered, washed with 20 ml. of water and air-dried to give 17.02 g. of 12. Additional product was obtained by evaporation of the filtrate. Total yield 18.22 g. (6k%) . M.p. 104-105° C 6-Mercaptotetrazolo [ . -b rldazlne (13) A mixture of 21.3 g. (0.137 mole) of 12 and 20 g. (0.25 mole) of potassium hydrosulfide in 200 ml. of ethanol was refluxed for 2 hours and evaporated to dryness. The residue was dissolved in 100 ml. of water and filtered to remove a small amount of insoluble material. The filtrate was acidified to pH 1 with dil. hydrochloric acid to precipitate 4, as colorless needles which were collected by filtration, washed with 20 ml. of water and dried. Yield 9.80 g. (47$). M.p. l4o-l4l° C. (dec).

IR: ^m?x 25∞' 15k0> lkk3> 1295' 840 ^'1· MR: 4¾°+K2C05 7.44 (l H, d, 10 Hz, pyridazine-H), 7.77 (1 H, d, 10 Hz, pyridazine-H).

Anal. Calcd. for C^H^S: C 31.37; H, 1.97; N, 45.72; S, 20.94.

Found: C, 31.52; 31.66; H, 1.70, I.69; N, 46.01, 46.01; s, to.95.

Preparation of β- (c-amlnomethylphenyl propionic acid - Method A β- (o-Aminomethylphenyl) propionic acid lactam (2 ) Preparation of β- (o-aminomethylphenyl) ropionic acid lactam (2 ) was carried out by a procedure which was a modification of ,ν' the method of Knunyants and Fabrichnyi. I. L. Knunyants and B. P. Fabrichnyi, Doklady Acad. Nauk, SSSR, 68 , 523 ( 19 9) ; C.A. 1^9 d ( 1950) .

To a stirred mixture of 30 g. (0 .2 mole) of β-tetralone and 14 g. (0.4 mole) of sodium azide in 400 ml. of chloroform was added dropwise 50 ml. of cone sulfuric acid at 30-40° C. When the addition was completed, the mixture was stirred for 2 hours at 30° C. The reaction mixture was poured into 400 ml. of ice water and extracted with three 200 ml. portions of chloroform. The extracts were washed with 50 ml. of water, dried with anhydrous sodium sulfate and treated with a small amount of active carbon. The filtration was evaporated under reduced pressure to give 31 g. of the residual solid which was a mixture of β- (o-aminomethylphenyl) propionic acid lactam 2, and o- (2-aminoethyl)phenylacetic acid lactam 3 · Isolation of the desired lactam 2 was carried out by the procedure described below.

Crude mixture 10 g. recrys. f. CHCl,-n-hexane ψ I evap. pure 3 2.47 g. m.p. 159-60° C. e cry \| ml. fraction Fr. 4-6 ψevap. recrys. f. CHCl,- ligroin pure 2 1.77 g. m.p. 118-122° C. β~ (o-Aminomethy1phenyl) propionic acid hydrochloride A solution of 1.77 g. (0.011 mole) of β- (o-aminomethyl-phenyl) propionic acid lactam (2) in 10 ml. of concentrated hydrochloric acid was refluxed for 4 hours and then evaporated to dryness. The residue was dissolved in 20 ml. of water, treated with a small amount of charcoal and filtered. The filtrate was concentrated to about 5 ml. An addition of 40 ml. of acetone to the concentrate gave β- (o-aminomethylphenyl) -propionic acid hydrochloride as colorless needles which were collected by filtration, washed with acetone (10 ml.) and dried. Yield 1.21 g. (51#) . M.p. 19,-1 8° C.

IR: Jj£ 33OO-260O, 1718, I6OO, 1540, 1440 cm"1.

NMR: Xppm°"d 2.4-4.0 (4 H, m, CH^-Ct^-CO) , 4.00 (2H, s, CHg-N), 7.0-7.40 (4H, m, phenyl-H) , 8.50 (3H, br-s, H^*) . Anal, calcd. for C10H"15N02*HC1: C, 55.69; H, 6.54; N, 6.49; CI, 16.44.

. Found: C, 55-31; H, 6.5Ο; N, 6.51; CI, 16.61.

Preparation of o-amlnomethylphenylpropionic acid ■ Method B o-Cvanophenylpropionlc acid A mixture of 8.23 g. (Ο.Ό45 mole) of o-nitrophenylcinnamic acid and 2.80 g. (0.043 mole) of sodium hydroxide in 50 ml. of water was hydrogenated with 2.0 g. of 10$ palladium on charcoal at 50 psi hydrogen pressure in a Parr apparatus until the theoretical volume of hydrogen was absorbed (about 3 hours) . The catalyst was removed by filtration. Sodium nitrite (2.97 g., 0.043 mole) was added to the cold filtrate and the mixture was added dropwise to 16.6 g. (ca. 0.17 mole) of concentrated hydrochloric acid at O-50 C. with vigorous stirring. To the of potassium carbonate in 10 ml. of water with stirring. This cold mixture was added to a mixture of 9. 10 g. (0.14 mole) of potassium cyanide and 6.3 g. (0.07 mole) of cuprous cyanide in 33 ml. of water with vigorous stirrin 'at 0-5° C. The mixture was stirred for 1 hour at 0-5° C, for 1 hour at room temperature and finally 1 hour at 50° C, and then filtered to remove insoluble material, the filtrate being washed with ethyl acetate ( 100 ml.), acidified with dil. hydrochloric acid to pH 3 and extracted with ethyl acetate ( 4 x 100 ml.). The combined extracts were washed with water ( 100 ml.), treated with a small amount of charcoal and dried on anhydrous sodium sulfate. After removing the solvent the residue was recrystallized from water ( 100 ml.) to give 4.31 g. (57 ) of o -cyanophenylpropionic acid as light brown prisms. M.p. 126-127° C.; lit. 136° C; see F. Mayer et al., Ber., 61 , 1966 ( 1928) .

IRr NMR: CH2-CH2), 7.0-7.7 (4H, m, benzene-H), 10 . 15 (1H, s, COOH) . o-Amlnomethylphenylproplonic acid hydrochloride A mixture of 2 .30 g. (0 .013 mole) of o-cyanophenylpropionic acid, 30 ml. of ethanol and 15 ml. of 6 hydrochloric acid was hydrogenated with 1.0 g. of 10$ palladium on charcoal at 50 psi hydrogen pressure. The theoretical amount of hydrogen was absorbed in 3 hours. The catalyst was filtered off and the filtrate was concentrated to give 2 .2 g. (77$) of o-aminomethyl-phenylproplonlc acid hydrochloride as colorless needles melting at 190- 1920 C. The product was identified with the authentic sample prepared by Schmidt rearrangement of β-tetralone followed by hydrolysis; see I. L. Knunyants and B. P. Fabrichnyi, B- [o- (t-Butoxycarbonylamlnomethyl) phenyl] propionic acid To an ice-cooled solution of 1 .08 g. ( 5 mmoles) of β-(o-aminomethylphenyl) propionic acid hydrochloride, 1.71 g. (17 mmoles) of triethylamine in 8 ml . of water was added dropwise a solution of 1.07 g. (17 mmoles) of t-butoxy-carbonylazide in 6 ml. of tetrahydrofuran with stirring.

