FI57420B - FORM OF ANTIBACTERIAL 7-SUBSTITUTE 3- (3-HYDROXY-PYRIDAZINE-6-YLTIOMETHYL) -3-CEFEM-4-CARBOXYL SYROR - Google Patents

Description

rBl advertisement cn / nrs mjEgr · IBJ <11> UTLÄGGNINGSSKIUFT / 420 C (45) '^ v ^ (51) Kv.ik? /int.ci.3 C 07 D 501/36 FINLAND— "FINLAND (21) Ptwnttlhukemui - Patuntaiwdknlng 2677/73 (22) Hak «ml * pIWi - An * eknlngadtg 28.08.73 (23) AlkupUvl — GHtlgh * t * daf 28.08.73

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Patents and Registration Ameion utitgd och utUkriftun pubiieand 30.0 ^ .80 (32) (33) (31) Requested «tolird — Begird prlorltet 31.08.72 06.09.72, 06.09.72 USA (US) 28576U, 286630 286792 (71) Bristol -ityers Company, 3U5 Park Avenue, New York, NY , USA (US) (72) Takayuki Naito, Tokyo, Jun Okumura, Yokohama, Japan-Japan (JP) (7 * 0 Oy Kolster Ab (5 ^) Method for antibacterial 7-substituted 3- (3-hydroxypyridazin-6-ylthiomethyl) This invention relates to a process for the preparation of novel 3-thiol-7-carboxylic acids. The present invention relates to a process for the preparation of new 3-thiolated 7-substituted 7-substituent 7-cephem-7-carboxylic acids. to prepare ~-acylamidocephalosporanic acid derivatives in which the substituent in position 7 is o-aminomethylphenylacetamido, o-aminomethyl-p-hydroxyphenylacetamido or o-sminomethylphenylthioacetamido and the thiol group in position 3 is 3-hydroxypyridazin-6-y. NHo r = <* oo / \ "/ S \ x — L \ - (S) m-CH2-C-NH-CH-CH CHg v_ / II i C _» C-CHo-S- / Voh / \ = / 0 L · 2 57420 wherein X is H or OH and m is 0 or 1, provided that m is 0 when X is OH, and for the preparation of these non-toxic pharmaceutically acceptable salts.

These new compounds have been found to be valuable as tactile toxins, animal feed supplements, drugs for mastitis in cattle, and drugs for the treatment of infectious diseases caused by several Gram-positive and Gram-negative bacteria in poultry and other animals, including humans.

7-Phenylacetamidocephalosporanic acid, also called the N-phenylacetyl derivative of 7-ACA, cephaloram, PACA and apparently phenazporine, is well known in the scientific literature. Publications describing the preparation and / or properties of this compound, which may or may not have substituents on the benzene ring, and the corresponding compounds in which the 3-acetoxymethyl group has been replaced by methyl, hydroxymethyl and / or pyridinium methyl include: the following:

Chaivette, R.R., et al. "Chemistry of Cephalosporin Antibiotics II. Preparation of a New Class of Antibiotics and the Relation of Structure To Activity", Journal of the American Chemical Society, 8U, 3 * + 01-3U02 (1962).

Chauvette, R.R., et al. "Structure-Activity of Relationships Among 7-Acylamidocephalosporanic Acids", Antimicrobial Agents and Chemotherapy - 1962, 687-69 ^.

Cocker, J.D. et al., "Cephalosporanic Acids. Part II. Displacement of the Acetoxy Group by Nuckleophiles", Journal of the Chemical Society, 5015-5031 (196 5). Cocker, J.D. et al., "Cephalosporanic Acids. Part IV. 7-Acylamidoceph-2-em-it-carboxylic Acids", Journal of the Chemical Society, IIU2-H51 (1966).

Culp, H.W., et al., "Metabolism and Absorption of the 7_ (Phenylacetamido-1-C1-4) - Cephalosporanic Acid", Antimicrobial Agents and Chemotherapy - 1963, 2U3-2U6.

Jago, M., "Antibacterial Activity of Some Derivatives of 7 ~ Aminocephalosporanic Acid Against Staphylococcus Aureus and Synergism Between these and Other Antibiotics", Brit. J. Pharmacol., 22, 22-23 (196U).

Loder, B., et. al., "The Cephalosporin C Nucleus (7-Aminocephalosporanic Acid) and Some of Its Derivatives", Biochemical Journal, 79 »* + 08- ^ 16 (1961).

Nishida, M., et al., "Studies on Microbial Degradation of Cephalosporin C Derivatives. II", The Journal of Antibiotics, 21, 375-378 (1968).

Nishida, M., et al. "Studies of Microbial Degradation of Cephalosporin C Derivatives I", The Journal of Antibiotics, 21, 165-169 (1968).

Spencer, J.L., et al., "Chemistry of Cephalosporin Antibiotics VIII. Synthesis and Structure-Activity Relationships of Cephaloridine Analogues", Antimicrobial Agents and Chemotherapy - 1966, 573-580.

3,57420

Stedman, R.J. et al., "7-Aminodesacetoxycephalosporanic Acid and its Derivatives", J.Med.Chem., 7 (1), 117-119 (196 * 0 · Sullivan, HR, et al., "Metabolism of Oral Cephalothin and Related Cephalosporins in the Rat ", Biochemical Journal, 102, 976-982 (1967).

Vymola, F., et al., "The Classification and Characteristics of Cephalosporin Antibiotics I. Systematic Study of the Quantitative Sensitivity of Some Pathogenic Microorganisms to Cephaloridine", Journal of Hyviene, Epidemiology, Microbiology and Immunology, 10, I8O-189 (1966 ).

There are reports in the patent literature of many other 7-acyl derivatives of 7-aminocephalosporanic acid, e.g. 7-N- (o'-aminoalkyl) phenylacetamido] cephalosporanic acids (U.S. Pat. No. 3,382 21 + 1), 7- (N-aminophenylthio) acetamido] cephalosporanic acid (U.S. Pat. No. 3 ** 22,100), 7- (halo- phenylthioacetamido) cephalosporanic acids (U.S. Pat. No. 3,335,136), 7- (To-aminomethylphenylthio) acetamidocephalosporinic acid (DE Application No. 2,112,058), and a myriad of derivatives included in this general group without further specification, e.g. 69/02013 (Farmdoc 39 172).

7- (p-aminophenylacetamido) cephalosporanic acid and the corresponding N-triphenylmethyl derivative are disclosed in U.S. Patent 3 ** 22,103; see also JA Patent Publication 27 112/67 (Farmdoc 25 ** 06).

U.S. Patent No. 3,219,662 discloses compounds of the formula R-CH 2 -CCO-ACA wherein R is phenyl, nitrophenyl (especially para-nitro), chlorophenyl, alkylphenyl and alkoxyphenyl, and the corresponding phenoxy or substituted compounds and corresponding compounds in which the 3-acetoxymethyl group has been replaced by a 3-pyridinium methyl group. A broader group of these compounds, including a series in which R is phenylthio and compounds in which R is benzyl / so. 7- (N-phenylpropionamido) cephalosporanic acid7, alkoxybenzyl, alkanoyloxybenzyl, aminobenzyl, etc. are disclosed, at least in general, for use as starting materials in GB Patents 1,012,9 ** 3 and 1,153 1 + 21 (Farmdoc 23,900 **); see. see also GB 1 001 1 * 78 and U.S. Patent 3 280 118. GB 1 082 9 ** 3 and 1 082 962 disclose 7-phenylacetamidocephalosporanic acids having as substituents in the benzene ring as starting materials e.g. hydroxy and amino group.

U.S. Patent No. 3 3 * + 1,531 describes 7- (o-, m- and p-carboxyamidomethylphenylacetamido) cephalosporanic acids and their betaines.

U.S. Patent 3 ** 31,259 (Farmdoc 27,715) discloses several 7- (halo-, dihalo-, nitro- and halonitrophenylacetamido) cephalosporanic acids for use as starting materials for the reaction with certain nucleophiles. Additional 7- (phenylacetamido) cephalosporanic acids having various substituents on the benzene ring are. 57420 is described in the following patents: JA patent publication 2712/67 (Farmdoc 25 l + 06), JA patent publication 26 105/69 (Farmdoc 1 * 0 36o), GB patent publication 1 178 U71 (Farmdoc 27 715 »see NL patent publication 67 / 100906) and JA Patent Publication 25,785/69 (Farmdoc 1 * 0 81 + 7).

