CH593989A5 - Antibacterial cephalosporins - prepd. from amino-tetrazolo-pyridazinyl-thiomethyl-cephem-carboxylic acids - Google Patents

Abstract

Prepn. of new cephalosporins of formula (I) comprises reacting a 7-amino-3-cephem-4-carboxylic acid deriv. of formula (II) with a reactive deriv. of an acid of formula (III) and deprotecting the prod. (where Q is OH, OM or a readily hydrolysable ester residue; M is a nontoxic cation; n = 1 or 2; B is a protected amino gp.). (I) are broad-spectrum antibiotics with high activity against penicillin- and cephalosporin-resistant bacteria, (e.g. E. coli and S. aureus) and low toxicity (LD50 >1000 mg/kg, i.v., mouse). In an example, (I; Q = CH, n = 1) is prepd. from (II; Q = OH, n = 1) and 2,4-dinitrophenyl o-t-butoxycarbonylaminomethylphenylacetate.

Description

  

  
 



   The present invention relates to the preparation of certain new 3-thiolated-7-acylaminocephalosporanic acid derivatives. These are valuable antibacterial agents and are suitable as additives to cattle feed and as agents for the treatment of mastitis in horned cattle and also as therapeutic agents for humans, mammals and poultry, in particular for the treatment of infectious diseases which are caused by some grief-positive and grief-stricken negative bacteria.

  These are in particular 7- (o-aminomethylphenylacetamido) -3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid, 7- (o aminomethylphenylpropionamido) -3 - (tetrazolo [4,5 -bj-pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid, the dimethanesulfonate derivatives of the acids mentioned and non-toxic pharmaceutically acceptable salts and easily hydrolyzable esters thereof. In the context of the process described, the preparation of a new intermediate of 7-amino 3- (tetrazolo [4,5-b] pyridazin-6-yl-thiomethyl) -3-cephem-4-carboxylic acid is described, its acylation in the specified Way the new active cephalosporin derivatives can be obtained.



   The literature on the cephalosporin antibiotics is extensive and largely known.



   The amphoteric compounds which can be prepared according to the invention have the formula
EMI1.1
 where n is 1 or 2. The compound occurs in the form of the zwitterion. It can be converted into its non-toxic pharmaceutically acceptable salts, including the sodium dimethanesulfonate salt, and into easily hydrolyzable esters.



   The nontoxic, pharmaceutically acceptable salts include the nontoxic salts derived from the carboxylic acid. It is z. B. to metal salts of sodium, potassium, calcium and aluminum, also the ammonium salts and substituted ammonium salts, z. B. Salts of such non-toxic amines as the trialkylamines, including triethylamine, procaine, dibenzylamine, N-benzyl-ss-phenethylamine, l-ephenamine, N, N'-dibenzylethylenediamine, dehydroabietylamine, N, N'-bis-dehydroabietylethylenediamine, N-lower alkylene diamine , e.g. B. N-ethylpiperidine, as well as salts with other amines, such as those used for the formation of salts with benzylpenicillin.

  Non-toxic acid addition salts (ie the amine salts) with mineral acids, such as the hydrochlorides, hydrobromides, hydroiodides, sulfates, sulfamates and phosphates, as well as the acid addition salts of amines, such as the maleates, acetates, citrates, oxalates, succinates, benzoates, tartrates, also come into consideration. Fumarates, malates, mandelates, ascorbates and the like.



   Particularly preferred salts of the amphoteric compounds which can be prepared according to the invention are the sodium dimethanesulfonate salts. These salts are water-soluble and consequently can be used particularly well for the production of injectable administration forms.



   Included in the scope of the invention is the preparation of the compounds (either used as intermediates or as metabolic precursors) in which the amino group is protected by substituents such as t-butoxycarbonyl, carbobenzyloxy, formyl, o-nitrophenylsulfenyl, ss, ss, ss-trichloroethoxycarbonyl, 4-oxo-2-pentenyl-2,1-carbo-ethoxy-1-propenyl-2 and the like. Of particular importance among the blocking groups of this type are the ketones (especially acetone) and aldehydes (especially formaldehyde and acetaldehyde), described e.g. In U.S. Patents 3,198,804 and 3,347,851, as well as diess-ketoesters and pss-diketones, described e.g. In U.S. Patent 3,325,479, and diess-ketoamides described in Japanese Patent Number 71/24714.



   The process according to the invention for the preparation of antibiotically active compounds of the formula I.
EMI1.2
  wherein n is 1 or 2, as well as the non-toxic pharmaceutically acceptable salts and easily hydrolyzable esters thereof, is characterized in that a compound of the formula II
EMI2.1
 or a salt or an easily hydrolyzable ester of the latter is reacted with an acylation-capable derivative of an acid of the formula III
EMI2.2
 wherein B is a protected amino group, to a compound of formula IV
EMI2.3
 or a salt or an easily hydrolyzable ester of the latter, whereupon the protective group is split off with formation of the compound I or the corresponding salt or ester.



   According to the invention, the amphoteric compounds are prepared by coupling with a special 3-thiolated-7-aminocephalosporanic acid of the designation II, i.e. H.



  the 7-amino-3- (tetrazolo [4,5 -b] pyridazin-6-ylthiomethyl) 3-cephem-4-carboxylic acid or a salt or easily hydrolyzable ester thereof (including, but not limited to, those such as mentioned in U.S. Patent No. 3,284,451 and British Patent 1,229,453, as well as any of the silyl esters described in U.S. Patent 3,249,622 for use with 7-aminopenicillanic acid and in British Patent 1,073,530 and in particular the pivaloyloxymethyl , Acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl and ss, ss, ss-trichloroethyl ester), an acid of the formula III or a functional equivalent of an acylating agent for primary amino groups being possible as coupling partner. After coupling, the protective group is split off and gives the desired product.

  In Formula III, B represents a protected amino group of the type used either in the peptide syntheses or in any of the numerous syntheses of ampicillin or cephaloglycine or cephalexin from 2-phenylglycine. Particularly beneficial protecting groups are a proton, as in the compound of the formula
EMI2.4
 or an ß-diketone or ß-ketoester as described in British Patent 1,123,333 and U.S. Patent Numbers 3,325,479 and 3,316,247, e.g. B. methyl acetoacetate or an β-ketoamide, as described in Japanese Patent 71/24714 (Farmdoc 47, 321 S), in which case the acid containing the blocked amino group is preferably converted into a mixed anhydride, e.g.

  B. with ethyl chloroformate, before the reaction with the compound II or a salt thereof takes place, whereupon the blocking group is split off after the reaction has taken place and the desired product I is obtained.



   The splitting off of the blocking group to the desired end product of the formula I can take place as follows: If it is, for. B. the ti-butoxycarbonyl group, this can be split off by treatment with formic acid or trifluoroacetic acid.



   The carbobenzyloxy group can be removed by catalytic hydrogenation. The 2-hydroxy-1-naphthcarbonyl group can be removed by acid hydrolysis. The trichloroethoxycarbonyl group can be removed by treatment with zinc dust in glacial acetic acid and the carboethoxy-1-propenyl-2 group is preferably split off by treatment with formic acid. Obviously, other functionally equivalent protecting groups can also be used for the amino group, their use falling within the scope of the present invention.