The stirring was continued for 19 hours and the temperature of the mixture was allowed to rise to room temperature. The reaction mixture was concentrated to remove tetrahydorfuran, washed with 50 ml. of ether, acidified to pH 3 with dilute hydrochloric acid and extracted with ethyl acetate (3 x 100 ml.). The combined extracts were washed with water (50 ml.) and dried on anhydrous sodium sulfate and filtered. Concentration of the filtrate afforded β- [o-(t-butoxycarbonylaminomethyl) -phenyl] propionic acid as colorless crystals which were collected by filtration. Recrystallization from ethyl acetate-n-hexane (1:1, 200 ml.) gave fine needles which melted at 117-117.5° C. Yield 1.01 g. (73$) .

NMR: \ CDC 13 1.40 ( 9 H, s, t-Butyl-H) , 2.4-3.1 (4H, m, Q ppm ¾-0Η2-0θ) , 4.25 (2 H, d, 6 Hz, GHg-N) , 7.10 (4 H, s, phenyl-H) , 8.^0 (2 H, br-s, NH and COOH) .

Anal. Calcd. for C^H^NO^: C, 64.50; H, 7 .58; N, 5 .01 .

Found: C, 64.64; H, 7 .56 ; N, 5 .08. 2. -Dlnitrophenyl[g-o- (t-butoxycarbonylaminomethyl)phenyl.- proplonate To a cooled (5° C.) solution of 0.98 g. (3.5 mmoles) of p-[o-(tert-butoxyca'rbonylaminomethyl) henyl] ropionic acid and O.65 g. (3.5 mmoles) of 2,4-dinitrophenol in 15 ml. of dry ethyl acetate was added 0.72 g. (3.5 mmoles) of Ν,Ν' -dicyclo- hexylcarbodiimide with stirring for one hour at room temperature, The mixture was filtered to remove Ν,Ν'-dicyclohexylurea which was washed with ethyl acetate (10 ml.). The filtrate was combined with the washing and evaporated to dryness to give yellow oily active ester 2,4rdinitrophenylI^-o-(t-butoxycarbonyl- aminomethyl)phenyl]propionate ( YQ^Q' 1770, 1710 cm"1). This active ester was used for the preparation of cephalosporanic acid derivatives without further purification.

The following examples are given in illustration of , but not in limitation of , the present invention . All temperatures are in degrees Centigrade . 7-Aminocepha losporanic acid is abbreviated as 7-ACA ; -ACA- represents the moiety having the structure - and thus 7-ACA can be represented as H-ACA-O-C-CH.

Description of the Preferred Embodiments Example 1 £0 \ Preparation of 7-amlno-3-(tetrazolo[4. -b]pyridazln-6-ylthio- methyl) -3-cephem-4-carboxylic acid 7-ACA 9 0 11 17 18 19 7-Amlno-3-( tetrazolo[ .5-b]pyridazln-6-ylthiomethyl) -3-cepheiTi-20 4-carboxyllc acid (14) 1 (i) To a hot solution (50-6o° C.) of 9.56 g. (O.062 mole) 22 of 1_ and 10.42 g. (0.124 mole) of sodium bicarbonate in 300 ml 23 of water was added carefully 16.86 g. (O.062 mole) of 7-ACA and 24 the mixture was heated at 8Ο-850 C. for 30 minutes. About 7 g. 25 of sodium bicarbonate was added to the reaction mixture to 26 dissolve insoluble material. The solution was treated with 27 active carbon, filtered and the filtrate was acidified to pH 5 28 with dil. hydrochloric acid. The precipitate was collected by - (ii) A stirred solution of l6.8 g. (0.11 mole) of JQ and l8.48 g. (0.22 mole) of NaHCO^ in 1 L of o.l phosphate buffer (pH 6.4) was heated at 50° C. and to the solution was added portiohwise 30 g. (0.11 mole) of 7-ACA. The mixture was heated at 80° C. for 2.5 hours, during which period insoluble material still remained. The reaction mixture was cooled to room temperature and the precipitated lj+ was collected by filtration, washed thoroughly with 200 ml. of water and air-dried.

Additional 14, was obtained from the filtrate and the washings by acidifying to pH 5 with dil. HC1. Total yield 32.9 g. (83 ). M.p. 245-250° C. (dec).

IR: 1800, l6l5 , 1538, 1360 cm"1.

UV: Xi»!!aHCa5 257 nm (ε 19500) , 275 nm ( ε12000) , 310 nm (sh) ( £ 5700) .

N R: i p2°+K2C03 3.35 (l H, d, l8 Hz, 2-H), 3.76 ( 1 H, d, l8 Hz, 2-H), 4.00 ( 1 H, d, 10 Hz, 3-QH2) , 4.48 (l H, d, 10 Hz, 3-CBg) 4 .93 ( 1 H, d, 4 Hz, 6-H), 5 .32 ( 1 H, d, 4 Hz,.7~H) , 7.46 ( 1 H, d, 10 Hz, pyridazine-&), 8.18 (l H, d, 10 Hz, pyridazine-H) .

Anal. Calcd. For ci2HllN7°3S2 0 , 39.44;; H, 3.03 N, 26.83 ; S, 17. 55 Found: C, 39.19 H, 2.71 ; N, 26.84; S, 17.35. t-Butoxycarbonyl azlde (15) To a cooled solution of 100 g. (Ο.76 mole) of t-butyl carbazate in 87 g. of glacial acetic acid and 120 ml. of water was added dropwise a solution of βθ g. (Ο.85 mole) of sodium nitrite in 50 ml. of water over a period of 40 minutes, the temperature being kept at 10-15° C. After the addition was completed the stirring was continued for an additional 20 min. at the same temperature. To the mixture was added 100 ml. of water and a separated oil was extracted with five 100 ml. portions of methylene chloride. The combined organic extracts were washed with 100 ml. of 10$ sodium bicarbonate solution and 100 ml. of water successively, and dried over anhydrous sodium, sulfate. The methylene chloride was removed under diminished pressure on a water bath maintained at 40-4 ° C. The residual azide was distilled and collected at 45°C./20 mm Hg. It weighed 92.7 g. (84$).. o- (t-Butoxyca bonylaminomethy1) phenylacetic acid (l6) To a solution of 70 g. (0.35 mole) of o-aminomethylphenyl-acetic acid hydrochloride (4.) and ll6 g. (1.15 moles) of tri-ethylamine (TEA) in 400 ml. of water was added dropwise a solution of 64 g. (Ο.45 mole.) of t-butoxycarbonyl azide (15) in 300 ml. of tetrahydrofuran (THF) under stirring at 0° C. After the addition was completed, the temperature was allowed to rise to room temperature and the stirring was continued for 20 hours.