The replacement of the 3-acetoxy group of cephalosporin with various heterocyclic thiols is disclosed in a) ZA patent publication 70/2290 / ks. see also NL patent publication 70/05519 (Farmdoc 80i88r £ 7 »in which the side chains were, for example, 7-α-aminophenylacetamido groups and typical heterocyclic thiols were 2-methyl-1,3,1 + -thiadiazole-5-thiol and 1 -methyl-1,2,3,10-tetrazole-5-thiol, and b) U.S. Patent 3,516,997 »wherein the side chain structure of position 7

O O O

was R - (alk) m-CO-NH- and R -S- (alk) m-CO-NH-, where R was one of several aromatic heterocyclic groups and among the numerous heterocyclic thiols at position 3 were e.g. 1-methyltetrazole -5-thiol and 2-methyl-1,3,1 + -thiadiazole-5-thiol, and c) U.S. Patent 3,563,930 ·

Numerous cephalosporins, including cephalosporin C, have been occasionally reacted with nucleophilic, aromatic mercaptans to give compounds of formula

S

X \ CH

Acyl-NH-CH-CH 2 O 2 O = i - i --- c <^ - CB 2 -S-Al ·

loOH

In U.S. Pat. No. 3,278,531, Ar denotes phenyl or certain substituted phenyls or certain heterocyclic rings named, for example, in column 5. Similar nucleophiles, e.g. 2-mercapto-pyrimidines, are described in the following patents published to Glaxo: US-3,261 , GB-1 101 1 + 22, US-3 bl9 350 and US-3 502 665. A similar description is given in Ciba GB Patent 1,109,525, e.g. in defining R 1 + a corresponding "h". Additional nucleophiles of this type are described in the following Fujisawa patent publications: BF-711 + 518 (Farmdoc 35 307, NL-68/06129 and ZA-2695/68), CA-818 501 (Farmdoc 38 81 + 5), GB-1 187 323 (Farmdoc 31,936, NL-67/11 + 888) and in particular US-3,516,997 (Farmdoc 3I + 328, NL-68/01799), which contains a compound called cefazoline having a tetrazolylacetyl side chain attached to the 7-amino group and 5 ^ a methyl-thiadiazolylthiomethyl group in position 3, and to some extent described in the scientific literature.

5,574,220 in the literature, e.g., Antimicrobial Agents and Chemotherapy 1969 »American Society for Microbiology, Betesda, Maryland, p. 236-2U3 and J. Antibiotics (Japan) 23 (3), 131-11 + 8 (19T0).

More recent reports of replacing the 3-acetoxy group of a cephalosporin with various heterocyclic thiols are found in U.S. Patent 3,563,900 and NL 70/05519 (Farmdoc 80,188R), in which the side chains were, for example, 7-o-.-Aminophenylacetamido groups and typical heterocyclic 1,2,1-thiadiazole-5-thiol and 1-methyl-1,2,3,11-tetrazole-5-thiol, the latter corresponding to U.S. Patent 3,6k,021, issued February 2, 1972, filed 1969-0 ^ -18. Further similar disclosures are described in the following patents: US-3,563,983, BE-771,189 (Farmdoc 12,817.T), JA-72/05550 (Farmdoc 12,921T) and JA-72/0551 (Farmdoc 12,922T).

Various cephalosporins with the structure / \

Acyl-NH-CH-CH CH

1 I J

O = C-N d-CH 8 -S-alkyl 1

U00H

wherein acyl represents various side chains, including o-aminophenylacetyl, is described in some of the above-mentioned patents and in the following patents published to Glaxo: BE-73I + 532 (Farmdoc 1 + 1 619) and BE-731 + 533 (Farmdoc 1 + 1 620) and U.S. Pat. -3,668,203.

Cephalosporins having the structure

/OF

Acyl-NH-CH-CH CH 2,

1 1 I

0 = C — N C-CHO ^ \ o / s ^ o I »tl where X is mm. -SC- and -SC-, are described in some of the above-mentioned patents as well as in U.S. Patents 3,239,515 »3,239,516, 3 2U3 1 * 35, 3 258 1 * 61, 3 1 + 31 259 and 3 1 + 1 + 6 803.

6 57420

Related publications in the scientific literature include e.g. J.Med. Chem. 8, 17 ^ -181 (1965) and J.Chem.Soc. (London) 1595-1005 (1965), 5015-5031 (1965) and 1959-1963 (1967).

There are several reports in the literature on methanesulfonates of antibiotics. BE Patent 7b2 728 (Farmdoc 11h66R) discloses ampicillin monomethane sulfonate. U.S. Patent Nos. 3,268,508 and 3,295,264 disclose the conversion of one or more of the four free amino groups of kanamycin to a methanesulfonate derivative. Bacitracin sodium methanesulfonate is described in U.S. Patent No. 3,205,137 and neomycin in J. Antibiotics (Japan) 12, 11U-115 (1959). The Merck Index quotes Koyama et al., JA-1 + 898 (1957). Colistimethate is described in detail in U.S. Pat. Dispensatory 26th Edition, J.B. Lippincott Company, Philadelphia,

Penna, ss. 315-320. It is described as the pentasodium salt of the Penta (methanesulfonic acid) derivative of Colist A in U.S.P. AVU, ss. 621-622. U.S. Patent 2,599,950 provides the following Example VII: "Polymyxin Formaldehyde Sodium Bisulfite Compound."

In vitro and in vivo experiments with 7- (o-aminomethyl) phenylthioacetamide-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-1-carboxylic acid = compound A, 7- (o-aminomethyl-p- -hydroxy-phenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid = compound B and 7-Zp-aminomethyl) phenylacetamido-3- (3-hydroxypyrazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid = Compound C has excellent in vitro and in vivo activity against several Gram-positive and Gram-negative bacteria, including bacteria resistant to cephalothin and cephaloridin.

The dimethanesulfonate addition compound of these cephalosporins is prepared as a water-soluble preparation suitable for injection and for studying absorption and excretion, as well as acute toxicity and pain on injection.

When tested for in vitro bacterial activity by the tube dilution or agar dilution method, the minimum inhibitory concentration of all three compounds was less than 1.0 mg / ml for almost all or almost all Staphylococcus aureus, Streptococcus pyrogenes, Diplococcus pneumoniae, Bacillus anthracis, Proteus rettgella Against strains of Salmonella enteritidis and Salmonella typhosa. Compound C was further effective against all or almost all strains of Bacillus mycoides and Enterobacter clocae with a minimum inhibitory concentration τ 57420 of less than 1.0 mg / ml. The minimum inhibitory concentration was generally less than 1+ mg / ml and in some cases less than 1.0 mg / ml against Escherichia coli, Klebsiella pneumoniae, Proteus mirabillis, compound B and surprisingly compound C against strains of Proteus vulgaris and surprisingly compound C against Proteus morganii. .

Compound A was completely stable in solution at 3 ° C at pH 7> k and had a half-life of more than 2 h.

The in vivo efficacy of Compound A was studied by subcutaneous injection of the compound into mice infected with 9 pathogenic bacteria, including penicillinase-positive S. aureus and cephalosporin kinase-positive E. coli. The bacteria used were S. aureus, S. pyrogenes, E. coli, KL. pneumoniae-, P. vulgaris-, Pr. morganii- and Pr. strains of the species mirabilis. The CD? Concentrations in mouse blood, both subcutaneously and intramuscularly, at doses as low as 10 mg / kg showed good absorption.

The in vivo efficacy of Compound B was studied by subcutaneous injection of the compound into mice infected with two pathogenic bacteria, penicillinase-positive S. aureus and cephalosporinase-positive E. coli. The CD50 was in both cases less than 10 mg / kg.

In in vitro experiments with Compound C, Gram-negative organisms comprised 1 cephalothin-resistant Enterobacteriacae strain (3 E. coli, 6 Proteus, 1 Enterobacter, 1 Shigella and 3 Serratia strains) that were not inhibitory by 100 mg / ml cephalothin and sometimes also not 100 mg / ml cephaloridin and cefapyrin. Compound C was remarkably active against these organisms, inhibiting 9 strains from 1 + + strain at a dose of 6.3 mg / ml or less. The minimum inhibitory concentration of Compound C was not substantially reduced even in the presence of human serum (e.g., up to 50%). The solution of the compound at 3T ° C was very stable at pH U, 6-8 and the half-life was over 2k h.

The in vivo efficacy of Compound C was studied by subcutaneous injection of the compound into mice infected with 10 pathogenic bacteria, including penicillinase-positive S. aureus and cephalosporinase-positive E. coli. The bacteria used were strains of S. aureus, S. pyrogenes, D. pneumoniae, E. coli, K. pneumoniae, P. vulgaris and S. marcescens. The CD 2 -q was never greater than 75 mg / kg; in only three cases did CD, -0 be above 20 mg / kg and in six cases 5.0 mg / kg or less and the minimum was 0.5 mg / kg. Concentrations of the substance in the blood of mice given by subcutaneous injection at doses as low as 5 mg / kg showed excellent absorption; urinary recovery was good (50%) and urinary paper chromatographic examination showed that the compound was the only bioactive substance present that was metabolically stable. When Compound C was administered at a concentration of 12.5% or less, no tissue irritation was observed after injection.

The present invention relates to a process for the preparation of antibacterial 7-substituted 3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acids of the formula [CH2 ™ 2] = \? ' y_L) - (S) -CH -C-NH-CH-CH CHO // m I i - \ _ (J N Hv

C- N .C - CH - S 0 \\ -. OH

// 2 0 I

COOH j wherein X is H or OH and m is 0 or 1, provided that m is 0 when X is OH, and to prepare these pharmaceutically acceptable salts.

Non-toxic pharmaceutically acceptable salts referred to above include non-toxic carboxylic acid salts, including non-toxic metal salts such as sodium, potassium, calcium and aluminum trialkyl amine salts, ammonium salt and substituted ammonium salts of ammonium salts, e.g. , dibenzylamine, N-benzyl-beta-phenethylamine, 1-efenamine, Ν, Ν'-dibenzylethylenediamine, dehydroabietylamine, Ν, Ν'-bis-de-hydroabiethylethylenediamine, N- (lower) alkylpiperidine e.g. N-ethyl- piperidine and other amines used to form salts with benzylpenicillin; and non-toxic acid addition salts (i.e., amine salts including a mineral acid addition salt such as hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate, and phosphate, and organic acid addition salts such as maleate, acetate, citrate, acalate, acalate, succinate, succinate, succinate, succinate, succinate, succinate, succinate, succinate, succinate, succinate, succinate, succinate, succinate). etc. Highly preferred salts are the sodium methanesulfonate salts of the compounds of formula I. Due to their water solubility, these salts can be used in injectable compositions.