 

   As suitable for reaction with the compound II functional derivatives of the acid III are, for. B. called the corresponding acid anhydrides, including the mixed anhydrides and especially the mixed anhydrides, which are available from stronger acids, such as the lower aliphatic monoesters of carbonic acid or alkyl and aryl sulfonic acids of sterically hindered acids, such as diphenylacetic acid. Furthermore, an acid azide or an active ester or thioester (for example with p-nitrophenyl, 2,4-dinitrophenyl, thiophenyl, thioacetic acid) can be used.

  The free acid itself can also be coupled with the compound II, after the free acid has first been reacted with N, N'-dimethylchloroformiminium chloride [compare British patent number 1 008 170 and Novak and Weichet, Experientia XXI, 6, 360, (1965 )] or by the action of enzymes or of an N, N'-carbonyldiimidazole or an N, N'-carbonylditriazole [see South African patent 63/2684] or of a carbodiimide reagent [especially N, N'-dicyclohexylcarbodiimide, N, N'- Diisopropylcarbodiimide or N cyclohexyl-N '- (2-morpholinoethyl) -carbodiimide, compare Sheehan and Hess, Journal of American Society 77, 1067 (1955)] or from an alkylamine reagent [compare R. Buijle and HG Viehe, Angewandte Chemie International Edition 3 , 582 (1964)] or an isoxazolium salt reagent [see RB Woodward, RA Olofson and H.

  Mayer, Journal of American Chemical Society 83, 1010 (1961)] or a ketenimine reagent [see C. L. Stevens and M. E. Mond, Journal of American Chemical Society 80, (4065)].



   Another equivalent of the acid III is a corresponding azolide; H. an amide of the acid in question, the amide nitrogen member of which is a quasi aromatic S-membered ring with at least two nitrogen atoms, such as. B. imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and their substituted derivatives. As an example of a general method for the preparation of an azolide, the reaction of N, N'-carbonyldiimidazole with a carboxylic acid in equimolar proportions at room temperature in tetrahydrofuran, chloroform, dimethylformamide or a similar inert solvent to form the carboxylic acid imidazolide in virtually quantitative yield with release may be given of carbon dioxide and one mole of imidazole. The carboxylic acids thus give diimidazolides.

  The resulting imidazole by-product precipitates and can be separated off and the imidazolide isolated, although this is not necessary.



  The method of carrying out this reaction for the preparation of a cephalosporin and the methods for isolating the cephalosporin are well known to those skilled in the art.



   The use of enzyme for coupling the free acid with the protected amino group with the compound II was mentioned above. It is within the scope of such methods to use an ester, e.g. B. the methyl ester, this free acid with enzymes such as are produced by various microorganisms, e.g. B. those described by T. Takahashi and coworkers in Journal of American Chemical Society, 94 (11), 4035-4037 (1972) and by T. Nara and coworkers, Journal of Antibiotics (Japan) 24 (5) 321 , 323 (1971).



   After the coupling reaction has been carried out and the protective group has been split off, the zwittesions obtained of the formula
EMI3.1
 if desired, are converted into the sodium dimethanesulfonate salt of the formula
EMI3.2

The conversion of the zwitterion-cephalosporanic acid into the dimethanesulfonate derivatives can take place in a manner known per se.



   A preferred method is to react the zwitterion with formaldehyde-sodium bisulfite (or a source of the latter such as sodium bisulfite and formalin) and water in the presence of a strong sodium base, e.g. B.



  Sodium 2-ethylhexanoate. Such a mixture is expediently warmed to above room temperature, e.g. B. to over 40 "C, preferably to 40 to 45" C, for a short period of time until the formation of the desired product has occurred. The substance falls out as a solid, e.g. B.



  by adding a water-soluble, essentially anhydrous, solvent, which is preferably an alcohol such as ethanol or isopropanol. If appropriate, the zwitterion products can also be converted into non-toxic pharmaceutically acceptable salts or into easily hydrolyzable esters by methods known per se.



   For the person skilled in the art it is obvious that the compounds of the formula I can alternatively also be prepared by first acylation of 7-aminocephalosporanic acid or a salt thereof with the desired 7-side chain acid or functional derivatives thereof, followed by the Replacement of the 3-acetoxy group by 6-mercaptotetrazolo [4,5-b] pyridazine. This alternative manufacturing method also falls within the scope of the present invention.



   The present inventive method is applied to a compound of the formula
EMI3.3
 or a salt or easily hydrolyzable ester thereof, produced by reacting 7-aminocephalosporanic acid or a salt thereof, with the thiol of the formula
EMI4.1
 or a salt thereof, preferably a sodium or potassium salt. If appropriate, the 7-amino-3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid thus obtained can be converted into a salt or into an easily hydrolyzable one by methods known per se Ester are converted. The replacement of an ester group, e.g. The 3-acetoxy group, the 7-aminocephalosporanic acid through a thiol group is in itself a well-known reaction and can be carried out in aqueous solution in the presence of a weak base such as sodium bicarbonate.

  This reaction is preferably carried out at a temperature in the range of about 50 to 100 "C.



   The 7-amino-3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid intermediate is found to be useful in the preparation of active 7-acylaminocephalosporins such as those of Formula I. .



   For the treatment of bacterial diseases in humans, the compounds which can be prepared according to the invention can be administered parenterally according to the usual methods for treatment with antibiotics in an amount of approximately 5 to 200 mg / kg / day and preferably 5 to 20 mg / kg / day, divided into several Cans, e.g. B. three to four a day. Appropriately they are administered in unit doses of z. B. 125, 250 or 500 mg of active substance with suitable physiologically compatible carrier substances or excipients. These dosage units can be used in the form of liquid preparations such as solutions or suspensions.

 

   The following examples illustrate the process according to the invention. All temperatures are in centigrades.



  7-Aminocephalosporanic acid is abbreviated as 7-ACA, where ACA describes the part of the following structure
EMI4.2
 and therefore 7-ACA can be written as
EMI4.3
 example 1
EMI4.4
   Preparation of 7-amino-3- (tetrazolo [4,5-b] pyridazine
6-ylthiomethyl) -3-cephem-4-carboxylic acid
EMI5.1
    7-Amino-3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (14)
I. 16.86 g = 0.062 mol of 7-ACA were carefully added to a solution of 9.56 g = 0.062 mol of substance 13 and 10.42 g = 0.124 mol of sodium bicarbonate in 300 ml of water, heated to 50 to 60 ° C. The mixture was heated to 80 to 85 ° C. for 30 minutes. 7 g of sodium bicarbonate were then added to the reaction mixture in order to dissolve the insoluble substance.

  The solution was treated with activated charcoal, filtered and the filtrate acidified to pH 5 with dilute hydrochloric acid. The resulting precipitate was filtered off, washed with water, dried in air and finally dried over phosphorus pentoxide in vacuo, 14.47 g (64%) of substance 14 being obtained.



  Melting point 238 to 250 C (decomposition).