The tetrahydrofuran was distilled off below 40° C. and the aqueous solution was washed with 200 ml. of ether, layered with 200 ml. of ethyl acetate and acidified with dil. hydrochloric acid to pH 3 under cooling at 0° C. The organic layer was separated and the aqueous la er was extracted with four - ~Y solution was washed with 200 ml. of water, dried over anhydrous sodium sulfate and concentrated jin vacuo . The concentrate was treated with 500 ml. of n-hexane to give 87 .9 g. (95$) of 16. as colorless needles melting at 114-116° C. 5 2.4-Dinitrophenyl o-t-butoxycarbonylamlnomethylphenylacetate (17_) 6 Dicyclohexylcarbodiimide ( 17 .72 g., 0 .086 mole) (DCC) was 7 added in one portion to a mixture of o-(t-butoxycarbonylamino- 8 methyl) henylacetic acid ( l6) (22 .73 g., 0 .086 mole) and 2 , 4- 9 dinitrophenol ( 15 .82 g., 0.086 mole) (2,4-DNP) in 250 ml. of 10 THF. The reaction mixture was stirred for 2 hours at room 11 temperature. The precipitated dicyclohexylurea was filtered off 12 and washed with 100 ml. of THF. The filtrate and washings were 13· combined and concentrated under reduced pressure below 50° to 1¾ give a viscous yellow oil which was triturated with n-hexane 15 ( 150 ml.) to afford 17 as yellow needles. Yield 34 .9 g. ( 94 ) . 16 M.p. 76-770 C. 17 IR: Y™£ 3340, 1785 , 1685 , 1610, 1540, 1530, 1500 , 1340 cm" 18 Anal. Calcd. For C20H21N3°8: C, 55 .68; H, 4 .91; N, 9-74. 19 Found: C, 55 .70; H, 5 .0 ; N, 9.93 . ^ 7-f o-t-Butoxycarbonylaminomethylphenylacetamido) -3- (tetrazolo- * [ .5-b]pyrldazin-6-ylthiomethyl) -3~cephem-4-carboxylic acid ( l8) 22 To a solution of 20.26 g. (O-.Okj mole) of Γ7_ in 150 ml. of * tetrahydrofuran was added in one portion a solution of l4 .4o g. l* (0 .039 mole) of 14 and 19 ^ 19 6. (0 . 19 mole) of TEA in 150 ml. of 25 50$ aqueous tetrahydrofuran at 0-5° C. The reaction mixture 2^ was stirred for l8 hours and concentrated under reduced pressure 27 to remove the tetrahydrofuran below 30° C. The aqueous 8 concentrate was washed with two 200 ml. portions of ether, were washed with two 100 ml. portions of water, dried with anhydrous sodium sulfate, treated with active carbon and filtered. Evaporation of the solvent gave a pale yellow oil which was triturated with ether to give 13.89 g. (58$) of 18. M.p. 166-173° C. (dec).

IR: vjjj 1770, 1710, 1690, 1535, 1370, 1255, 1170 cm"1.

Anal. Calcd for C, 49.51; H, 4.79; N, 17.77; S, 10.17.

Found: C, 49. 8; 49.6 ; H, 4.15; 4.59; N, 17.41; 17.66; S, 10.14. 7-(o-Amlnomethylphen lacetamido)-3- (tetrazolo[4.5-b ]-Pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (20 To 20 ml. of trifluoroacetic acid was added in one portion I3.8O g. (0.022 mole) of l8 and the mixture was stirred for 45 minutes at 0-10° C. To the reaction mixture was added 300 ml. of ether to give 20 as the trifluoroacetate, which was collected by filtration and washed with ether. The trifluoroacetate was dissolved in 20 ml. of water and adjusted to pH 5 with ammonium hydroxide to give the zwltterion as a gummy oil which was separated by decantatlon and triturated with water. The solid product was collected by filtration, washed with 20 ml. of water and 200 ml. of acetonitrile successively and dried in vacuo on phosphorus pentoxide to yield 5-10 g. (45$) of 20 which was quite pure but amorphous.

Recrystallization of 20 The amorphous powder (3. Ο g.) was dissolved in 400 ml. of 50$ aqueous tetrahydrofuran with heating at 60-700 C. under vigorous stirring. The solution was treated with a small amount of active carbon. The filtrate needles melting at I90-1930 C. (dec).

Anal. Calcd. for 1 2H20: C, 46.7 ; H, 4.30; N, 20.77 S, II.89. Found: C, 47.18, 4? 37; H, 4.08, 3.88; N, 20.93, 20.23; S, 12.03.

Fxample 2 Sodium o-( l-Ethoxycarbonyl-l-propen-2-ylamlnoTnethyl) phenyl- acetate (1Q) To a alcoholic sodium ethoxide solution (metallic sodium 5.75 g. (0.25 atom) and absolute ethanol 500 ml.) were added 41.26 g. (0.25 mole) of o-aminomethylphenylacetic acid (obtained by neutralization of the hydrochloride 4. with aqueous ammonia) and 52.5 g. (0.25 mole) of ethyl acetoacetate successively. The mixture was refluxed for 6 hours and treated with active carbon and filtered through dlatomaceous earth " " to near dryness and cooled to 0° to give 19 as colorless needles which were collected by filtration, washed with 200 ml. of ethanol and dried in. vacuo over ?2°5 * ^e solld _12 weighed 44.51 g. An additional amount of 23. was obtained by concentration of the mother liquor. Total yield 56 .51 g. (76$) .

M.p. 231-232° C. (dec.) . 7- (o-Aminomethylphenylacetamido) -3~ (tetrazolo[ .5-b .py idazln-6-ylthlomethyl) -3-cephem-4-carboxyllc acid (20) Ethyl chlorofor ate (6.87 g.> Ο.ΟΟ63 mole) was added in one portion to a stirred suspension of 17.44 g. (0 .057 mole) of sodium o- ( l-ethoxycarbonyl-l-propen-2-ylaminomethyl) phenyl-acetate (19) in 200 ml. of dry THF containing 1 ml. of N,N-dimethylbenzylamine at - 15° . Stirring was stopped and a cooled solution of 20 .80 g. (Ο. Ο57 mole) of 14 and 9 . 0 g. (0 .095 mole) of triethylamine in 200 ml. of 50 aqueous THP was added slowly along the wall. The mixture was stirred vigorously for 0 min. at 0- 15° , treated with active carbon and filtered through "Dicallte". The bed was washed with 50 ml. of 50$ aqueous THF containing 8 ml. of triethylamine. Formic acid ( 3 ml.) was added to a combined solution of the filtrate and the washings to precipitate unreacted 7-ACA (2 .5 g.) which was filtered off. The filtrate was mixed with 200 ml. of ether and then 15 ml. of formic acid.

The mixture was stirred for 10- 15 min. at room temperature and the resulting precipitate was collected by filtration, washed with 100 ml. of ether and 500 ml. of water successively and dried in vacuo on Pg0^ to yield 23 .05 g. (79$) of 20 , m.p. 180-186° (dec).