Compounds in which the amino group is protected by a substituent, such as tert-butoxycarbonyl, benzyloxycarbonyl, formyl, o-nitrophenylifulphenyl-, I, J *, J *, - trichloroethoxycarbonyl,? -Oxo-2-pentenyl- With 2,1-ethoxycarbonyl-1-propenyl-2, are intermediates or metabolic precursors used in the preparation. Such protecting groups include, in particular, ketones (especially 9,574,20 acetone) and aldehydes (especially formaldehyde and acetaldehyde), which are described, for example, in U.S. Patent Nos. 3,198,8ok and 3,300 + 7,851, and β-ketoesters and β-diketones, e.g. U.S. Patent No. 3,325-79 and IMcetoamides described in Japanese Patent Publication No. 71 / 2U71 (Farmdoc 1 + 7 321S).

The process according to the invention is characterized in that a compound of the formula / S χ H .N - CH-CH CH „2 I I 12 N-v is obtained.

xc - 1 ^ cxc-CH ^ -w> -0H

COOH 1Ί or a salt thereof for the reaction with an acylation derivative of the formula ch2nhb

X- (.) - (S) -CH0-COOH

// m 2 where B represents an amino protecting group and m and X are the same as above to give a compound of formula

CH2NHB

/ x 2 / \ X - (\ J- (S) -CH -C-NH-CH- CH CHO V / m \ _y n - n

Iv COOH

wherein B, X and m are the same as above, or a salt thereof, and then the amino protecting group is removed to give the desired compound of formula I or a pharmaceutically acceptable salt thereof.

, 0 5 74 20

Amphoteric compounds of formula I are prepared by attaching to the amino group of 7-amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid of formula II or a salt thereof an acylating reagent which is an acid of formula III or a functional group thereof. equivalent. After coupling, the amino protecting group B is removed to give the desired product. Amino protecting group B is a protecting group used either in peptide synthesis or in any of the numerous syntheses for the preparation of ampicillin or cephaloglycine or cefalexin from 2-phenylglycine. Highly valuable protecting groups include a proton as in the formula CH_NH0.HCl / 22 X -? Γ- (S) m -CH2-2-Cl or β-diketone or β-ketoester as in GB 1,123,333 and U.S. Pat. No. 3,325 1 * 79 and 3,316,211 *, e.g. methyl acetoacetate, or a ketoamide as in JA patent 71/2 ^ + 71 (Farmdoc 1 * 7 321S), in which case the acid containing the amino protecting group is most suitably converted to a mixed anhydride e.g. with ethyl chloroformate before the compound II or a salt thereof to give the desired product I after deprotection.

The coupling reaction is followed by deprotection to give the products of this invention, e.g. the tert-butoxycarbonyl group can be removed by treatment with formic acid or trifluoroacetic acid, the benzyloxycarbonyl group can be removed by catalytic hydrogenation, the 2-hydroxy-1-naphthocarbonyl group can be removed by trichlorolylation with zinc dust in glacial acetic acid and the 1-ethoxycarbonyl-1-propenyl-2-group is most preferably removed with formic acid. Of course, other functionally equivalent amino protecting groups may be used and such groups are considered to be within the scope of the invention.

ί 57420

With respect to the acylation reagent of formula III, functional equivalents include e.g. corresponding acid halides, including acid anhydrides, mixed anhydrides, and in particular mixed anhydrides prepared from stronger acids such as lower aliphatic monoesters of carboxylic acid or alkyl or aryl sulfonic acids and weaker acids such as diphenylacetic acid. In addition, an acid azide or an active ester or thioester (e.g., formed with p-nitrophenyl, 2, U-dinitrophenol, thiophenol, thioacetic acid) may be used or the free acid itself, as long as it has first reacted with N, N'-dimethylchloroforminium chloride, may be combined to compound II / see GB 1 008 170 and Novak, Weichet, Experientia XXI, 6, 360 (1965) 7 or the combination can be performed using enzymes or Ν, Ν'-carbonyldiimidazole or N, N'-carbonylditrazole / see. ZA Patent 63 / 268hj or carbodiimide reagent / especially N, N'-dicyclohexylcarbodiimide, Ν, Ν'-diisopropylcarbodiimide or N-cyclohexyl-N '- (2-morpholinoethyl) carbodiimide; see. Sheelan, Hess, J.Amer.Chem.Soc., 77, 1067 (1955) .7 or alkyllylamine reagent / see. R. Buijle, H. G. Vienhe, Angev Chem. International Edition 3, 582, (196H ^ 7 or isocheatolium salt reagent / see RB Woodward, RA Olofson, H. Mayer, J.Amer.Chem.Sbc., 83, 1010 (1961) 7 or keteneimine reagent / see. CL Stevens, ME Mond, J.Amer.Chem.Soc., 80,.

57420

The acid equivalent is furthermore the corresponding azolide, i.e. an amide of the corresponding acid having an amide nitrogen as a member in a quasi-aromatic ring containing at least two nitrogen atoms, which are imidazole, pyrazole, triazoles, benzimidazole, benzotriazole and substituted derivatives thereof. The azolid is prepared, for example, by reacting W, N'-carbonyldiimidazole equimolarly with a carboxylic acid at room temperature in tetrahydrofuran, chloroform, dimethylformamide or the like in an inert solvent to give a substantially quantitative amount of the carboxylic acid imidazole and liberate the carboxylic acid imidazolide. Dicarboxylic acids give rise to diimidazolide. The by-product, imidazole, precipitates and can be separated to leave the imidazolide, but this is not necessary.

Methods for performing wedding cephalosporin preparation reactions as well as methods for separating the resulting cephalosporin are well known.

the use of enzymes was mentioned above when it is desired to combine the amino-protected free Nappo with compound II. Such methods include the use of said free acid ester, e.g., net ester, with enzymes produced by various microorganisms, e.g., the enzymes in question have been described by T. Takahashi et al., Aner.Chem.3oc., 9 ^ (11), 1+ 035- 0 037 and T. Hara et al., J. Antibiotics (Japan) 2U (5), 321-323 (1971).

After the coupling reaction and deprotection, the compound of formula I can, if desired, be converted into its non-toxic pharmaceutically acceptable salts by known methods. Zwitterionic products of formula _W_. s / \

X— (y-CH2 C Nil-- | N-N

0 ^ CII2 ~ S -OH

in the positions X and m are the same as above, can be converted to their sodium salts of dimethanesulfonate of the formula 3,57420 x-^ / ~ ck0; i (ch 30, Ha).

/ £ 2? 3 2 COONa

Cephalosporanic acids in the zwitterionic form can be converted to dimethanesulfonate derivatives by known methods. It is preferred to react the zwitterion with sodium formaldehyde bisulfite (or a source thereof such as sodium bisulfite and formalin) and water in the presence of a strong sodium base, e.g. sodium 2-ethylhexanoate. This mixture is heated above room temperature, e.g. above i * 0 ° C, preferably at about U0-1.5 ° C, for the short time required to form a solution of the desired product. The product is recovered by stirring, e.g. by adding a water-soluble substantially anhydrous solvent, preferably an alcohol such as ethanol or isopropanol.

The starting material used in the process according to the invention is a compound of the formula y-fH-f SxfH2 »-h o ^ c-vcy-cvs - <^ j> -OH

COOH

or a salt of this compound. The compound of formula II can be prepared by reacting 7-aminocephalosporanic acid or a salt thereof with a thiol of formula

N-N

Π3- ^ ^ -OH

or a salt thereof, preferably a sodium or potassium salt, and, if desired, modifying the resulting 7-amino-3 * (6-hydroxypyridazin-3-ylthiomethyl) -3-cephem

1L

57420 L + carboxylic acid to a salt.

Replacement of an ester group of a 7-aminocephalosporanic acid, e.g. a 3-acetoxy group, with a thiol is a known reaction which can be carried out in aqueous solution in the presence of a weak base such as sodium bicarbonate. The substitution reaction is most conveniently carried out at a temperature of about 50-100 ° C.

A novel intermediate of formula II, 7-amino-3- (6-hydroxypyridazin-3-ylthiomethyl) -3-cephem-U-carboxylic acid, can be used to prepare active cephalosporin compounds of formula I or other potentially valuable novel cephalosporin derivatives.

It will be apparent to those skilled in the art that the compounds of formula I may alternatively be prepared by first acylating 7-aminocephalosporanic acid or a salt thereof with a side chain acylation reagent at position 7, followed by nucleophilic replacement of the 3-acetoxy group with 3-hydroxy-6-mercaptopyridazine, desired product.