   II. A stirred solution of 16.8 g = 0.11 mol of substance 13 and 18.48 g = 0.22 mol NaHCO> in one liter of 0.1N phosphate buffer (pH 6.4) was heated to 50.degree 30 g = 0.11 mol of 7-ACA were added to the solution in portions. The mixture was heated to 80 ° C. for 2.5 hours, during which time insoluble substance still remained. The reaction mixture was cooled to room temperature and the precipitated substance 14 was filtered off. It was washed thoroughly with 200 ml of water and dried in air Substance 14 was obtained from the filtrate and the washing solutions by acidification with dilute hydrochloric acid to pH 5. Total yield 32.9 g (83%), melting point 245 to 250 ° C. (decomposition).



   IR: v max 1800, 1615, 1538, 1360 cm.



   UV: 1 A. "HC03 237 nm (± 19,500), 275 nm (E 12,000), 310 nm (sh), (e 5700).



   NMR: <3 D2O + K2co3 3.35 (1H, d, 18 Hz, 2-H), 3.76 (1H, d, 18 Hz, 2-H), 4.00 (1H, d, 10 Hz, 3-CH2), 4.48 (1H, d, 10 Hz, 3-CH2), 4.93 (1H, d, 4 Hz, 6-H), 5.32 (1H, d, 4 Hz, 7- H), 7.46 (1H, d, 10 Hz, pyridazine-H), 8.18 (1H, d, 10 Hz, pyridazine-H).



  Analysis for C12HIIN703S2: Calculated: C 39.44 H 3.03 N 26.83 S 17.58% found: C 39.19 H 2.71 N 26.84 S 17.35% t-butoxycarbonylazide (15)
To a cooled solution of 100 g = 0.76 mol of t-butyl carbazate in 87 g of glacial acetic acid and 120 ml of water was added dropwise a solution of 60 g = 0.85 mol of sodium nitrite in 50 ml of water in the course of 40 minutes, the temperature was kept at 10 to 15 C. After the addition was complete, stirring was continued for 30 minutes at the same temperature. 100 ml of water were then added to the mixture and a separated oil was extracted with 5 portions of 100 ml of ethylene chloride each time. The combined organic extracts were washed with 100 ml of 10% sodium bicarbonate solution and then with 100 ml of water, followed by drying over anhydrous sodium sulfate.



  The methylene chloride was removed under reduced pressure on a water bath which was kept at 40 to 45 ° C. The remaining azide was distilled and obtained at 450 ° C./20 mm Hg. It weighed 92.7 g (84%).



     O- (t-butoxycarbonylaminomethyl) phenylacetic acid (16)
To a solution of 70 g = 0.35 mol of o-aminomethylphenylacetic acid hydrochloride (4) and 116 g = 1.15 mol of triethylamine (TEA) in 400 ml of water, a solution of 64 g = 0.45 mol of t-butoxycarbonylazide ( 15) in 3 ml of tetrahydrofuran (THF) was added at 0 ° C. with stirring.



  After the addition was complete, the temperature was allowed to rise to room temperature and stirred for an additional 20 hours. The tetrahydrofuran was distilled off at 40 C and the aqueous solution washed with 200 ml of ether, covered with 200 ml of ethyl acetate and acidified with dilute hydrochloric acid to pH 3 while cooling to 0 C.



  The organic layer was separated and the aqueous layer extracted with four 200 ml portions of ethyl acetate. The combined ethyl acetate extracts were washed with 200 ml of water, dried over anhydrous sodium sulfate and concentrated in vacuo. The concentrate was treated with 500 ml of n-hexane and gave 87.9 g (95%) of substance 16 as colorless needles which melted at 14 to 116.degree.



   2,4-Dinitrophenyl-o-t-butoxycarbonylaminomethyl phenyl acetate (17)
17.72 g = 0.086 mol of dicyclohexylcarbodiimide (DCC) were added in a proportion to a mixture of 22.73 g = 0.086 mol of o- (t-butoxycarbonylaminomethyl) phenylacetic acid (16) and 15.82 g = 0.086 mol 2, 4-Dinitrophenol (2,4-DNP) in 250 ml THF. The reaction mixture was stirred for two hours at room temperature. The precipitated dicyclohexylurea was filtered off and washed with 100 ml of THF. The filtrates and washing solutions were combined and concentrated under reduced pressure below 50 ° C. until a yellow viscous oil remained, which was driven off with 150 ml of n-hexane, the substance 17 being obtained in the form of yellow needles. Yield 34.9 g (94%), melting point 76 to 77 "C.



     IR AKmBarx 3340, 1785, 1685, 1610, 1540, 1530, 1500,
1340 cm-1.



  Analysis for C20H21N308: Calculated: C 55.68 H 4.91 N 9.74% found: C 55.70 H 5.05 N 9.93%
7- (o-t-Butoxycarbonylaminomethylphenylacetamido) 3- (tetrazolo [4,5- b] pyridazin-6-ylthiomethyl) -3-cephem
4-carboxylic acid (18)
A solution of 20.26 g = 0.047 mol of the substance 17 in
150 ml of tetrahydrofuran was added all at once to a solution of 14.40 g = 0.039 mol of substance 14 and 19.19 g =
0.19 mol of triethylamine in 150 ml of 50% strength aqueous tetrahydrofuran at 0 to 5 "C. The reaction mixture was stirred for 18 hours and then concentrated under reduced pressure and below 30 ° C. to remove the tetrahydrofuran.

  The aqueous concentrate was washed with two portions of 200 ml each of ether, acidified to pH 2 with dilute hydrochloric acid and extracted with five portions of 200 ml each of ethyl acetate. The combined extracts were washed with two 100 ml portions of water, dried over anhydrous sodium sulfate, treated with activated charcoal and filtered. Evaporation of the solvent gave a light yellow oil which, on trituration with ether, gave 13.89 g (58%) of substance 18. Melting point 166 to 1730 C (decomposition).



      IR: max 1770, 1710, 1690, 1535, 1370, 1255, 1170 cm Analysis for C26H28N806S2 H2O: calculated: C 49.51 H 4.79 N 17.77 S 10.17% found: C 49.58 H 4, 15 N 17.41 S 10.14%
C 49.65 H 4.59 N 17.66
7- (o-aminomethylphenylacetamido) -3- (tetrazolo- [4,5 -b] pyridazin-6-ylthiomethyl) -3 -cephem-4-carboxylic acid (20)
Substance 18 in an amount of 13.8 g = 0.022 mol was added to 20 ml of trifluoroacetic acid and the mixture was stirred for 45 minutes at 0 to 10 C.



  300 ml of ether were added to the reaction mixture, the substance 20 being formed as trifluoroacetate, which was filtered off and washed with ether. The trifluoroacetate was dissolved in 20 ml of water and the pH value was adjusted to 5 with ammonium hydroxide, the zwitterion being formed as a resinous oil which was decanted off and triturated with water. The solid product was filtered off, washed with 20 ml of water and 200 ml of acetonitrile and finally dried over phosphorus pentoxide in vacuo. This gave 5.10 g (45%) of substance 20, which was completely pure but amorphous.

 

   Recrystallization of the substance 20
The amorphous powder weighing 3.50 g was dissolved in 400 ml of 50% aqueous tetrahydrofuran with heating to 60 to 70 ° C. and vigorous stirring. The solution was treated with a small amount of activated charcoal. The filtrate was cooled, scraped with a Glass rod and left to stand in the refrigerator overnight, giving 2.27 g of substance 20 as fine needles which melted at 190 to 193 ° C. with decomposition.