Recrystallization of 20 The amorphous product 5 g. of active carbon and filtered. The filtrate was seeded and stored in a refrigerator overnight to give 7 .80 g. of 20 as fine needles. .p. 186-189° (dec).

Example "3 A suspension of the zwitterionic form of 7~ (.o-aminomethyl-phenylacetamido) -3-(tetrazolo[ 4 , 5-b]pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (O.361 g.) in 3 ml. of methanol is cooled in ice and treated with a few drops of concentrated hydrochloric, acid until a clear solution is obtained. 7-(o-Aminomethylphenylacetamido) -3- ( tetrazolo[ , 5-blpyridazin-6-yl-. thiomethyl) -3-cephem-4-carboxylic acid hydrochloride precipitates as a pale brown colored solid upon the addition of ether and is collected by filtration and dried in vacuo over P2°5 * . Example To a stirred suspension of the zwitterionic form of 7~(o.-aminomethylphenylacetamido) -3- (tetrazolo[ 4 , 5-b]pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (O.361 g.) is added I N aqueous sodium hydroxide at room temperature until a clear solution (pH. 10.8) is obtained. This solution is immediately freeze-dried to give impure, solid, sodium 7~ (jo-aminomethylphenyl-acetamido) -3~(tetrazolo[il,5-b]pyridazin-6-ylthiomethyl) -jj-cephem-4-carbox late. 1 Preparations of Dlmethanesulf onate 2 xample 5 •at 2 (30# SEH In acetone) l 26 27 ' Procedure: Put 2.0 moles (about 1025 g. on an anhydrous basis) of 7-(o-aminomethylphenylacetamido-3-(tetrazolo[4,5-b]-pyridazin-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 5 0 g. of sodium formaldehyde bisulfite (4.05 moles), 5000 ml. of water and 2700 ml. (4.87 moles) of 30$ SEH (sodium 2-ethylhexanoate) in acetone in a suitable tank and with stirring heat the mixture to 40-45° C. The mixture dissolves in about 10 min. to a yellow solution.

After 15 minutes of heating add 50 g. of decolorizing charcoal ("Darco KB") to the solution and stir 15 minutes more at 40-45° C.

Filter through diatomaceous earth ("Dicallte") after heating the reaction at 40-45° C. for a total of 30 minutes.

Wash the carbon cake with 2000 ml. of 50 ethanol-water .

Combine the filtrates, adjust to 25° C. and add the solution at 25° C to. 112 liters of rapidly stirred 100$ ethyl alcohol. A fine white amorphous precipitate of the di(sodium-methane sulfonate) of sodium 7_ (o-aminomethylphenylacetamido-3- (tetrazolo[4,5-b]pyridazin-6-ylthiomethyl)-3-cephem-4-carboxyiate forms, Stir the suspension for about 10 minutes and then filter and wash the cake with 15 liters of 100$ ethyl alcohol.

Dry the cake at 50-55° C. in an oven with air circulating for about 2 hours and then under vacuum at 4-6 mm. for 24 hours.

The yield is about 1200-1400 g. of amorphous, white, solid di (sodium-methane sulfonate) of sodium 7_ (o,-aminomethylphenyl- acetamido-3-(tetrazolo[4,5-b]pyridazin-6-ylthiomethyl) -3-cephem-4- carbox late. The product usually contains several percent meth l)aminomethylphenylacetamido] -3-(tetrazolo[4, 5-b3pyridazin- 6-ylth.iomethyl) -3-cephem-4-carboxylate .

Example 6 The following slurry is prepared: 2 . 19 grams of sodium-formaldehyde bisulfite (2 equivalents). 3.5 g. of 7-(o-aminomethylphenylacetamido) -5-(tetrazolo[ 4 , 5-b] pyridazin-6-ylthiomethyl)-3-cephem-4-cartoxylic acid zwltterion (100-200 mesh) . 25 ml. of water (volume can be varied). 14 ml. of 20$ SEH-isopropanol.

A near solution is obtained in about 0 .5 hour of rapid stirring at 24° C. The- temperature of the mixture is raised rapidly to 40-43° C. This is maintained for about two minutes and then rapidly cooled to 20-23° C.

The solution is filtered to remove some insolubles (total time in solution should not exceed two hours) .

The pH 7 .3 solution is added over a minute period to βθθ ml of very rapidly stirring absolute ethanol (other alcohols such as anhydrous isopropanol may be used) . An amorphous precipitate of · the di( sodium-methane sulfonate) of sodium 7~ (o.-aminomethylphenyl- acetamido)-3- (tetrazolo[4, 5-b ]pyridazin-6-ylthiomethyl) -3-cephem- 4-carboxylate forms. The mixture is stirred for 5 minutes. The precipitate is collected by filtration, washed with 60 ml. of ethanol (or isopropanol) and vacuum dried at 50° C. for 24 hours. The yield is about 4 .3 g.