In the treatment of bacterial infections in humans, the compounds of this invention are administered parenterally using standard methods and doses of antibiotic therapy in an amount of about 5 to 200 mg / kg / day, preferably about 5 to 20 mg / kg / day, and divided into the dose is accumulated, for example, 3 ~ h times a day from sub-doses. The treatment is carried out in single doses containing, for example, 125, 250 or 500 mg of active ingredient in admixture with suitable physiologically acceptable carriers or excipients. Single doses are in liquid form such as solutions or suspensions.

The following examples illustrate this invention. All temperatures are given in degrees Celsius. 7 ~ aminocephalosporanic acid is abbreviated as 7-ACA, -ACA- represents a group having the structure 15 57420 r »

-NH-CH - CH CH

i i i

c - nn .c - ch2-0 I

COOH

Example 1 - 7-Amino-3- (O-hydroxypyridazin-3-ylthiomethyl) -3-cephem-1-carboxylic acid

A mixture of 0.60 g (0.00 * + 7 moles) of 3 'hydroxy-6-mercaptopyridazine, 1.27 g (0.001 + 7 moles) of 7-aminocephalosporanic acid, 0.78 g (0.009 * + moles) ) sodium bicarbonate and 25 ml of 0.1 M phosphate buffer (pH 6.1 *) were heated at 60 ° C for 5 hours. After filtering off the remaining insoluble matter and adjusting the pH of the filtrate to 5 ° with acetic acid, a brown precipitate was obtained, which was collected by filtration, washed successively with water and acetone and dried in vacuo to give 1.03 g (71%) of 7-amino-3 - ( 6-hydroxypyridazin-3-ylthiomethyl) -3-cephem-1-carboxylic acid, m.p. 290-300 ° C (decomposes).

IR: y / '* 2? 1805, l680, 1650, 1580, lUl5 cm ”1

niEUCS

UV: λ max 8 + 9 nm (£ 19 * + 00) ruro L DoO-CoCO.

ppm δ 3.22 (1H, d, 19 Hz) »3.37 μl» d »^ Hz) * 3.65 (1H, d, 11 + Hz), 3.72 (1H, d, 19 Hz) 1+ .90 (1H, d, 1 + Hz), 5.30 (1H, d, 1 + Hz), 6.75 (1H, d, 10Hz), 7.30 (1H, d, 10Hz).

Analysis: C 12 H 12 W 2-1 / 2 H 2 ° calculated C 1 + 1.25 H 3.75 H 16.0k S 18.36 found C 1 + 1.1 + 5 H 3.70 N 15.83 S 18.03 .

Example 2 A. 7 '(2-tert-Butoxycarbonylaminomethyl-1 + -hydroxy-phenylacetamido) -3- (6-hydroxypyridazin-3-yl-thiomethyl) -3-cephem-1 + -carboxylic acid.

To a solution of 1.7 g (6 mmol) of 2-tert-butoxycarbonylaminomethyl-1 + -hydroxyphenylacetic acid, 1.2 g (6.6 mmol) of 2,1 + -dinitrophenol a and 1 + 0 ml of tetrahydrofuran were added in one portion 1.35 S (6.6 mmol) of dicyclohexylcarbodiimide, and the mixture was stirred for 1.5 hours at room temperature. The separated urea was filtered off. To the filtrate was added in one portion a solution of 2.0 g (5.9 mmol) of 7-amino-3- (6-hydroxypyridazin-3-ylthiomethyl) -16,574,20 3-cephem-4-carboxylic acid, 1.7 mL (ca. 12 mmol) of triethylamine and 30 ml of water. After stirring for 20 hours at room temperature, the mixture was washed with ethyl acetate (3 x 150 ml) and acidified with 6N hydrochloric acid to give a precipitate which was collected by filtration, washed with water (50 ml) and dried. The precipitate was dissolved in a liter of methanol at 50 ° C and the insoluble matter was filtered off. The filtrate was treated with activated carbon and evaporated to dryness. The residue was triturated with ether (50 ml) to give 920 mg (25%) of 7- (2-tert-butoxycarbonylaminomethyl-1-hydroxyphenylacetamido) -3- (6-hydroxypyridazin-3-ylthiomethyl) -3-cephem-H-carboxylic acid. , sp. 190-200 ° C (decomposes).

IR: ν max: 177 ° »17 ° °» 167 ° »15 ° °» 1250 °

Analysis: C 26 H 29 N 5 O 8 S 2 * H 2 O calculated C 50.23 II 5.03 Å 11.27 found C 50.63 HU, 69 μl 10.9¾ B. 7- (2-Aminomethyl-1 * -hydroxyphenylacetamido) -3- (6 -hydroxypyridazin-3-ylmethyl) -3-cephem-1-carboxylic acid 2 ml of trifluoroacetic acid and 850 mg (1.0 mmol) of 7- (2-tert-butoxycarbonylaminomethyl-k-hydroxyphenylacetamido) - The 3- (6-hydroxypyridazin-3-yl-thiomethyl) -3-cephem-4-carboxylic acid was stirred together at 0-5 ° C and the mixture was stirred for one hour. Dilution of the mixture with 150 ml of ether gave the desired cephalosporin, 7- (2-aminomethyl-k-hydroxyphenylacetamido) -3- (6-hydroxypyridazin-3-ylthiomethyl) -3-cephem-¾-carboxylic acid, trifluoroacetate, which filtered and suspended in 30 ml of water.

The pH of the suspension was adjusted to 6 with dilute ammonium hydroxide and diluted with one liter of acetonitrile with stirring. The solid was filtered off, washed with acetonitrile (3 x 10ϋ ml) and dried in vacuo to give 5¾ mg (77%) of 7- (2-aminomethyl-U-hydroxyphenylacetamido) -3- (6-hydroxypyridazine-3 -ylthiomethyl) -3-cephem-¾-carboxylic acid, m.p. 210 ° C (decomposes).

IR: ν max 1770 * 1660 »158 °, 1390 cm -1

Analysis: C 21 H 21 N 5 O 6 S 2 * 3H 2 O calculated C k 3.82 II k, 73 H 12.50 found C ¾ 3.85 H 2 28 N 13.12.

17 57420

Example 3 7- (o-Aminomethyl-p-hydroxy-phenyl-acetamido) -3 '- (3-hydroxy-pyridazin-6-yl-thiomethyl) -3-cephem-1'-carboxylic acid.

To a stirred suspension of 2.1 mmol of sodium o- (1-ethoxycarbonyl-1-propen-2-ylaminomethyl) -p-hydroxyphenylacetate, 5 ml of dry acetonitrile and one drop of N, Ν'-dimethylbenzyl amine, 0.25 g (2.3 mmol) of ethyl chloroformate are stirred at -15 ° C and stirring is continued for 20 minutes at -10 to 15 ° C. A solution of 0.71 g (2.1 mmol) of 7-amino-3 '(3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-1'-carboxylic acid, 0.21 g ( 2.1 mmol) of triethylamine, 3 ml of acetonitrile and 3 ml of water, and the mixture is stirred for one hour at 0 ° C. The reaction mixture is treated with a small amount of activated carbon and filtered. 0.5 ml of formic acid is added to the filtrate with shaking, and unreacted 7-amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-1 * carboxylic acid is filtered off from the mixture. 100 ml of acetonitrile are added to the filtrate and the mixture is allowed to stand for one hour at room temperature. The precipitate is collected by filtration, washed with 10 ml of water and dried in vacuo to give about 0.5 g of 7 '(o "aminomethyl-p-hydroxyphenylacetamido) -3- (3" hydroxypyridazin-6-ylmethyl) - 3 ~ cephem-U-carboxylic acid.

Example b 7-O-Aminomethyl-p-hydroxy-phenyl-acetamido-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-1 * -carboxylic acid

Into a 1 liter three-necked round-bottomed flask fitted with a drying tube, stirrer and thermometer, 0.08 moles of finely chopped potassium ro- (1-carbomethoxypropen-2-ylaminomethyl) -p-hydroxyphenyl acetate were added to 350 ml of dry tetrahydrofuran (THF) and a few drops of N , N-dimethylbenzylamine a. The suspension is stirred vigorously and cooled to -U0 ° C in an acetone dry ice bath. 13.6 g (0.06 mol) of isobutyl chloroformate are added to the suspension in one portion and the temperature is kept below -35 ° C during the addition.

After 2.5 minutes, the acetone-dry ice bath is removed and a solution of 0.036 mol of 7-amino-3- (3'-hydroxypyridazin-6-ylthiomethyl) -3-cephem-1 * carboxylic acid and 5.0 g (0.050 mol) of ) N-methylmorpholine in 250 ml of water. The reaction mixture is stirred until its temperature reaches room temperature (1.2 hours). The THF is removed under reduced pressure. The aqueous solution is layered with 100 ml of ethyl acetate and acidified with concentrated hydrochloric acid (pH 1.7 ~ 2.0). The mixture is treated with 3.5 g of activated carbon ("Darco KB") and filtered. The layers are separated and the aqueous phase is extracted with ethyl acetate (2 x 100 ml). The aqueous phase is filtered through filter paper and the pH is adjusted to U, 0. After scraping, the solution is left overnight at 5 "to 7 ° C to crystallize. The product is filtered off, washed with a small amount of water and dried in a vacuum desiccator. The yield of 7- (o-aminomethyl-p-hydroxyphenylacetamido) -3- (3-hydroxypyridazin-6-ylmethyl) -3-cephem-U-carboxylic acid is about 30%,

Example 5

A suspension of 0.361 g of J- (o-aminomethyl-p-hydroxyphenylacetamido) ~ 3 '(3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid in zwitterionic form in 3 ml of methanol is obtained, cooled in ice and cooled. drop in concentrated hydrochloric acid until a clear solution is obtained. Upon addition of ether, 7- (o-aminomethyl-p-hydroxyphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3 ** cephem-4-carboxylic acid hydrochloride precipitates as a light brown precipitate which is collected by filtration and dried in vacuo over PgO 2. above.