  Analysis for C21H20N804S2 11/2 H2O calculated: C 46.74 H 4.30 N 20.77 S 11.89% found: C 47.18 H 4.08 N 20.93 S 12.03%
C 47.37 H 3.88 N 20.23 Example 2
EMI6.1
  
Sodium o- (1-ethoxycarbonyl-1-propen-2-ylaminomethyl) phenyl acetate (19)
To an alcoholic sodium ethoxide solution (prepared from 5.75 g = 0.25 gram atom of sodium and 500 ml of absolute ethanol) were added 41.26 g = 0.25 mol of o-aminomethylphenylacetic acid (obtained by neutralizing the hydrochloride 4 with aqueous ammonia) and 32 , 5 g = 0.25 mol of ethyl acetoacetate. The mixture was refluxed for 6 hours and then treated with activated charcoal, whereupon it was filtered through diatomaceous earth (Dicalite). The filter bed was washed with 200 ml of hot ethanol.

  The combined filtrates and washing solutions were evaporated to near dryness and cooled to 0 ° C., substance 19 being obtained as colorless needles. They were filtered off, washed with 200 ml of ethanol and dried in vacuo over phosphorus pentoxide. The solid substance 19 weighed 44.51 g. An additional amount of substance 19 was obtained by concentrating the mother liquor. Total yield 56.51 g (76%), melting point 231-232 ° C. (decomposition).



      7- (o-Aminomethylphenylacetamido) -3- (tetrazolo [4,5-b] - pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (20)
6.87 g = 0.0063 mol of ethyl chloroformate were added in one go to a stirred suspension of 17.44 g = 0.057 mol of sodium o- (l-ethoxycarbonyl-l-propen-2-yl-aminomethyl) -phenyl acetate (19) in 200 ml of dry tetrahydrofuran, which contained 1 ml of N, N-dimethylbenzylamine, at a temperature of - 150 C. Stirring was interrupted and a cooled solution of 20.80 g = 0.057 mol of substance 14 and 9.60 g = 0.095 mol Triethylamine in 200 ml of 50% strength aqueous tetrahydrofuran is allowed to flow slowly along the wall.

  The mixture was then stirred vigorously for 30 minutes at 0 to 15 ° C., treated with activated charcoal and filtered through Dicalite. The filter bed was washed with 50 ml of 50% strength aqueous tetrahydrofuran which contained 8 ml of triethylamine. 3 ml of formic acid were then added to the combined filtrate and the washing solutions to precipitate unreacted 7-ACA (2.5 g), which was filtered off. The filtrate was mixed with 200 ml of ether and additionally with 15 ml of formic acid. The mixture was stirred for 10 to 15 minutes at room temperature, after which the filtrate obtained was filtered off, washed successively with 100 ml of ether and 500 ml of water and finally brought to dryness in vacuo over phosphorus pentoxide.

  Yield 23.05 g (79%) of substance 20, melting point 180 to 186 ° C. (decomposition).



   Recrystallize from 20
The amorphous compound weighing 12.93 g was dissolved in 1.2 liters of 50% strength aqueous tetrahydrofuran at 50 to 60 ° C. with vigorous stirring, treated with 5 g of activated charcoal and filtered. The filtrate was seeded and kept in a refrigerator overnight, whereby 7.80 g of the substance 20 were obtained in the form of small needles. Melting point 186 to 1890 C (decomposition).



   Example 3
A suspension of the zwitterion form of 7- (o-amino methylphenylacetamido) -3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (0.361 g) in 3 ml of methanol was washed with ice cooled and treated with a few drops of concentrated hydrochloric acid until a clear solution resulted. The 7- (o-aminomethylphenylacetamido) -3- (tetra zolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid hydrochloride precipitates out as a light brown solid substance with the addition of ether and is filtered off and dried in vacuo over P2O5.



   Example 4
1N sodium hydroxide solution is added at room temperature to a stirred suspension of the zwitterion form of 7- (o-aminomethylphenylacetamido) -3- (tetrazolo [4,5-b] -pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (0.361 g) added until a clear solution of pH 10.8 is obtained. This
Solution is immediately freeze-dried and gives impure solid sodium 7- (o-aminomethylphenylacetamido) -3 (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate.



  Manufacture of dimethanesulfonate
Example 5
EMI7.1

EMI7.2
 (30% in acetone)
EMI7.3
 1. Warming up in water 2. Treating with activated charcoal and filtering 3. Add the filtrate to an anhydrous alcohol
EMI8.1
   + H2O + 2-ethyl-hexanecarboxylic acid
Procedure
Give 2.0 moles (about 1025 g, based on anhydrous substance) 7- (o-aminomethylphenylacetamido-3- (tetrazolo [4,5 -h] pyridazin-6-ylthiomethyl) -3 -cephem-4-carboxylic acid, 540 g Formaldehyde-sodium bisulfite (4.03 mol), 3000 ml of water and 2700 ml (4.87 mol) of 30% total sodium 2-ethylhexanoate in acetone in a suitable container and heat the mixture to 40 to 45 "C. while stirring Mixture dissolves to a yellow solution in approximately 10 minutes.



   After 15 minutes of heating, add 50 g of activated charcoal (Darco KB) to the solution and stir for another 15 minutes at 40 to 45 "C.



   Filter through diatomaceous earth (Dicalite) after heating the reaction mixture to 40 to 45 "C for a total of 30 minutes.



   Wash the filter cake with 2000 ml of 50% ethanol / water. Combine the filtrates, bring them to 25 C and add them at 25 C to 112 liters of vigorously stirred, 100% ethyl alcohol. A fine amorphous precipitate of disodium methanesulfonate of sodium 7- (o-aminomethylphenylacetamido) -3- (tetrazolo [4,5 -b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate precipitates out.



   Stir the suspension for about 10 minutes and then filter. Then wash the filter cake with 15 liters of 100% ethyl alcohol.



   Dry the cake at 50 to 55 "C in a drying cabinet with air circulation for two hours and then in a vacuum at 4 to 6 mm for 24 hours.



   The yield is approximately 1200 to 1400 g of amorphous white solid disodium methanesulfonate of sodium 7- (o aminomethylphenylacetamido) -3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate. The product usually contains several percent water and possibly a trace of ethanol.



   This product is also referred to as sodium 7 (o-N, N-bis (sodium sulfomethyl) aminomethylacetamido) -3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate.



   Example 6
The following slurry is made:
2.19 grams of formaldehyde sodium bisulfite (two equivalents).



  3.50 g of 7- (o-aminomethylphenylacetamido) -3 - (tetrazolo- [4,5- b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid zwitterion (0.15-0.075 mm mesh size). 25 ml of water (the volume can vary). 14 ml of 30% sat. Sodium ethyl hexanoate in isopropanol.



   Almost complete dissolution is achieved in about 1/3 hour rapid stirring at 24 C. The temperature of the mixture is rapidly increased to 40 to 43 "C. Here it is held for about 2 minutes and then rapidly cooled to 20 to 23" C. .