The product is soluble in water at about pH 7 to the extent of at least 200 mgm./ml. Such a solution is stable for at least s 1 Example 7 2 Sodium 7-fo-N.'N-blsf sodiosulfomethyl)amlnomethylphenylacetamido3- 3 3-tetrazolo[ . -b.-pyridazln-6-.ylthio ethyl)- -·cephem^- - 4 carboxyiate 5 A . Preparation using hvdroxymethanesulfonate 6 A mixture of 1 g. (1.95 mmole) of 7~ (o-aminomethylphenyl- γ acetamldo-3- (tetrazolo [ 4 ,5-b ]-pyridazln-6-ylthlomethyl) -3-cephem- 8 4-carboxylic acid, 1.52 g. (10 mmole) of sodium hydroxymethane- 9 sulfonate monohydrate, 6 ml. (6 mmole) of 1 M SEH solution in 0 ethyl acetate, 10 ml. of isopropanol and 10 ml. of water was 1 stirred at room temperature for 3.5 hr. The resulting solution 12 was treated with 1 g. of active carbon and poured under stirring 1¾ into 300 ml. of abs. ethanol and the mixture was stirred at room temperature for 30 min. to give the crystalline product which was 15 collected by filtration, washed with three 50 ml. portions of abs. 16 ethanol and dried over PgO,. at mm. for 20 hr. to give ΐγ 1.51 g. of sodium 7"[o-N,N-bis(sodiosulfomethyl)aminomethylphenyl-lQ acetamido ]-3- (tetrazolo [4 ,5"b ] -pyr idazin-6-ylthlomethyl) -3-cephem-ig 4-carboxylate which was readily soluble in water ( ^> 1 g./ml.). go M.p. >270°. 21 m'm ^ 17β0' 1660-1620, I605 , 1540, l400, 1200, 1040 cm"1 22 UV: λ¾χ 242 nm (εΐ8,000), 271 nm (£.11,600, sh) , 312 nm 23 24 Hz, 25 , 4.30 2 (4H, s, N-pHg-S05Na), 4.92 (1H, d, 4.5 Hz, 6-H) , 5.42 (1H, d, 27 4.5 Hz, 7-H), 7.Ο5-7.3Ο (4H, phenyl-H), 7.40 (1H, d, 10 Hz, 1 2 B. Preparation using formalin and sodium bisulfite 3 (a) To a solution of 1 ml. (10 mmole) of 30 formalin and 4 1 g. of sodium bisulfite in 10 ml. of water was added successively 5 1.026 g. (2 mmole) of 7~ (.o-aminomethylphenylacetamido-3- 6 (tetrazolo[ ,5-b]-pyridazin-6-ylthiomethyl)-3-cephem-4-carbox lic 7 acid, 6 ml. of 1 M SEH solution and 10 ml. of isopropanol. 8 The mixture was stirred for 2.5 hours at room temperature and poured into 300 ml. of ethanol. The resulting sodium 7-[o-N,N- 10 bis (sodiosulfomethyl)aminomethylphenylacetamido ]-3- ( tetrazolo- 11 [4,5-b]-pyridazin-6-ylthiomethyl)-3-cephem-4-carboxylate was \2 collected by filtration, washed with three 50 ml. portions of 13 ethanol and dried In v acuo. Yield 1.6l g. lJ* (b) To a mixture of 1.026 g. (2 mmole) of 7-(o-aminomethyl- *5 phenylacetamido-3-(tetrazolo[ll 5-b]-pyridazin-6-ylthiomethyl)-3- * cephem-4-carboxyllc acid, 6 ml. of 1 SEH in ethyl acetate, 17 io ml. of isopropanol and 10 ml. of water was added 1 ml. *8 (10 mmole) of 30$ formalin. The mixture was stirred for 2 hours *9 at room temperature to give a clear solution with a small amount 20 of oily precipitate. After 1 g. of sodium bisulfite was added, 21 the solution was stirred for an additional 2 hours, during which 22 time the oily precipitate dissolved in the solution. The reaction 23 mixture was poured into 300 ml. of ethanol under vigorous stirring 21* to give 1.6l g. of sodium 7~[o-NiN-bis(sodiosulfomethyl)amino- 25 methylphenylacetamido]-3-(tetrazolot ,5-b]-pyridazin-6-ylthio- 26 methyl) -3~cephem- -carboxylate which was collected by filtration, 27 washed with three 0 ml. portions of ethanol and dried in vacuo.

Example 8 2 A.7- [ β- (o-t-Butoxycarbonylaminomethylphenyl) propionaTnido 3 -3- 3 tetrazolot^ . - 3pyrlda2ln-6-ylthiomethyl)~5-cephem-1l-carboxyIic ^ acid. 5 Ν,Ν'-Dicyclohexylcarbodiimide (0.4l g., 2 mmole) was added 6 to a mixture of β- (o-t-butoxycarbonylaminomethylphenyl) ropionic 7 acid (Ο.56 g., 2 mmole) and 2,4-dinitrophenyl (0.37 g.> 2 mmole) 9 in 5 ml. of THF and the mixture was stirred for 1 hr. at room 9 temperature. The precipitated urea was filtered off. To 10 the filtrate was added a solution of 7-amino-3-(tetrazolo&,5-b-- 11 pyridazin-6-ylthiomethyl)-3-cephem- -carboxylic acid (0.73 g.» 1 2 mmole) and triethylamine (0.8l g., 8 mmole) in 10 ml. of 0# ^ aqueous THF and the mixture was stirred for 18 hr. at room temperature. The reaction mixture was washed with ether (2x20 ml) 15 and the aqueous layer was acidified to pH 2 with dil. HC1 and 16 extracted with ethyl acetate (3 x 50 ml.). The combined extracts 17 were washed with water (30 ml.), treated with active carbon, porated under reduced pressure d with 50 ml. of ether to give ylphenyl) propionamido3-3- omethyl)-3"cephem-4-carboxyllc acid as a colorless solid which was collected by filtration and 23 dried in vacuo on P 0c. 2k 25 Yield 0.33 g. (2656 . M.p. 110-120° (dec). 2 26 IR: ^max 1780' 1710' ΐ690' 1530' 1370' 1250 ^'1· 270 nm (sh) ( ε.12500) , 29 NN^ ^DDm0^6 1·57 (9*Η' s' t-Bu"H)» 4·96 (1 Η, d, 4 Hz, -H), 5.55 (1 H, d-d, 4 & 8 Hz, 7-H) , 6.99 (4 H, s, phenyl-H) , 7.5V (1 H, d, 10 Hz, pyridazine-H), 8.38 (1 H, d, 10 Hz, pyridazine-H). .69 (1 H, d, 8 Hz, CONH) Anal. Calcd. for 1/2 H20: C, 51.02; H, 4.91; N, 17.65.

Found: C, 1.74; H, 4.85; N, 17.88.

B. 7- [B-fo-Aminomethylphenyl) propionamido] -5- (tetrazolof 4 ,5-bl - pyridazin-6-ylthiomethyl)-5-cephem-4-carboxylic acid Trif luoroacetic acid (0.5 ml.) and 7- [p- (o-tert-butoxy- carbony laminomethy lphenyl) propionamido ] -3- ( t etra zolo [ 4 , -b ] - pyridazin-6-ylthiomethyl)-3-cephem-4-carboxylic acid (0.28 g., 0.46 mmole) were mixed under cooling at 0° and stirred for 30 min. Ether (50 ml.) was added to the mixture to give the trif luoroacetate of 7-[p-(o-aminomethylphenyl)propionamido]-3- ( tetrazolo [4, -b ]pyridazin-6i-ylthiomethy]^-3-cephem-4-carboxy lie acid which was separated by decantatlon, washed with ether, dissolved in water (l ml.) and adjusted at pH 6 with dil..

NH^OH. The product 7- ίβ- (o-amlnome thy lphenyl) propionamido] -3- ( tetrazolo [4, 5-b 3 pyridazin-6-ylthiomethyl)-3-cephem-4-carboxy lie acid was collected by filtration and dried in vacuo on P2°5* Yield 0.10 g. (420). M.p. 190-197° (dec).

KBr IR: V> max 1760, 1665, 1600, 1 3 cm -1 Anal. Calcd. for C22H22NQ01|S2 · 1/2 HgO: C, 49.33; H, 4.33; N, 20.92; S, 12.18.