Example 6

To a stirred suspension of 0.361 g of 7- (o-aminomethyl-p-hydroxyphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid is added 1N aqueous sodium hydroxide solution at room temperature until a clear solution (pH 10.8). The solution is freeze-dried immediately to give a solid, crude sodium 7 '(o-aminomethyl-p-hydroxyphenylacetamido) -3- (3-hydroxypyridazin-6-ylmethyl) -3'-cephem-1-carboxylate. .

19 57420

Example 7 ΟΗΛ _ / = (_ I / s \ \ JT I I I2 * - «

--n \ V — 0H

c

COOH

F5 '+ HOCH ^ SO ^ Na + CH ^ CH ^ CH-COONa (30% SEH acetone solution) 1. Heat in water 2. Treat with activated carbon and filter ^ 3 · The filtrate is added to anhydrous alcohol CH2N (CH2SO3Na) 2 1 ^ S \ HO- (') - CH „-C-NH-CH - CH CH0„ „\ = / II I2 // \\

/ -N x CVS '\ __ y — 0H

COONa + HgO + Jc-ethylhexyl acid 20 57420

Procedure:

2.0 moles (about 1010 g calculated on the anhydrous basis) of 7- (o-aminomethyl-p-hydroxyphenyl) ethamido-3- (3-hydroxypyridazin-6-ylthiomethyl) -3'-cephem-U-carboxylic acid, 5.0 g of sodium formaldehyde bisulphite (U, 03 moles), 3000 ml of water and 2700 ml (?, 87 moles) of a 30% acetone solution of sodium 2-ethylhexanoate (SEH) in a suitable tank, and the mixture is heated to 1 * 0-1.5 ° C with stirring. After heating for 15 minutes, add 50 g of activated carbon ("Darco KB") and stir for a further 15 minutes at U0-1 * 5 ° C. After stirring the reaction mixture for a total of 30 minutes at L0-U5 ° C. It is filtered through diatomaceous earth ("Dicalite"), the layer is washed with 2000 ml of an ethanol-water mixture (50%), the filtrates are combined, the temperature is adjusted to 25 [deg.] C. and the solution is added at 25 [deg.] C. % ethanol Nitrium-7 '(o-aminomethyl-p-hydroxyphenylacetamido-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylate) the ylate di (sodium methanesulfonate) forms a fine white amorphous precipitate. The suspension is stirred for about 10 minutes, after which it is filtered and the precipitate is washed with 15 liters of 100% ethanol. The precipitate is dried in an oven at 50-55 ° C with circulating air for about 2 hours and then vacuum at a pressure of k-6 mm for 2k hours. About 1200-11 * 00 g of amorphous white solid sodium 7- (o-aminomethyl-p-hydroxyphenylacetamido-3- (3H-hydroxypyridazin-6-ylthiomethyl) -3'-cefen-carboxylate di (sodium ethanesulfonate) are obtained. The product usually contains several percent water and possibly residues from ethanol, also called sodium 7- / O-N, Libis (sodium sulfanethyl) aminomethyl-p-hydroxyphenylacetamido> 7 "3 ** (3-hydroxy-pyridazin-6-ylthiomethyl) ~ -3-cephem-U-carboxylate.

Example 8

Prepare a slurry containing 2.19 g (2 equivalents) of sodium formaldehyde bisulfite, 3.5 g of 7- (o-aminomethyl-p-hydroxyphenylacetamido) -3 '(3-hydroxypyridazin-6-ylthiomethyl) -3-cephem -L-carboxylic acid zwitterion (100-200 mesh), 25 ml of water (volume can be changed) and 10 ml of 30% SEH-isopropanol. Dissolution occurs almost completely when stirred vigorously for 0.5 hours at 2k ° C. The temperature of the mixture is rapidly raised to U0-U3 ° C. The temperature is maintained here for about two minutes, after which it is rapidly lowered to 20-23 ° C. The insoluble matter is removed by filtration (the total residence time should not exceed two hours). A solution with a pH of 7.3 is added over 5 minutes to 600 ml of very efficiently stirred absolute ethanol (21,574,420) (other alcohols such as anhydrous isopropanol can also be used). Sodium 7- (o-aminomethyl-p-hydroxyphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3'-cephem-U-carboxylate di (sodium methomethyl sulfate) is formed as an amorphous as a precipitate. The mixture is stirred for 5 minutes. The precipitate is filtered off, washed with 60 ml of ethanol (or isopropanol) and dried for 2 hours at 50 ° C under vacuum. The yield is about U, 3 g). The solubility of the product in water at about pH 7 is at least 200 mg / ml. Such a solution remains at room temperature for at least 2 hours, more dilute solutions are more stable. The product has the same bacterial spectrum as the corresponding zwitterion and is fully biologically active whether it is hydrolyzed back to the zwitterion or not.

Example 9

Sodium 7-O-N, N-bie (sodium sulfomethyl) aminoastyl p-hydroxyphenylacetamido-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate.

Preparation of hydroxymethane sulfonate.

A mixture of 2.05 mmol of 7- (o-aminomethyl-p-hydroxyphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid zwitterion, 1.56 g ( 10 mmol) of sodium hydroxymethanesulfonate monohydrate, 6 ml (6 mmol) of a 1 M solution of SEH in ethyl acetate, 10 ml of isopropanol and 10 ml of water are stirred at room temperature for 3.5 hours. After treatment with 1 g of activated carbon, the mixture is poured into 300 ml of absolute ethanol with stirring and stirring is continued at room temperature for 30 minutes to give a crystalline product which is filtered off, washed with absolute ethanol (3 x 50 ml) and dried at 2 ° C. »5" to 52 ° C / 1 mm for 20 hours to give about 1.5 g of sodium 7-N, N-bis (sodium sulfomethyl) aminomethyl-p-hydroxyphenylacetamide-3 - ((3'-hydroxypyridazin-6-ylthiomethyl) ) -3-cephem-1 * -carboxylate which is readily soluble in water ("> 1 g / ml).

Example 10

Sodium r-7β-N, N-bis (sodium sulfomethyl) aminomethyl-p-hydroxyphenylacetamido? -3- (3'-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylate. Preparation using formalin and sodium bisulphite.

(a) To a solution of 1 ml (10 mmol) of 30% formalin and 1 g of sodium bisulfite in 10 ml of water is added successively 2 mmol of 7- (o-aminomethyl-p-hydroxyphenyl acetamido) -3- (3-hydroxypyrrole). data in-6-yl thiomethyl) -3-cephem-β-carboxylic acid zwitterion, 6 ml of 1 M SEH solution and 10 ml of isopropanol. The mixture is stirred for 2.5 hours at room temperature, after which it is poured into 300 ml of 22,574,420 ethanol. The resulting sodium, N, N-bis (sodium urazomethyl) aminomethyl p-hydroxy-8-phenylacetamido-3- (3-hydroxypyridazin-6-ylthiomethyl-3-cephem-U-carboxylate is filtered off and washed with ethanol (3 x 50 ml) and dried in vacuo to give a yield of about 1.5 g.

(b) To a mixture of 2 mmol of 7 "(o-aminomethyl-2-hydroxyphenylacetylamino) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem- U-carboxylic acid kha or s-ion, 6 ml of 1 M ethyl acetate solution, 10 ml of isopropanol and 10 ml of water, 1 ml (10 mmol) of 30% formalin are added, and after stirring for 2 hours at room temperature, a clear solution is obtained. with a slight oily precipitate.

When 1 g of sodium bisulfite is added and stirred for a further 2 hours, an oily precipitate dissolves. Pouring the reaction mixture into 300 ml of ethanol with vigorous stirring gives about 1.8 g of sodium 7-β-β, N-bie (sodium sulfomethyl) aminomethyl-p-hydroxyphenylacetamide-7-3 ”(3-hydroxypyridazine-6- ylthiomethyl) -3-cephem-U-carboxylate as a precipitate which is collected by filtration, washed with ethanol (3 x 50 ml) and dried in vacuo.

Example 11 A. 7- (o-tert-Butoxycarbonylaminomethyl-phenylthioacetamide) -3- (3-hydroxypyridazin-6-yl-thiomethyl) -3-cephem-1'-carboxylic acid.