   The solution is filtered to remove insolubles (the total time the solution has existed should not exceed two hours).



   The pH 3 solution should be added over 5 minutes to 600 ml of very rapidly stirred absolute alcohol (other alcohols, such as anhydrous isopropanol, can also be used). It exhibits an amorphous substance, namely the disodium methanesulfonate of sodium 7- (o-aminomethylphenylacetamido) -3 - (tetrazolo [4,5 -b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate. The mixture is stirred for 5 minutes. The precipitate is filtered off, washed with 60 ml of ethanol (or isopropanol) and dried in vacuo at 50 ° C. for 24 hours. The yield is approximately 4.3 g.



   The product is soluble in water at around pH 7 in a proportion of at least 200 mg / ml. Such a solution is stable for at least two hours at room temperature; more dilute solutions are also stable for longer.



  The product shows the same antibacterial properties as the related zwitterion and is completely biologically active, regardless of whether it has been hydrolyzed back to the zwitterion or not.



   Example 7
Sodium 7- [o-N, N-bis (sodium sulfomethyl) aminomethylphenylacetamido] -3-tetrazolo [4,5- b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate
A. Manufactured using hydroxy methanesulfonate
A mixture of 1 g = 1.95 mmol of 7- (o-aminomethylphenylacetamido) -3 - (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid, 1.52 g = 10 mmol of sodium hydroxymethanesulfonate monohydrate, 6 ml = 6 mmol of an 1 molar solution of sodium ethylhexanoate in ethyl acetate, 10 ml of isopropanol and 10 ml of water was stirred at room temperature for 3 1/2 hours.

  The resulting solution was treated with 1 g of activated charcoal and poured into 300 ml of absolute ethanol with stirring, after which the mixture was stirred at room temperature for 30 minutes. A crystalline product was obtained which was filtered off. It was then washed three times with 50 ml of absolute ethanol each time and dried over P2O5 at 45 to 52 ° C / 1 mm for 20 hours. This gave 1.51 g of sodium 7 [ON, N-bis (sodium sulfomethyl) aminomethylphenylacetamido ] -3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate, which was easily soluble in water (more than 1 g per ml), melting point above 270 C.



     IR: v max: 1760, 1660-1620, 1605, 1540, 1400, 1200,
1040 cm-l.



  UV: RHa2 242 nm (e 18,000), 271 nm (± 11,600, sh), 312 nm (E 5,100, sh).



     ONLY: 8 pDp2m 3.34 (1H, d, 18 Hz, SCH2), 3.76 (1H, d, 18 Hz,
S-CH2), 3.85 (2H, s, CO-CH2-), 4.04 (4H, N-CH2- and 3-CH2-S), 4.30 (4H, s, N-CH2SO3Na) , 4.92 (1H, d, 4.5 Hz, 6-H), 5.42 (1H, d, 4.5 Hz, 7-H), 7.05-7.30 (4H, phenyl-H ), 7.40 (1H, d, 10 Hz, pyridazine-H), 8.11 (1H, d, 10 Hz, pyridazine-H).



   B. Preparation using formalin and
Sodium sulfite a) To a solution of 1 ml (10 mmol) of 30% formalin and 1 g of sodium bisulfite in 10 ml of water, 1.026 g = 2 mmol of 7- (o-aminomethylphenylacetamido) -3 tetrazolo [4,5-b] pyridazine were added in succession -6-ylthiomethyl) -3-cephem-4-carboxylic acid, 6 ml of an 1 molar solution of sodium ethylhexanoate and 10 ml of isopropanol were added. The mixture was stirred for 21/2 hours at room temperature and then poured into 300 ml of ethanol. The obtained sodium 7- [ON, N-bis (sodium sulfomethyl) aminomethylphenylacetamido] -3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3 - cephem-4-carboxylate was filtered off, washed with three times 50 ml each of ethanol and dried in vacuo. Yield 1.61g.



   b) To a mixture of 1.026 g = 2 mmol of 7- (o-aminomethylphenylacetamido) -3 - (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid, 6 ml of 1 molar sodium methylhexanoate, 10 ml of isopropanol and 10 ml of water were added to 1 ml = 10 mmol of 305Wiges formalin. The mixture was stirred for two hours at room temperature and gave a clear solution with a small amount of an oily precipitate. 1 g of sodium bisulfite was added and the solution was stirred for a further two hours, during which time the oily precipitate went into solution.

  The reaction mixture was poured into 300 ml of ethanol with vigorous stirring and gave 1.61 g of sodium 7 [ON, N-bis (sodium sulfomethyl) aminomethylphenylacetamido] -3- (tetrazolo [4,5-b] pyridazine-6 -ylthiomethyl) -3 cephem-4-carboxylate, which was filtered off, washed three times with 50 ml of ethanol each time and finally dried in vacuo.



   Example 8
A. 7- [ss- (o-t-Butoxycarbonylaminomethylphenyl) propionamido] 3- (tetrazolo [4,5- b] pyridazin-6-ylthiomethyl) -3-cephem4-carboxylic acid
0.41 g = 2 mmoles of N, N'-dicyclohexylcarbodiimide were added to a mixture of 0.56 g = 2 mmoles of ss- (ot butoxycarbonylaminomethylphenyl) propionic acid and 0.37 g = 2 mmoles of 2,4-dinitrophenol in 5 ml Tetrahydrofuran and the mixture was stirred at room temperature for one hour. The precipitated urea was filtered off. A solution of 0.73 g = 2 mmol of 7-amino-3 - (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid and 0.81 g = 8 mmol was added to the filtrate Triethylamine in 10 ml of 50% aqueous tetrahydrofuran, and the mixture was stirred at room temperature for 18 hours.



  The reaction mixture was washed with twice 20 ml of ether and the aqueous layer was acidified to pH 2 with dilute hydrochloric acid, followed by extraction with three times 50 ml of ethyl acetate. The combined extracts were washed with 30 ml of water, treated with activated charcoal, dried over anhydrous sodium sulfate and evaporated under reduced pressure until an oil remained. The oil was triturated with 50 ml of ether and gave the 7- [ss- (ot-butoxycarbonylaminomethylphenyl) propionamido] -3 - (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3 -cephem-4-carboxylic acid as a colorless solid substance, which was filtered off and dried in vacuo over P2O5. Yield 0.33 g (26%), melting point 110 to 1200 ° C. (decomposition).



     IR: 1780, 1710, 1690, 1530, 1370, 1250 cm- '.



     
1 NaHCO3 UV: 2 NaHC03 240 nm (± 19 400), 270 nm (sh) (e 12 500),
310 nm (sh) (e 5000).



   nMSO6 NMR: 6 "Oa6 1.37 (9H, s, t-Bu-H), 4.96 (1H, d, 4 Hz,
6-H), 5.55 (1H, d-d, 4 and 8 Hz, 7-H), 6.99 (4H, s,
Phenyl-H), 7.57 (1H, d, 10 Hz, pyridazine-H), 8.38 (I, d, 10 Hz, pyridazine-H), 8.69 (1H, d, 8 Hz, CONH) .