Found: C, 49.04; H, 4.26; N, 20.17; S, 11.96. 8 9 1 A. Example Q Sodium B-[o-( l-Ethoxycarbonvl-l-propen-2-vlaminomethyl) phenyl 1-5 propionate Ί To an alcoholic sodium ethoxide solution (metallic sodium 5 5.75 g. (0.25 atom) and absolute ethanol 500 ml.) are added 6 0.25 mole of β-to-aminomethylphenyl]propionic acid (obtained by 7 neutralization of its hydrochloride with aqueous ammonia) and 8 32.5 g. (Ο.25 mole) of ethyl acetoacetate successively. The 9 mixture is refluxed for 6 hours and treated with active carbon 0 and filtered through diatomaceous earth ("Dicalite") . The filter. 1 bed is washed with 200 ml. of hot ethanol. The combined filtrate 2 and washings are evaporated to near dryness and cooled to 0° to 3 give sodium β- (o-(l-ethoxycarbonyl-l-propen-2-ylaminomethyl) phenyl3- 4 propionate as colorless needles which are collected by filtration, 5 washed with 200 ml. of ethanol and dried %n vac.uo over PG05 « AN 6 additional amount is obtained by concentration of the mother liquor, γ Total yield about. 0 g. 18 B. 19 7- [ 3- (o-Amlnomethv Phenv 1) propionamldo 1 -3- (tetrazolo[ . ¾-b ]- 0 Pyrldazln-6-ylthlomethyl) -3-cephem-4-carboxyllc acid 1 Ethyl chloroformate (6.87 g., 0.0063 mole) is added in one 2 portion to a stirred suspension of 0.057 mole of sodium β-[ο- ( 1- 2¾ ethoxycarbonyl-l-propen-2-ylaminomethyl)phenyl]propionate in 200 ml. 2 of dry THP containing 1 ml. of Ν,Ν-dimethylbenzylamine at -15° . 25 Stirring is stopped and a cooled solution of 20.80 g. (0.057 mole) 26 of 7-amino-3-(tetrazolo[ ,5-b3pyridazin-6-ylthiomethyl) -3-cephem- 27 4-carboxylic acid and 9.60 g. (0.095 mole) of triethylamine in 28 200 ml. of 50$ aqueous THP is added slowly along the wall. The 29 mixture is stirred vigorously for 30 min. at 0-15° , treated with 1 with 50 ml. of 0$ aqueous THF containing 8 ml. of triethylamine . 2 Formic acid (3 ml.) is added to a combined solution of the filtrate ' ■ and the washings to precipitate unreacted 7-ACA (2 .5 g.) which is filtered off. The filtrate is mixed with 200 ml. of ether and then 5 15 ml. of formic acid. The mixture is stirred for 10- 15 in. at 6 room temperature and the resulting precipitate is collected by 7 filtration, washed with 100 ml. of ether and 500 ml. of water 8 successively and dried In vacuo on to yield 7~[β~ (o-amino- 9 meth lphenyl) proplonamido] -3- (tetrazolo[ 4 , 5-b_py idazin-6-ylthio-10 methyl) -3-cephem-4-carboxyllc acid, about 20 g.

H Reerystallization of 7~[p-i{o-aminomethylphenyl)propionamido]-3- 12 (tetrazolo[ 4 , 5-b]pyridazin-6-ylthiomethyl ) -3""cephem-4-carboxylic * acid The amorphous product described above (13 g.) is I1* dissolved in 1 .2 1 of 50$ aqueous THF at 50-60° under vigorous 15 stirring, treated with g. of active carbon and filtered. The 1 filtrate is seeded and stored in a refrigerator overnight to give 7 about 8 g. as fine needles. 1® Example 10 19 A suspension of the zwitterionic form of 7- [β- (o-aminomethy - 20 phenyl)propionamido]-3-(tetrazolo[ 4 , 5-b]pyridazih-6-ylthiomethyl)- 21 3-cephem-4-carboxylic acid (O.361 g.) in 3 ml. of methanol is 22 cooled in. Ice and treated with a few drops of concentrated 23 hydrochloric acid until a clear solution is obtained. 7_[β-(ο- 21* aminomethy 1phenyl) propionamido ] -3- (tetrazolo [ 4 , 5-b ]pyridazin-6- 25 ylthiomethyl) — 3-cephem-4-carboxylic acid hydrochloride precipitates 26 as a pale brown colored solid upon the addition of ether and Is 27 collected by filtration and dried in vacuo over P2° ' 28 29 Example 1 To a stirred suspension of the zwitterionic form of 7~ [β~ (o-aminomethylphenyl) propionamido] -5" ( tetrazolo [ 4, 5-b ] - pyridazin-6-ylthiomethyl) -3**cephem-4-carboxylic acid (O.361 g.) is added 1 i£ aqueous sodium hydroxide at room temperature . ntil a clear solution (pH 10.8) ie obtained. This solution is immediately freeze-dried to give impure, solid sodium 7"ίβ- (o-aminomethylphenyl) - propionamido]-J- (tetrazolo[4, 5-b ]pyridazin-6-ylthiomethyl) -3~ 9 cephem-4-carboxylate. 0 1 ■ * 2 3 4 5 6 7 18 χ9 20 21 22 23 24 25 26 27 28 29 Preparations of Dlmethanesulf onate Example 12 (50j6 SEH in acetone) 1 Procedure: Put 2.0 moles (about 1053 g. on an anhydrous 2 basis) of 7-[β- (o-aminomethylphenyl)propionamido]-3- (tetrazolo- 3 [4,5-b]pyridazin-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 540 g. 4 of sodium formaldehyde bisulfite (4.03 moles), 3000 ml. of water 5 and 2700 ml. (4.87 moles) of 30 SEH (sodium 2-ethylhexanoate) 6 in acetone in a suitable tank and with stirring heat the mixture 7 to 40-45° C. The mixture dissolves in about 10 min . to a yellow 8 solution. 9 After 15 minutes of heating add 50 g. of decolorizing 10 charcoal ("Darco KB") to the solution and stir 15 minutes more U at 40-45° C. 12 Filter through diatomaceous earth ("Dicalite") after heating ¾ the reaction at 40-45° C. for a total of 30 minutes. 14 Wash the carbon cake with 2000 ml. of 0$ ethanol-water . 15 Combine the filtrates, adjust to 25° C. and add the 6 solution at 25° C. to 112 liters of rapidly stirred 100$ ethyl ΐγ alcohol. A fine white amorphous precipitate of the di(sodium- 8 methane sulfonate) of sodium J- [β- (o-aminomethylphenyl) propionamido]- 19 3~(tetrazolo[4,5-b]pyridazin-6-ylthiomethyl) -3_cephem-4-carboxylate 20 forms . 21 Stir the suspension for about 10 minutes and then filter and 22 was the cake with 15 liters of 100$ ethyl alcohol. 2¾ Dry the cake at 50-55° C. in an oven with air circulating 2 for about 2 hours and then under vacuum at 4-6 mm. for 24 hours. 25 The yield is about 1200-1400 g. of amorphous, white, solid 26 di (sodium-methane sulfonate) of sodium 7~.β~ (o-aminomethylphenyl) - 27 propionamido]~3- (tetrazolo[4,5-b ]pyridazin-6-ylthiomethyl) -3-cephem-4 28 carboxylate. The product usually contains several percent water 29 and possible a trace of ethanol.

I methyl)aminomethylphenyl]propionainido^ -3- (tetrazolo[H, 5-b] pyridazin-6-ylthiomethyl) -3_cephem-4-carboxylate . 3 Example 4 The following slurry is prepared: 5 2. 19 grams of sodium-formaldehyde bisulfite (2 equivalents). 6 3.5 g. of 7~ί β- (o-aminomethylphenyl) ropionamido] -3~ (tetrazolo- 7 [ 4 , 5-b]-pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid & zwitterion ( 100-200 mesh) . 9 25 ml. of water (volume can be varied). 10 14 ml. of 30$ SEH-isopropanol.