To a solution of 2.19 g (U, 7 mmol) of 2,4-dinitrophenyl (o-tert-butoxycarbonylaminomethylphenylthio) acetate in 50 ml of tetrahydrofuran was added at 0 ° C a solution of 1.02 g ( 5 mmol) -7-amino-3- (3-hydroxypyridatein-6-ylthiomethyl) -5-cephem-4-carboxylic acid, 50 ml of triethylamine, 50 ml of tetrahydrofuran and 20 ml of water. The reaction mixture was stirred at room temperature for 17 hours, after which it was treated with activated carbon and filtered. The tetrahydrofuran was evaporated in vacuo and the concentrate was washed with ether (5x100 ml). The aqueous solution was layered with 100 ml of ethyl acetate and acidified (pH 2) with dilute hydrochloric acid at 0 ° C with vigorous stirring. The aqueous layer was extracted with ethyl acetate (5 x 100 mL), the organic layer was combined with ethyl acetate extracts, dried over anhydrous sodium sulfate and treated with activated carbon. Evaporation of the filtrate under reduced pressure at 30 ° C gave an oily product which was triturated with 100 ml of ether to give 7- (o-tert-butoxycarbonylaminomethylphenylthioacetamido) -3- (β-Hydroxypyridate-6-ylthionethyl) -3-cephem. -4-kerboxylic acid as a pale yellow solid. The precipitate was collected by filtration and washed with 100 ml of ether to give 0.71 g of 23,57420 (38%) of 7 '(o-tert-butoxycarbonylaminomethylphenylthioacetamido) -3- (3'-hydroxypyridazin-6-ylthiomethyl) -3' U-cephem-carboxylic acid.

B. 7 '(o-Aminoethyl phenylthioacetamido) -3' (3-hydroxypyridazin-6-yl-thiomethyl) -3-cephem-U-carboxylic acid 0.50 g (0.8 mmol) of 7- (o-tert. -butoxycarbonylaminomethyl phenylacetamido-3- (3- (3-hydroxypyridazin-6-ylthiomethyl) -3'-cephem-U-carboxylic acid was mixed in one portion with 3 ml of trifluoroacetic acid at 0 ° C. Stirring was continued for one hour at 0-5 ° C. Addition of 100 mL of ether gave a white precipitate. The precipitate was collected by filtration and suspended in U ml of water at room temperature. The pH of the suspension was adjusted to 5 with dilute ammonium hydroxide. When the precipitate was collected, 7 - (o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid was obtained as a brown powder which was washed successively with water and acetone. and dried in vacuo over phosphorus pentoxide. The product weighed 220 mg (52%), m.p. 200-210 ° C (decomposes).

IR: 1770, I670, 1580, 1210 cm -1

1% NaHCO

λ max 250 nm (6 20800), 270 nm (1 »* 900)

Analysis for C 21 H 21 N 5 O 5 S 3 * 1/2 H 2 ° calculated C 1 H, 67 H U, 6U N 12, U0 S 17.0U found C UI *, 1 * 3 H 3.85 N 10.8U S 17.89.

Example 12 A. 7 “(o-tert-Butoxycarbonylaminomethylphenylthioacetamido) -3” (3 “hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid To a cooled solution of 1.85 g (U mmol) 2 , U-dinitrophenyl (o-tert-butoxycarbonylaminomethylphenylthio) acetate (prepared as in U.S. Patent No. 3,657,232) in 50 mL of tetrahydrofuran was added at 0 ° C to a solution of 1.02 g (3 mmol) of 7- amino-3- (3-hydroxypyrid-4-azin-6-ylthiomethyl) -3-cephem-U-carboxylic acid and 0.85 ml (about 6 mmol) of triethylamine in a mixture of 30 ml of tetrahydrofuran, 30 ml of acetonitrile and 50 ml of ml of water. The temperature of the mixture was allowed to rise slowly to room temperature, after which the mixture was stirred for 12 hours. The reaction mixture was treated with a small amount of activated carbon and filtered. The organic solvent was evaporated from the filtrate in vacuo below 35 ° C. The resulting aqueous solution was washed well with ether (U x 30 mL), covered with 50 mL of ethyl acetate, and diluted hydrochloric acid was stirred at 0-5 ° C to lower the pH to 3. The precipitate that appeared between the layers was filtered off and collected. The precipitate weighed 0.6 g. This was the desired 7- (o-tert-butoxycarbonylaminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid. Additional product was obtained from the two-layer filtrate. The aqueous layer was extracted with ethyl acetate (3 x 100 mL). The organic layer was combined with organic extracts, dried over anhydrous sodium sulfate and evaporated to dryness. The residue was washed well with 100 ml of ether and dried in vacuo over phosphorus pentoxide. The product weighed 0.1 * 2 g. A total of 1.02 g (55%) of 7 '(o-tert-butoxycarbonylaminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-1'-carboxylic acid was obtained, m.p. 150-160 ° C (decomposes).

IR: ^ Sks 1770 »1690» 1590 · 1535 »1175 a *" 1 UV: Λ1 * NeHCO 3 250 nm (i 21700), 270 nm (sh) (£ 15,500) mus

Analysis: Calculated for C 11 H 9 O 3 .1 / B H 9 O C 1 * 9.67 HU, 97 H 11.13 S 15.30 Found C 1 * 9.95 H 1 *, 60 N 10.70 S 15.52 .

B. 7- (o-Aminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-yl-thiomethyl) -3'-cephem-1-carboxylic acid, ppo A mixture of 0.96 g (1.56 mmol) butoxycarbonyl-protected cephalosporin, 7 '(o-tert-butdecycarbonylaminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthionethyl) -3-cephem-U-carboxylic acid, and 1 * ml of trifluoroacetic acid, one hour. Addition of 100 mL of ether gave a white precipitate which was collected and washed with 100 mL of ether. When the precipitate was suspended in 10 mL of water and the pH was adjusted to 5 with dilute ammonia to give the desired cephalosporin, 7- (o-aminomethylphenylthioacetamido) - 3- (3-Hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid, which was collected by filtration, washed with 50 ml of acetonitrile and dried in vacuo. The yield was 0.1 * 6 g (56%)> The product was dental (IR) with the product obtained in Example 11.

Example 13 7- (o-Aminomethyl-phenylthioacetamido) -3- (3-hydroxypyrimidin-6-ylthiomethyl) -3-cephem-U-carboxylic acid

To a stirred suspension of 2.1 mmol of sodium o- (1-ethoxycarbonyl-1-propen-2-ylaminomethyl) phenylthioacetate, 5 ml of dry acetonitrile and one drop of Ν, Ν-dimethylbenzylamine is stirred at -15 ° C. in 0.25 g (2.3 mmol) of ethyl chloroformate and stirring is continued for 20 minutes at -15 ° C.

25 57420

To the mixture is added in one portion a solution of 0.71 g (2.1 mmol) of 7-amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid and 0.21 g (2.1 mmol) of mmol) of triethylamine in 3 ml of acetonitrile and 3 ml of water, and the mixture is stirred for one hour at 0 ° C. The reaction mixture is treated with a small amount of activated carbon and filtered. 0.5 ml of formic acid is added to the filtrate and the mixture is shaken and the unreacted 7-amino-3- (3-hydroxypyrimidin-6-ylthiomethyl) -3-cephem-U-carboxylic acid is filtered off. 100 ml of acetonitrile are added to the filtrate and the mixture is allowed to stand at room temperature for one hour. When the precipitate is collected by filtration, washed with 100 ml of water and dried in vacuo, 0, 7 g of 7 - (o-aminomethylphenylthioacetate) -3 "(3" hydroxy hydroxide) are obtained. -yl) thiomethyl) -3-chem-U-carboxylic acid.

„Example 1¾ 7-O-Aminomethylphenylthioacetamido-3- (3-hydroxypyridazin-6-yl-thiomethyl) -3-cephem-1-carboxylic acid

To a 1 liter three-necked round bottom flask fitted with a drying tube, stirrer and thermometer is placed 0.08 moles of finely chopped potassium o- [1-carbomethoxypropen-2-ylaminomethyl] phenylthioacetate in 350 ml of dry tetrahydrofuran with U drops of N, N-dimethyl. The suspension is stirred vigorously and cooled to -U0 ° C in an acetone dry ice bath. 13.6 g (0.06 mol) of isobutyl chloroformate are added in one portion to the suspension and the temperature is kept below -35 ° C at all times. After 2.5 minutes, the acetone-dry ice bath is removed and a solution of 0.036 mol of 7'-amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid and 5.0 g (0.050 mol) of ) N-methylmorpholine in 250 ml of water. The reaction mixture is stirred until its temperature reaches room temperature (1-2 hours). Tetrahydrofuran is removed under reduced pressure. The aqueous solution is layered with 100 ml of ethyl acetate and acidified (pH 1.7 ~ 2.0) with concentrated hydrochloric acid. The mixture is treated with 3.5 g of activated carbon ("Darco KB") and filtered. The layers are separated and the aqueous layer is extracted with ethyl acetate (2 x 100 mL). The aqueous layer is filtered through filter paper and the pH is adjusted to U, 0. After scraping, the solution is left overnight at 5 ~ 7 ° C to crystallize. The product is filtered off, washed with a small amount of water and dried over a vacuum desiccator, the yield of 7-o-aminomethylphenylthioacetamido-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-1'-carboxylic acid is about 30%.

26 57420

Example 15

A suspension of 0.3 g of 7- (o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid in zwitterionic form in 3 ml of methanol is cooled in ice and concentrated by dropwise addition. hydrochloric acid until a clear solution is obtained. Upon addition of ether, 7- (o-aminomethyl-phenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-1 * -carboxylic acid hydrochloride precipitates as a light brown solid which is collected by filtration and dried over P 2 O 3. tllä.

Example 16

To a stirred suspension of 7- (o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid (0.361 g) is added 1N aqueous sodium hydroxide solution at room temperature until a clear solution (pH 10.8) is formed. ). Immediate freeze-drying of this solution gives crude sodium 7- (o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylate a.