  Analysis for C27H30N606S2 1/2 H2O: calculated: C 1.02 H 4.91 N 17.63% found: C 51.74 H 4.83 N 17.88%
B. 7- [ss- (o-aminomethylphenyl) propionamido] -3 (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl)
3-cephem-4-carboxylic acid
0.5 ml of trifluoroacetic acid and 0.28 g = 0.46 mmol of 7- [ss (o-tert-butoxycarbonylaminomethylphenyl) propionamido] - 3-tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3 - cephem-4 carboxylic acid were mixed while cooling to 0 C and stirred for 30 minutes. 50 ml of ether were then added to the mixture, the trifluoroacetate of 7- [ss- (o aminomethylphenyl) propionamido] -3- (tetrazolo [4,5-b] -pyridazin-6-ylthiomethyl) -3-cephem-4 -carboxylic acid was formed.



  It was separated by decantation, washed with ether, dissolved in 1 ml of water and the pH of the solution adjusted to 6 with dilute ammonia. The 7- [ss- (o-aminomethylphenyl) -propionamido] -3- (tetrazolo- [4,5-b] pyridazine-6-ythiomethyl) -3-cephem-4-carboxylic acid obtained was filtered off and dried in vacuo over phosphorus pentoxide . Yield 0.10 g (42%), m.p. 190 to 1970 C (decomposition).



     IR: v max 1760, 1665, 1600, 1536 cm -1.



  Analysis for C22H22N804S2 1/2 H2O: Calculated: 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%
Example 9
A. Sodium ss- [o- (1-ethoxycarbonyl-1-propen-2 ylaminomethyl) -phenyl] -propionate
To an alcoholic sodium ethoxide solution, prepared from 5.75 g of 0.25 gram atom of sodium and 500 ml of absolute ethanol, 0.25 mol [o-aminomethylphenyl] propionic acid (obtained by neutralizing its
Hydrochloride with aqueous ammonia) and 32.5 g = 0.25 mol of ethyl acetoacetate. The mixture was refluxed for 6 hours and treated with activated charcoal, followed by filtration through diatomaceous earth (Dicalite). The filter bed was washed with 200 ml of hot ethanol.

  The combined filtrates and washing solutions were evaporated to near dryness and cooled to 0 ° C., sodium ss- [o- (l ethoxycarbonyl-l-propen-2-ylaminomethyl) phenyl] propionate being obtained as colorless needles. It was filtered off, washed with 200 ml of ethanol and dried in vacuo over P205. An additional portion was obtained by concentrating the mother liquor. Total yield 50 g.



   B. 7- [ss- (o-aminomethylphenyl) propionamido] -3 (tetrazolo [4,5- b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid
6.87 g = 0.0063 mol of ethyl chloroformate were added all at once to a stirred suspension of 0.057 mol of sodium ss- [o- (1-ethoxycarbonyl-1-propen-2-ylaminomethyl) phenyl] propionate in 200 ml of dry tetrahydrofuran , which contained 1 ml of N, N-dimethylbenzylamine, at a Tempe temperature of - 15 C. Stirring was interrupted and a cooled solution of 20.80 g = 0.057 mol of 7-amino-3- (tetra zolo [4.5- b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid and 9.60 g = 0.095 mol of triethylamine in 200 ml of 50% aqueous tetrahydrofuran allowed to flow slowly along the wall.

  The mixture was stirred vigorously for 30 minutes at 0 to 15 ° C., treated with activated charcoal and filtered through Dicalite. The filter bed was washed with
50 ml of 50% aqueous tetrahydrofuran containing 8 ml of triethylamine. 3 ml of formic acid were then added to the washing solutions combined with the filtrate in order to precipitate unreacted 7-ACA (2.5 g), which was then filtered off. The filtrate was mixed with 200 ml of ether and then with 15 ml of formic acid. The mixture was stirred for 10 to 15 minutes at room temperature and the resulting precipitate was filtered off. It was washed with 100 ml of ether and 500 ml of water and then im
Vacuum dried over phosphorus pentoxide.

  About 20 g of 7- [ss- (o-aminomethylphenyl) propionamido] - 3- (tetrazolo [4,5 -b] pyridazine-6-ylthiomethyl-3-cephem-4 carboxylic acid were obtained.



   Recrystallization of the 7- [ss- (o-aminomethylphenyl) propionamido] -3 - (tetrazolo [4,5 -b] pyridazin-6-ylthiomethyl) -3 - cephem-4-carboxylic acid. The amorphous product described above (13 g) was dissolved in 1.2 liters of 50% aqueous
Tetrahydrofuran at 50 to 60 C with vigorous stirring, be 10 15 20
25 lands with 5 g of activated charcoal and filtered. The filtrate was seeded and stored in a refrigerator overnight, where about 8 g of fine needles were formed.



   Example 10
A suspension of the zwitterion form of 7- [ss- (o-aminomethylphenyl) propionamido] -3 - (tetrazolo [4,5 -b] pyridazin6-ylthiomethyl) -3-cephem-4-carboxylic acid (0.361 g) in 3 ml of methanol was cooled with ice and treated with a few drops of concentrated hydrochloric acid until a clear solution resulted. The 7- [ss- (o-aminomethylphenyl) -propionamido] -3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid hydrochloride precipitates in a light brown form on addition of ether and can be filtered off and dried in vacuo over P2O5.



   Example 11
To a stirred suspension of the zwitterion form of 7- [ss - (o-aminomethylphenyl) propionamido] -3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (0.361 g) is added in sodium hydroxide solution at room temperature until a clear solution of pH 10.8 results.



  This solution is immediately freeze-dried and gives impure sodium 7- [ss- (o-aminomethylphenyl) propionamido] -3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3 - cephem-4- carboxylate.



  Manufacture of dimethanesulfonate
Example 12
EMI10.1

EMI10.2
 (30% in acetone)
EMI10.3
 1. Warming up in water 2. Treatment with activated charcoal and filters 3. Adding the filtrate to anhydrous alcohol
EMI10.4


<tb> <SEP> CH2N (CH2SO <SEP> 3Na) <SEP> 2
<tb> <SEP> 0 <SEP> 3
<tb> fixed <SEP> / \ CH2-CH2 - NH-cH-H <SEP> CH2 <SEP> II <SEP> N
<tb> <SEP> --N <SEP> y¸N
<tb> <SEP> OONa
<tb> + <SEP> H2O <SEP> + <SEP> 2-ethylhexanecarboxylic acid <SEP> COONa
<tb>
Procedure
Give 2.0 mol (about 1053 g, based on anhydrous substance) 7- [ss- (o-aminomethylphenyl) -propionamido] -3 (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3 -cephem- 4-carboxylic acid, 540 g formaldehyde sodium bisulfite (4.03 mol),

   Place 3000 ml of water and 2700 ml (4.87 mol) of 30% sodium 2-ethylhexanoate in acetone in a suitable vessel and heat with stirring to 40 to 45 ° C. The mixture dissolves into a yellow solution in about 10 minutes.



   After heating for 15 minutes, add 50 g activated charcoal (Darco KB) to the solution and stir for a further 15 minutes at 40 to 45 C. After the reaction mixture has been heated to 40 to 45 "C for a total of 30 minutes, filter through diatomaceous earth (Dicalite) .