II A near solution is obtained in about 0 .5 hour of rapid 12 stirring at 24° C. The- temperature of the mixture is raised 13 rapidly to 40-43° C. This is maintained for about two minutes I1» and then rapidly cooled to 20-23° C. 15 The solution is filtered to remove some insolubles (total 16 time in solution should not exceed two hours) . 17 The pH 7 .3 solution is added over a 5 minute period to 600 ml. 18 of very rapidly stirring absolute ethanol (other alcohols such as 19 anhydrous isopropanol may be used) . An amorphous precipitate of 20 · the di(sodium-methane sulfonate) of sodium 7- ίβ~ (o-aminomethylphenyl 21 propionamido] -3" (tetrazolo[ 4 , 5-b]pyridazin-6-ylthiomethyl) -3-cephem- 22 4-carboxylate forms. The mixture is stirred for 5 minutes. The 23 precipitate is collected by filtration, washed with 60 ml. of 24 ethanol (or isopropanol) and vacuum dried at 50° C. for 24 hours. 25 The yield is about 4 g. 26 The product is soluble in water at about pH 7 to the extent 27 of at least 200 mgm./ml. Such a solution is stable for at least 28 two hours at room temperature; more dilute solutions are stable 29 even longer. The product shows the same antibacterial spectrum 1 Example 2 Sodium 7" {6~-0-N.N-bls(sodiosulfomethyl)aminomethylphenyl]- 3 propionamldo^ -3-(fcetrazolo[ 4 , -b ] pyridazln-6-ylthiomethyl -3- 4 cephem-4-carboxylate . 5 A. Preparation using hvdroxymethanesul onate 6 A mixture of 1 .95 mmole of 7~ t β~ (o-aminomethylphenyl) - propionamido] -3_ (tetrazolo[ 4 , 5-0 ]pyridazin-6-ylthiomethyl) -3- 8 cephem-4-carboxylic acid, 1 .52 g. ( 10 mmole) of sodium hydroxy- 9 methanesulfonate monohydrate, 6 ml. (6 mmole) of 1 M SEH solution 0 in ethyl acetate, 10 ml. of isopropanol and 10 ml., of water is 1 stirred at room temperature £or 3 .5 nr. The resulting solution 2 is treated with 1 g. of active carbon and poured under stirring 13 into 300 ml. of abs. ethanol and the mixture is stirred at room 4 temperature for 30 min. to give the crystalline product which is 15 collected by filtration, washed with three 0 ml. portions of abs. 16 ethanol and dried over at 45- 2 mm. for 20 hr. to give Yj about 1 .5 g. of sodium 7" (P-[o-N,N-bis(sodiosulfomethyl)aminomethyl lQ phenyl]propionamido^-3"(tetrazolo[ 4 , 5-b]-pyridazin-6-ylthiomethyl) -3 i cephem-4-carbox late which is readily soluble in water ( ^>1 g./ml.) 20 21 22 23 24 25 26 27 28 29 Β· Preparation using formalin and sodium bisulfite (a) To' a solution of 1 ml. ( 10 mmole) of 30 formalin and 1 g. of sodium bisulfite in 10 ml. of water is added successively 2 mmole of J- [β- (o-aminomethylphenyl) propionamido ] ->- (tetrazolo-[ 4 , 5-b]pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid, 6 ml. of 1 M SEH solution and 10 ml. of isopropanol. The mixture is stirred for 2 .5 hours at room temperature and poured into 300 ml. of ethanol. The resulting sodium 7- ^-[o-N,N-bis(sodiosulfomethyl)-aminometh lphenyl] propionamido -3-(tetrazolo[ 4 , 5-b]pyridazin-6-ylthlo-methyl)-3-cephem-4-carboxylate is collected by filtration, washed with, three 0 ml. portions of ethanol and dried in vacuo . Yield about 1 .5 g. (b) To a mixture of 2 mmole of 7-[ -(o-aminomethylphenyl)-propionamido ] -3~ (tetrazolo[ 4 , 5- lpyridazin-6-ylthiometh l) -3-cephem-4-carboxylic acid/ 6 ml. of 1 M SEH in ethyl acetate, 10 ml. of isopropanol and 10 ml. of water is added 1 ml. ( 10 mmole) of 0$ formalin. The mixture is stirred for 2 hours at room temperature to give a clear solution with a small amount of oily precipitate. After 1 g. of sodium bisulfite is added, the solution is stirred for an additional 2 hours, during which time the oily precipitate dissolves in the solution. The reaction mixture is poured into 300 ml. of ethanol under vigorous stirring to give about 1 .5 g. of sodium 7~ ^-[o-N,N-bis(sodiosulfomethyl)aminomethyl- phenyl ] -propionamido -3"(tetrazolo [ 4 , 5~b ]pyr ldazin-6-ylthiomethyl) -3-cephem-4-carbox late which is collected by filtration, washed with three 0 ml. portions of ethanol and dried in vacuo .

^ In vitro and In vivo Studios 7- [ (ο,-Aminomethyl) phenylpropionamidol-3- (tetrazoloi* , 5-b]- pyridazin-6-ylthiomethyl)-j5-cephem-4-carboxylic acid, is a new broad-spectrum semi-synthetic cephalosporin having the structure shown below. Θ It has been shown to possess excellent in vit o and In vivo activities against a wide variety of Gram-positive and Gram- negative bacteria including those which are resistant to cephalothin and cephaloridine . Its dimethane-sul onate adduct. having the structure is prepared as a water-soluble preparation useful for injection and for studies of absorption and excretion, Initial studies of in vitro antibacterial activity by the tube dilution method or agar dilution method of this new cephalosporin showed Minimum Inhibitory Concentrations (M.I.C.'s) of less than 1.0 meg./ml. for all or nearly all strains tested of Staphylococcus aureus. Streptococcus pyogenes and Diplococcus pneumoniae and M.I.C.'s usually less than 4 meg. /ml. and often less than 1.0 meg./ml. against various strains of Salmonella enteritldis and Enterobacter cloacae .

* . ■■ The in vivo efficacy of this compound by subcutaneous injection was studied in mice having an experimental infection caused by pathogenic, penicillinase-positive S. aureus. The median curative dose (CD^0) was about 1.6 mgm./kg. 1 2 ■a 7-[ (jo-Aminoir.eth l)phenylacetamido]-3- (tetrazolof1! ,5-b ]■ pyridazin-6-ylthiomethyl) -^-cephem-^-carboxylic acid, is a new broad-spectrum semi-synthetic cephalosporin having the structure shown below. 8 5 6 It has been shown to possess excellent in vitro and in iv 7 activities against a wide variety of Gram-positive and Gra 18 negative bacteria Including those which are resistant to 19 cephalothin and cephaloridine . Its dimethane-sulf onate 0 adduct. having the structure 27 28 was prepared as a water-soluble preparation useful 29 for injection and for studies of absorption and excretion, Initial studies of in vitro antibacterial activity by the tube dilution method or agar dilution method of this new cephalosporin showed Minimum Inhibitory Concentrations (M.I.C.'s of less than 1.0 meg./ml.for all ov nearly all strains tested of Staphylococcus aureus . Streptococcus pyogenes .