57420 27

Preparation of dimethanesulfonate

Example 17 CH_NH-A i (^ jA ~ s-ch2-Lh-ch-A Xch2

C-N / C-CH_-S - (/ \\ - OH

/ ύ

COOH

P

+ HOCHgSO 4 Na + CH 2 CH 2 ^ CH-COONa (30j 30% SEH acetone solution) 1. Heat in water 2. Treat with activated carbon and filter 3. Add the filtrate to anhydrous alcohol v CHgNiCHgSO4 aJg / r ~ i X ^ s \

Solid // \\ - S-CH-C-NH-CH-CH CH_ \ = / I I I / A {\

.C-N .C-CH -S </ N) —OH

/ 2 \ = / COONa + HgO + o ^ -ethylhexyl acid 28____ 57420

Procedure:

2.0 moles (about 1020 g on the anhydrous basis) of 7 * (o-eminomethyl-phenylthioacetamido-3 '(3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid, 5U0 g of sodium formaldehyde bisulphite (U, 033) are added. moles), 3000 ml of water and 2700 ml (1+, 87 moles) of a 30% solution of SEH (sodium 2-ethylhexanoate) in acetone in a suitable tank and the mixture is heated to U0-U5 ° C with stirring. After heating for 15 minutes, 50 g of activated carbon ("Darco KB") are added and stirring is continued for a further 15 minutes at U0-1 * 5 [deg.] C. After the reaction mixture has been stirred at U0-U5 [deg.] C. for a total of 30 minutes, it filtered through diatomaceous earth ("Dicalite"), the carbon layer is washed with 2000 ml of a 50% ethanol-water mixture, the filtrates are combined, the temperature is adjusted to 25 ° C and the solution is added at 25 ° C to 112 liters of vigorously stirred 100% ethanol. 7 "(o aminometyylifenyylitioasetamido) -3 '(3-hydroxy-pyridazin-6-ylthiomethyl) -3" U-cephem-carboxylase di (sodium methanesulfonate) as a fine, white, amorphous precipitate. The suspension is stirred for about 10 minutes, after which it is filtered and the elderly is washed with 15 liters of 100% ethanol. The precipitate is first dried in an oven at 50-55 ° C with circulating air for 2 hours and then under vacuum (U-6 mm) for 2 hours. About 1200-100 g of amorphous white solid sodium 7- (o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylate di (sodium methanesulfonate) are obtained. The product usually contains several percent water and possibly some ethanol. The product is also called sodium 7 ', β-N, N-bis- (sodium sulfomethyl) aminomethyl phenylthioacetamide] -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylate.

Example 18

Prepare a slurry of 2.19 g (2 equivalents) of sodium formaldehyde-1-bisulfite, 3.5 g of 7- (o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephemurecarboxylic acid as a zwitterion (100 -200 mesh), 25 ml of water (volume can be changed) and 10 ml of 30% SEH isopropanol solution. When stirred vigorously for about 0.5 hours at 2 ° C, dissolution occurs almost completely. The temperature of the mixture is rapidly raised to U0-U3 ° C. The temperature is maintained here for about 2 minutes, after which it is rapidly cooled to 20-23 ° C. The insoluble matter is removed from the solution by filtration (the total residence time should not exceed two hours). The solution (pH 7 * 3) is added over 5 minutes to 600 ml of very well stirred absolute ethanol (other alcohols such as isopropanol can be used). Sodium 7- (o-allylmethylphenylthio-) 25- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate di (sodium methanesulfonate) is formed as an amorphous precipitate. 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 [deg.] C. for 2 hours. The yield is about U g. The solubility of the product in water at pH 7 is at least 200 mg / l. This solution remains at room temperature for at least 2 hours, dilute solutions are more stable. The product has the same bacterial spectrum as the corresponding zwitterion and is fully biologically active whether it is hydrolyzed back to the duodenum or not.

Example 19

Sodium 7-o-N, N-bis (sodium sulfomethyl) aminomethylphenylthioacetamido] 3- (3'-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylate. Manufacture from hydroxy imethanesulfonate.

A mixture of 2.05 nmoles of zwitterion of 7- (o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid, 1 * 56 g (10 mmol) of sodium hydroxide phonate monohydrate, 6 ml (6 mmol) of IM SEH ethyl acetate solution, 10 ml of isopropanol and 10 ml of water are stirred at room temperature for 3.5 hours. Treatment of the resulting solution with 1 g of activated carbon and stirring with 300 ml of absolute ethanol and stirring of the resulting mixture at room temperature for 30 minutes gave a crystalline product which was collected by filtration, washed with absolute ethanol (3 x 50 ml) and dried over PgO 4 (U5 ~ 52 ° C / 1 mm) in 20 hours to give sodium 7 - [(N, N-bis (sodium sulfomethyl) -eminomethylphenylthioacetemidyl] -3- (3-hydroxypyridazin-6-ylthiamethyl) -3 "cephem). -U-carboxylate, which is highly soluble in water (> 1 g / ml).

Example 20

Sodium 7- [O-N, N-bis (sodium sulfomethyl) aminomethylphenylthioacetainido] -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylate. Preparation using formalin and sodium bisulphite.

(a) To a solution of 1 ml (10 mmol) of 30% formalin and 1 g of sodium bisulfite in 10 ml of water is added 2 mmol of 7- (o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridazine-5 -ylthiomethyl) -3-cephem-U-carboxylic acid zwitterion, 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-N, N-bis (sodium sulfomethyl) aminomethylphenylthioacetamide-3- (3'-hydroxypyridazin-6-ylthiomethyl) -3'-cephem-U-carboxylate is collected by filtration, washed with ethanol (3 x 50 ml) and dried. in vacuo.

30 5 74 2 0 (b) To a mixture of 2 mmol of 7 '(o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid zwitterion, 6 ml of IM SEH ethyl acetate , 10 ml of isopropanol and 10 ml of water, 1 ml (10 mmol) of 30% formalin are added. After stirring for 2 hours at room temperature, a clear solution with a small amount of oily precipitate is obtained. When 1 g of sodium bisulfite is added and the solution is stirred for 2 hours, the oily precipitate dissolves. Pouring the reaction mixture into 300 ml of ethanol with vigorous stirring gives a solid sodium 7- [N, N-bis (sodium sulfomethyl) aminomethylphenylthioacetamido] -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-b-carboxylate. The mixture is filtered off, washed with ethanol (3 x 30 ml) and dried in vacuo.

Example 21 A. 7- (o-tert-Butoxycarbonylaminomethyl-phenyl-acetamido) -3- (3-hydroxy-pyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid.

To a stirred solution of 1.9 g (3.75 mmol) of 2, U-dinitrophenyl-o-tert-butoxycarbonylaminomethylphenyl acetate in 15 mL of tetrahydrofuran was added at 0 ° C in one portion, a cooled solution of 1.1b g ( 3.3 mmol) of 7-amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-b-carboxylic acid and 0.62 g (6.6 mmol) of triethylamine in 15 ml of acetonitrile and 5 ml of: in water. The temperature of the reaction mixture was allowed to slowly rise to room temperature and the mixture was stirred overnight. The organic solvent was evaporated under reduced pressure below 30 ° C and the remaining aqueous solution was washed with ether (3 x 10 ml). The aqueous layer was covered with 20 ml of ethyl acetate, and the pH of the mixture was lowered to 2.5 by stirring with dilute hydrochloric acid, and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (3 x 100 mL). When the organic matter formed during the extraction was collected and washed with a small amount of water, 7 “(o-tert-butoxycarbonylaminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid was obtained as a pale yellow flour (0, 30 g) (A). The combined organic extracts were treated with a small amount of activated carbon and filtered. When the filtrate was dried over anhydrous sodium sulfate and the solvent was evaporated, an oily substance was obtained, which was washed well with 50 ml of ether to give 0.59 g of 7 '(o-tert-butoxycarbonylaminomethylphenylacetaido) -3- (3-hydroxypyridazin-6-ylthiomethyl) 3-Cephem-b-carboxylic acid as a pale yellow flour (B). Both A and B had an eama IR spectrum. The total yield was 0.89 g (U6%), m.p. 160-170 ° C (decomposes).

31 57420 IR: ν max 1780, 1710, λmax, 1680, 1530 cm-1 m / z UV:? R ° 3 KaHCO 2 (ε 18U00) NMR 55 (2H, s) U, 02 (2H, s), 3.3-1 * .7 (5H, no PP® +

well), U, 95 (1H, d, 1 Hz), 5.50 (1H, d-d, 9, 2 Hz), 6.61 * (1H, d, 10 Hz), 7.0U

(1 * H, s), 7.18 (1H, d, 10 Hz), 8.90 ** (1H, d, 9 Hz), 12.60 ** (1H, br-s). (* double peak (J = 1 * Hz) when D 2 O is added, ** disappears when D 2 O is added).