   Wash the filter cake with 2000 ml of 50% ethanol / water. Combine the filtrates, bring them to 25 C and add the solution at 25 C to 112 ml of rapidly stirred 100% ethyl alcohol. A fine amorphous powder of disodium methanesulfonate of sodium 7- [ss- (o aminomethylphenyl) propionamido] -3- (tetrazolo [4,5-b] -pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate is formed .



   Stir the suspension for 10 minutes and then filter, wash the filter cake with 15 liters of 100% ethyl alcohol.



   Dry the cake at 50 to 55 "C in a circulating air oven for about two hours and then in the oven at 4 to 6 mm for 24 hours. The yield is about 1200 to 1400 g of amorphous white solid dimethanesulfonate of sodium 7 [ss - (o-aminomethylphenyl) -propionamido] -3- (tetrazolo- [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate The substance usually contains a few percent water and possibly a trace Methanol.



   The substance is also called sodium 7- {ss- [N, N bis- (natriumsulfomethyl) .aminomethylphenyl] -propionamido} -3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3- cephem-4-carboxylate.



   Example 13
The following slurry is produced:
2.19 grams of formaldehyde sodium bisulfite (2 equivalents).



   3.5 g of 7- [ss- (o-aminomethylphenyl) propionamido] -3 (tetrazolo [4,5- b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid zwitterion (0.15-0.075 mm screen mesh size).



  25 ml of water (the volume can vary).



   14 ml of 30% sodium 2-ethylhexanoate in isopropanol.



   If the mixture is stirred vigorously at 24 C for about half an hour, almost complete dissolution occurs. The temperature of the mixture is increased rapidly to 40 to 43 "C.



  This temperature is maintained for about two minutes, after which it is quickly cooled to 20 to 230 ° C.



   The solution is filtered to separate some insoluble matter (the total time of existence of the solution should not exceed two hours).



   The pH 3 solution is added over 5 minutes to 600 ml of very rapidly stirred absolute ethanol (other alcohols such as anhydrous isopropanol can also be used). An amorphous precipitate of the disodium methanesulfonate of sodium 7- [ss (o-aminomethylphenyl) propionamido] -3 - (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate is formed. The mixture is stirred for 5 minutes. The precipitate is then filtered off, washed with 60 ml of ethanol (or isopropanol) and dried in vacuo at 50 ° C. for 24 hours.



  The yield is approximately 4 g.



   The product is soluble in water at around pH 7 to the extent of at least 200 mg / ml. Such a solution is stable for at least two hours at room temperature. More dilute solutions are even more stable.



  The product shows the same antibacterial properties as the related zwitterion and is completely biologically active, regardless of whether it has been hydrolyzed back to the zwitterion or not.



   Example 14 Sodium 7- (ss- [oN, N-bis- (sodium sulfomethyl) -aminomethylphenyl] -propionamido} -3- (tetrazolo [4,5-b] -pyridazin-6-ylthiomethyl) -3-cephem-4 carboxylate
A. Manufacture Using
Hydroxymethanesulfonate
A mixture of 1.95 mmol of 7- [ss- (o-aminomethyl phenyl) propionamido] -3- (tetrazolo [4,5-b] pyridazin-6-yl-thiomethyl) -3-cephem-4-carboxylic acid, 1.52 g = 10 mmol sodium hydroxymethanesulfonate monohydrate, 6 ml = 6 mmol of a 1 molar solution of sodium 2-ethylhexanoate in ethyl acetate, 10 ml of isopropanol and 10 ml of water is stirred at room temperature for 31/2 hours. The resulting solution is treated with 1 g of activated charcoal and poured, with stirring, into 300 ml of absolute ethanol, whereupon the mixture is stirred at room temperature for 30 minutes.

  The resulting crystalline product is filtered off, washed three times with 50 ml of absolute ethanol and dried over P2O5 at 45 to 52 ° C./1 mm for 20 hours
1.5 g of sodium 7- {ss- [ON, N-bis (sodium sulfomethyl) amino methylphenyl] propionamido} -3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem -4-carboxylate, which is easily soluble in water (greater than 1 g / ml).



   B. Preparation using formalin and sodium bisulfite a) To a solution of 1 ml = 10 mmol and 30% formalin
1 g of sodium bisulfite in 10 ml of water is successively added 2 mmol of 7- [ss- (o-aminomethylphenyl) propionamido] 3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4- carboxylic acid, 6 ml of an 1 molar solution of sodium 2-ethylhexanoate and 10 ml of isopropanol. The mixture is stirred for 2 1/2 hours at room temperature and poured into 300 ml of ethanol. The obtained sodium 7- (P- [o-N, N-bis (sodium sulfomethyl) aminomethylphenyl] propionamido} -3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-ce Phem-4-carboxylate is filtered off, washed with three times
50 ml of ethanol and dried in vacuo. Yield about 1.5 g.



   b) To a mixture of 2 mmol of 7- [ss- (o-aminomethylphenyl) propionamido] -3 - (tetrazolo [4,5 -b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid, 6 ml 1 molar sodium 2-ethylhexanoate in ethyl acetate, 10 ml of isopropanol and
1 ml = 10 mmol of 30% formalin is added to 10 ml of water. The mixture is stirred for two hours at room temperature and gives a clear solution with a small proportion of an oily precipitate. After one hour, sodium bisulfite is added, the solution is stirred for a further two hours, during which time the oily precipitate dissolves in the solution.

  The reaction mixture is then poured into 300 ml of ethanol with vigorous stirring and gives about 1.5 g of sodium 7- {ss- [ON, N-bis- (natriumsulfomethyl) -aminomethylphenyl] -propionamido} -3- (tetrazolo- [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate.



  This is filtered off, washed with three times 50 ml of ethanol and dried in vacuo.



   Investigations in vitro and in vivo
7- [(o-Aminomethyl) -phenylpropionamido] -3- (tetrazolo [4,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid is a novel, semisynthetic, broad spectrum cephalosporin of the formula below
EMI12.1

This compound is shown to have excellent in vitro and in vivo activities against a wide variety of gram positive and gram negative bacteria, including bacteria which are resistant to cephalothin and cephaloridin. Its dimethanesulfonate adduct with the formula
EMI12.2
 can be converted into water-soluble preparations which can be injected and which are suitable for testing absorption and excretion, acute toxicity and pain tolerance during injection.



   Initial studies of the in vitro antibacterial effectiveness according to the tube dilution method or according to the agar dilution method of this new cephalosporin showed minimal inhibitory concentrations (MIC) of less than 1.0 mcg / ml against all or almost all strains of Staphylococcus aureus and Streptococcus pyogenes tested , Diplococcus pneumoniae, various types of bacillus, including Bacillus anthracis, mycoides and cereus, Klebsiella pneumoniae, Proteus rettgeri, Shigella and especially Shigella flexneri, Salmonella enteriiidis and Salmonella typhosa as well as MIC values usually of less than 4 mcg / ml and often less than 4 mcg / ml than 1.0 mcg / ml against various strains of Escherichia coli, Klebsiella unspecified, Proteus vulgaris, Proteus morganii, Proteus mirabilis and Eterobacter cloacae.