Dlplococcus pneumoniae . various Bacillus species including Bacillus anthracls . mycoides and cereus , Klebsiella pneumoniae. Proteus rettgeri. Shigella and especially Shigella flexneri. Salmonella enterltidi3 and Salmonella typhosa and M.I.C.'s usually less than 4 mcg"./ml. and often less than 1.0 meg./ml. against various strains of Escherichia coll. Klebsiella unspeciated, Proteus vulgaris . Proteus morgan!! .· Proteus mlrabllls and Enterobacter cloacae .

This series of Gram-negative test organism included 14 strains of cephalothln-resistant Enterobacteriacae (3 EL*, coll . 6 Proteus. 1 Enterobacter . 1 Shigella and 3 Serratia) which were not inhibited by 100 meg./ml. of cephalothin and in many cases not inhibited by 100 meg./ml. of cephaloridine and cephapirin. The compound of this invention was remarkably active against these organisms, inhibiting 12 of the 14 strains at 6.3 meg./ml. or less.

The M.I.C.'s of this compound were not substantially reduced in the presence of moderate concentrations of human serum, e.g. up to 50$. The compound in solution at 37° C. was highly stable at pHs ranging from 4.6 to 8.4 with half- lives exceeding 24 hours.

The Xn vivo efficacy of this compound by subcutaneous injection was studied in mice having experimental infections caused by 10 pathogenic bacteria including penicillinase- 1 The bacteria used wore strains of S. aureus. S pyogenes. 2 XL. Pneumoniae . F.t coll . . pneumoniae . P. vu'J ^arls and .S . ·; arcescens . The median curative dose (CD^Q) never exceeded 4 20 mgm . Ag . , only twice exceeded 10 mgm .Ag . and in seven 5 Instances was 5 .2 mgm . /kg . or less with a minimum of 0.2 6 mgm .Ag . γ Blood levels in mice after subcutaneous injection of this g compound at doses as low as 5 mgm . Ag . were determined and showed excellent absorption; urinary recovery was good 10 (about 60#) and paper chromatography of the urine showed this 11 compound to be the only bloactive substance present, indicating ^2 metabolic stability. yi Acute toxicity tests in mice by intravenous administration 14 of doses as high as 1000 mgm. Ag . showed no deaths. Two tests }ij designed to measure local tissue irritation after injection showed no irritation at concentrations of 12.5 or less. 17 18 19 20 21 22 23 24 25 26 27 28 2

Claims (1)

1. CLAIMS 1. A process for the preparation of the compounds having the formula I wherein n is 1 or 2, and nontoxic pharmaceutically acceptable salts and easily hydroiyzed esters thereof which process comprises reacting a compound of the ormula II or a salt or easily hydroiyzed ester thereof with an acylating derivative of an acid having the formula III 1 iherein B is an amino-protecting group and n is as 2 above to produce a compound having the formula 3 9 10 I 11 12 wherein Ji and n are as above, or a salt or easily 13 hydrolyzed ester thereof, and subsequently removing the 14- amino-protecting group B to produce the desired compound 1 of formula I or a nontoxic ph rmaceutically acceptable 16" salt or easily hydrolyzed ester thereof. 17 18 19 20 21 22 23 24 25 26 27 28 29 30 2. The process of Claim 1 wherein the acylating agent of formula III is the acid halide, mixed anhydride or activated ester. 3. The process of Claim 1 or Claim 2 wherein the amino-protecting group B is t-butoxycarbonyl or 1-carboethoxy-l-propenyl-2. 4. The process of Claim 1 wherein the acylating agent is a compound of the formula wherein n is 1 or 2. 5. The process Of any of Claims 1, 2 or 3 wherein the amino-protecting group is removed by treatment with formic acid or trifluoroacetic acid. 6, The process of any of Claims 1-5 wherein the dlmethanesulfonate sodium salt having the formula in which n is 1 or 2 is prepared by reacting the zwitterion of the formula Θ with water, sodium formaldehyde bisulfite or a source the and a strong sodium base. 7. The process of Claim 6 wherein the strong sodiu base is sodium 2-ethylhexanoate. 8. A process for the preparation of the compound of the formula or a salt or easily hydrolyzed ester thereof; which process comprises reacting 7-aminocephalosporanic acid or a salt thereof with a thiol of the formula * or a salt thereof *\nd, if desired, converting by methods 5 known per se the so-Droduced 7-amino-3- (tetrazoloHi, 5-b]- 6 pyridazin-6-ylthiomethyl)-3-cephem- -carboxylic acid to a salt 7 or easily hydrolyzed ester thereof. 8 9 10 H 12 13 i^ 15 16 17 . 18 19 20 21 22 . 23 2 25 26 27 28 29 30 43361/2 Compounds of the formula COOH wherein X is H or in which R is H or the group -CH^O^ a, and n is 1" or 2, and nontoxic pharmaceutically acceptable salts and easily hydrolyzed esters thereof. 10. Compounds of the formula wherein n is 1 or 2, and nontoxic pharmaceutically acceptable salts and easily hydrolyzed esters thereof. 11. Compounds of the formula 12. The compounds of the formula and nontoxic pharmaceutically acceptable salts thereof. The zwitterionic form of the compound of Claim 12. l . The sodium or potassium salts of the compound of Claim 12. 15. The hydrochloride of the compound of Claim 12. l6. The compound of the formula COOH and nontoxic pharmaceutically acceptable salts thereof. 17. The zwitterionic. form of the compound of Claim l6. 18. The sodium or potassium salts of the compound of Claim 16. 19. The hydrochloride of the compound of Claim 16. The compound of the formula COOH and nontoxic pharmaceutically acceptable salts and easily hydrolyzed esters thereof. 21. The sodium or potassium salts of the compound of Claim 20. 22. The process of Claim 1 substantially as hereinbefore described with reference to the Examples. 23. A compound as claimed in any of Claims 9-2l when prepared according to the process of any of Claims 1-8. 24. A pharmaceutical composition comprising a compound or salt as claimed in any one of Claims 10-19 in association with at least one pharmaceutical carrier or excipient. 25. A method of treating animals ,for diseases caused by. Gram-positive or Gram-negative bacteria which method comprises administering an effective amount of a compound or salt as claimed in any one of Claims 10-19 or a composition as claimed in Claim 24. 43361/2 26. A method of treating cattle for mastitis which method comprises administering an effective amount of a compound or salt as claimed in any one of Claima 9-21 or a composition of Claim 24· 27. A pharmaceutical composition as claimed in Claim 24 substantially as described herein. 28. A method as claimed in Claims 25 or 26 substantially as described herein. 29. 2he process of Claim 8 substantially as hereinbefore described with particular reference to Example

1. ND:dn