   The series of gram-negative test organisms comprised 14 strains of cephalothin-resistant Enterobacteriacae (3 E. coli, 6 Proteus, 1 Enterobacter, 1 Shigella and 3 Serratia), which were not inhibited by 100 mcg / ml cephalothin and in some cases were not inhibited by 100 mcg / ml cephaloridin and cephapirin. The compound prepared according to the present invention was remarkably active against these organisms and inhibited 12 of the 14 strains at concentrations of 6.3 mcg / ml or less.



   The MIC values of the new compounds were essentially not reduced in the presence of moderate concentrations of human serum, e.g. B. up to 50%. The compound in solution was highly stable at 37 C at pH values ranging from 4.6 to 8.4 with halfway times exceeding 24 hours.



   The in vivo efficacy of the new compound when injected subcutaneously was studied in mice with experimental infections caused by 10 pathogenic bacteria, including penicillinase-positive S. aureus and cephalosporinase-positive E. coli. The bacteria used were strains of S. aureus, S. pyogenes, D. pneumoniae, E. coli, K. pneumoniae, P. vulgaris and S. marcescens. The mean healing dose (CD50) was never above 20 mg / kg, only twice above 10 mg / kg and in 7 cases 3.2 mg / kg or less than a minimum of 0.2 mg / kg.

 

   The blood levels in mice after subcutaneous injection of the new compound in doses as low as 5 mg / kg were determined and showed excellent absorption. The urine recovery was good (about 60%) and paper chromatography of the urine indicated that the compound was the only bioactive substance present, indicating its metabolic stability.



   The acute toxicity tests by intravenous administration to mice at doses up to 1000 mg / kg revealed no deaths. Two tests, carried out to measure local tissue irritation as a result of the injection, showed no irritation at concentrations of 12.5% or less.



   PATENT CLAIM 1
Process for the preparation of antibiotically active compounds of the formula
EMI12.3
 

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



   

 

Claims (1)

** WARNING ** Beginning of CLMS field could overlap end of DESC **. EMI12.1 This compound is shown to have excellent in vitro and in vivo activities against a wide variety of gram positive and gram negative bacteria, including bacteria which are resistant to cephalothin and cephaloridin. Its dimethanesulfonate adduct with the formula EMI12.2 can be converted into water-soluble preparations which can be injected and which are suitable for testing absorption and excretion, acute toxicity and pain tolerance during injection. Initial studies of the in vitro antibacterial effectiveness according to the tube dilution method or according to the agar dilution method of this new cephalosporin showed minimal inhibitory concentrations (MIC) of less than 1.0 mcg / ml against all or almost all strains of Staphylococcus aureus and Streptococcus pyogenes tested , Diplococcus pneumoniae, various types of bacillus, including Bacillus anthracis, mycoides and cereus, Klebsiella pneumoniae, Proteus rettgeri, Shigella and especially Shigella flexneri, Salmonella enteriiidis and Salmonella typhosa as well as MIC values usually of less than 4 mcg / ml and often less than 4 mcg / ml than 1.0 mcg / ml against various strains of Escherichia coli, Klebsiella unspecified, Proteus vulgaris, Proteus morganii, Proteus mirabilis and Eterobacter cloacae. The series of gram-negative test organisms comprised 14 strains of cephalothin-resistant Enterobacteriacae (3 E. coli, 6 Proteus, 1 Enterobacter, 1 Shigella and 3 Serratia), which were not inhibited by 100 mcg / ml cephalothin and in some cases were not inhibited by 100 mcg / ml cephaloridin and cephapirin. The compound prepared according to the present invention was remarkably active against these organisms and inhibited 12 of the 14 strains at concentrations of 6.3 mcg / ml or less. The MIC values of the new compounds were essentially not reduced in the presence of moderate concentrations of human serum, e.g. B. up to 50%. The compound in solution was highly stable at 37 C at pH values ranging from 4.6 to 8.4 with halfway times exceeding 24 hours. The in vivo efficacy of the new compound when injected subcutaneously was studied in mice with experimental infections caused by 10 pathogenic bacteria, including penicillinase-positive S. aureus and cephalosporinase-positive E. coli. The bacteria used were strains of S. aureus, S. pyogenes, D. pneumoniae, E. coli, K. pneumoniae, P. vulgaris and S. marcescens. The mean healing dose (CD50) was never above 20 mg / kg, only twice above 10 mg / kg and in 7 cases 3.2 mg / kg or less than a minimum of 0.2 mg / kg. The blood levels in mice after subcutaneous injection of the new compound in doses as low as 5 mg / kg were determined and showed excellent absorption. The urine recovery was good (about 60%) and paper chromatography of the urine indicated that the compound was the only bioactive substance present, indicating its metabolic stability. The acute toxicity tests by intravenous administration to mice at doses up to 1000 mg / kg revealed no deaths. Two tests, carried out to measure local tissue irritation as a result of the injection, showed no irritation at concentrations of 12.5% or less. PATENT CLAIM 1 Process for the preparation of antibiotically active compounds of the formula EMI12.3 wherein n is 1 or 2, as well as non-toxic pharmaceutically acceptable salts and easily hydrolyzable esters thereof, characterized in that a compound of the formula EMI13.1 or a salt or an easily hydrolyzable ester of the latter is reacted with an acylation-capable derivative of an acid of the formula 3 CH23 (CII2) ¯C00fI III wherein B is a protected amino group to a compound of the formula EMI13.2 or a salt or an easily hydrolyzable ester of the latter, whereupon the protective group is split off with formation of the compound I or the corresponding salt or ester. SUBCLAIMS 1. The method according to claim I, characterized in that an acid halide or mixed anhydride of acid III is used as the acylating agent. 2. The method according to claim I and dependent claim 1, characterized in that the amino-protecting group is t-butoxycarbonyl or 1-carboethoxy-1-propenyl-2. 3. The method according to claim I, characterized in that the acylating agent is a compound of the formula EMI13.3 wherein n is 1 or 2 is used. 4. The method according to claim I and the dependent claims 1 and 2, characterized in that the amino-protecting group is split off by treatment with formic acid or trifluoroacetic acid. 5. The method according to claim I and the dependent claims 1 to 4, characterized in that in the form of the zwitterion of the formula EMI13.4 compound obtained by reacting with water, formaldehyde sodium bisulfite and a strong sodium base into the dimethanesulfonate sodium salt of the formula EMI13.5 <tb> <SEP> CH2I '<SEP> (Cli2 <SEP> SO <SEP> 3t) <SEP> 2 <tb> <SEP> 0 <tb> <SEP> II <SEP> n <SEP> o <tb> <SEP> 2n <tb> <SEP> Cli <SEP>) <SEP> -C-: ci --- t <tb> is transferred to <SEP>. <SEP> C- <SEP> $ T <tb> is transferred to <SEP>. <SEP> C <SEP> OOlTa <tb> 6. The method according to dependent claim 5, characterized in that sodium 2-ethylhexanoate is used as the strong sodium base. PATENT CLAIM II Application of the process according to claim I to a compound of formula II EMI14.1 or a salt thereof obtained by reacting 7-aminocephalosporanic acid or a salt thereof with a thiol of the formula EMI14.2 or a salt thereof is obtained.