Analysis calculated for C 52 H 11 O 3 .1 / ^ Η ,, Ο calculated C 52.3 * »H 5.07 N 11.7 ** S 10.75 found C 52.03 H 1 *, 98 N 11.37 S 10.09 B. 7- (o-Aminomethyl-phenyl-acetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-1-carboxylic acid 7- (o-tert-butoxycarbonylaminomethyl-phenyl-acetamido) -3 '( 3 “Hydroxy-pyridazin-6-ylthiomethyl) -3-cefen-1 * -carboxylic acid (500 mg, 0.87 mmol) was stirred at 0 ° C in 3 mL of trifluoroacetic acid and stirring was continued for an additional hour at below 0 ° C. . To the reaction mixture was added 100 ml of ether, and the resulting precipitate was filtered. The precipitate was suspended in 10 ml of water and dilute ammonium hydroxide was stirred in the suspension so that the pH rose to 5. The desired product, 7- (o-aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem- The U-carboxylic acid was filtered off, washed successively with 5 ml of water and 5 ml of acetonitrile and dried in vacuo over phosphorus pentoxide. The precipitate weighed 286 mg (67%), m.p. 222-230 ° C (decomposes).

IR: 1770 '1665' 16k0 · 1500 »1395, 1005 cm_1 UV: ^ mLs ^ 00 · 3 250" "(£ 181 * 00)» 270 nm (sh) (£ jLl * 600) TIMRfD50-KC ° Opp 3 3 »70 (2H» U »15 (2H» s) »3.0-1: .3 (1 * H, does not appear well), H, 90 (1H, d, U Hz), 5.1 * 5 ( 1H, d, 1 »Hz), 6.63 (1H, d, 9 Hz), 7.15 UH, s), 7.20 (1H, d, Hz).

Analysis: 0 82.1 1/2 E ^ calculated C 1 * 9.02 HU, 71 N 13.61 S 12.1 * 6 found C 1 * 8.66 HU, 52 N 13, Oi * S 12.1 * 3 .

Example 22 A. 7- (o-tert-Butoxycarbonylaminomethyl-phenylacetamide) -3- (3-hydroxy-pyridate-6-ylthiomethyl) -3-cephem-1 * -carboxylic acid

To a solution of 1.0 g (3.7 mmol) of o-tert-butoxycarbonylaminomethylphenylacetic acid and 0.69 g (3.7 mmol) in 6 mL of dry ethyl acetate was added in one portion 0.78 g of N, U '- dicyclohexylcarbodiimide, and the mixture was stirred at room temperature for one hour. When the separated dicyclohexylurea was filtered off and the filtrate was evaporated under reduced pressure, the active ester was obtained as a yellow oil. The active ester was dissolved in 30 ml of tetrahydrofuran and cooled to 0 ° C. To the solution was added in one portion a mixture of 1.02 g (3 amoles) of 7 'amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid, 0.85 ml of triethylamine, 30 ml tetrahydrofuran and 10 ml of water. The reaction mixture was stirred at room temperature for 16 hours, after which it was treated with activated carbon. The filtrate was diluted with 50 mL of water and washed with ether (2 x 100 mL). The aqueous solution was covered with 100 ml of ethyl acetate and acidified (pH 2) with dilute hydrochloric acid using efficient stirring at 5-10 ° C. The aqueous layer was extracted with ethyl acetate (2 x 100 mL). The organic layer was combined with ethyl acetate extracts, dried over anhydrous sodium sulfate, treated with activated carbon and filtered. Evaporation of the solvent gave 7- (o-tert-butoxycarbonylaminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3'-cephem-U-carboxylic acid as a pale yellow oily solid which was triturated with 100 ml of ether. . The butoxycarbonyl-protected product was collected by filtration, washed with 30 ml of ether and dried in vacuo. The product weighed 0.95 β (5U%) · B. 7- (o-aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid A cooled mixture of 0.95 g (1 * 6 mmol) of butoxycarbonyl-protected cephalosporin, 7- (o-tert-butoxycarbonylaminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid, and 6 ml of trifluoroacetic acid stirred for one hour at 0-5 ° C. To the reaction mixture was added 100 ml of ether, and the resulting precipitate was collected by filtration and washed with 100 ml of ether. The precipitate was suspended in 10 ml of water and the pH was adjusted to 5 with dilute ammonium hydroxide. Desired product, 7- (o-aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U carboxylic acid, filtered, washed with 50 ml of acetonitrile and dried in vacuo over phosphorus pentokide. The product was seeded at 0.50 g (6.1%).

Example 23 A. 7- (o-tert-Butoxycarbonylaminomethyl-phenyl-acetamido) -3- (3-hydroxy-pyridazin-6-yl-thiomethyl) -3-cephem-U-carboxylic acid To a cooled (-3 to 0 ° C) and stirred solution of was 93 g of 2, U-di-nitrophenyl-o-tert-butoxycarbonylaminomethylphenylacetate in 600 ml of 55.57420 tetrahydrofuran, a cooled solution of 62 g (0.18 mol) of 7-amino-3- (3- hydroxypyridazin-6-ylthiomethyl) -3'-cephem-1-carboxylic acid and U6 g (0.1 U6 moles) of triethylamine in 250 ml of water. The reaction mixture was stirred overnight and allowed to warm to room temperature. The mixture was treated with 10 g of activated carbon and filtered. The filtrate was concentrated to about 300 mL under reduced pressure below 10 ° C. The concentrate was washed with ether (5 x 200 mL) and treated again with about 20 g of activated carbon. Covering the filtrate with one liter of ether and acidifying (pH 3.5) with dilute hydrochloric acid while stirring vigorously gave crude 7- (o-tert-butoxycarbonylaminomethylphenylacetamido) -3 '(3-hydroxypyridazin-6-ylthiomethyl) -3- the cefem-U-carboxylic acid, which was filtered off, was washed successively with 2 liters of ether, 2 liters of water and 0.5 liters of ether. The product weighed 90 g.

- The product was divided into two parts. A second portion (50 g) was used in the next step without further purification.

A second portion (10 g) was dissolved in 1 liter of methanol to remove dark insoluble matter (3.9 g) and the filtrate was concentrated to about 50 ml. To Kim's concentrate was added 300 mL of acetonitrile to give 17.1 g of a pale yellow precipitate, which was collected and washed with 100 mL of acetonitrile. The Kim mother liquor was concentrated to give an additional 12.5 g of product.

B. 7- (o-Aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3'-cephem-1-carboxylic acid (a) 50 g of crude 7- (o-tert-butoxycarbonylaminomethylphenylacetamide) -3- (3-Hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid was dissolved in 100 ml of cooled trifluoroacetic acid. The solution was stirred for 1.5 hours at 0-5 ° C. A liter of ether was added to the solution to give the desired cephalosporin trifluoroacetate, which was filtered and washed with 300 mL of ether. When the trifluoroacetate was dissolved in 100 ml of water and the pH of the solution was adjusted to U with 10% ammonium hydroxide, a colored product (26.6 g) was obtained, which was washed successively with 300 ml of water and 300 ml of acetonitrile and dried in vacuo. . When the crude product was dissolved in 50 ml of formamide at room temperature and 150 ml of water was added dropwise to the solution, a small amount of colored precipitate formed which was filtered off. Addition of 1.5 liters of acetonitrile to the filtrate gave 7 '(o-aminomethylphenylacetamido) -3- (3-hydroxypyridatein-6-ylthiomethyl) -3-cephem-4-carboxylic acid as a pale yellow powder. The powder weighed 9.8 g.

m 57420 (b) Two 7- (o-tert-butoxycarbonylaminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-1 * carboxylic acid fractions (17.1 * g and 12.5 g) were treated as described above and 12.3 g of the desired 7- (o-aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid were obtained.

The IR spectra and MIC values of both (a) and (b) were almost the same. The products were combined and analyzed to give m.p. was 208 ° C (decomposes).

Analysis: c 21 H 21 N 5 O 5 S 2 · 2 ^ 2 H 2 °

calculated C 1 * 7.36 H 1 », 92 N 13.15 S 12.0U

found C 1 * 7.30, 1 * 7.56 H U, 37. U, 50 N 13.67, 13.71 S 12.38.

Example 2k 7- (o-Aminomethyl-phenyl-acetamido) -3- (3-hydroxy-pyrimidin-6-yl-thiomethyl) -3-cephem-1 * -carboxylic acid

To a stirred suspension of 0.63 g (2.1 mmol) of sodium o- (1-ethoxycarbonyl-1-propen-2-ylaminomethyl) phenyl acetate in 5 ml of dry acetonitrile with one drop of Ν, Ν ' -dimethylbenzylamine, 0.25 g (2.3 mmol) of ethyl chloroformate was stirred at -15 ° C and stirring was continued for 20 minutes at -10 to -15 ° C. To the mixture was added in one portion a solution of 0.71 g (2.1 mmol) of 7-amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-1 * carboxylic acid and 0.21 g (2.1 mmol) of ) triethylamine in 3 ml of acetonitrile and 3 ml of water and the mixture was stirred for one hour at 0 ° C. The reaction mixture was treated with a small amount of activated carbon and filtered. 0.5 ml of formic acid was added to the filtrate and the mixture was shaken and a small amount of unreacted 7-amino-3- (3-hydroxypyrimidin-6-ylthiomethyl) -3-cephen-1-carboxylic acid was filtered off. To the filtrate was added 100 ml of acetonitrile, and the mixture was allowed to stand at room temperature for one hour. The precipitate was collected by filtration, washed with 10 ml of water and dried in vacuo to give 0.1 * 9 g (U8%) of 7 '(o-aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3 -cephem-U-carboxylic acid, m.p. 200-210 ° C (decomposes).

Example 25 7- (o-Aminomethylphenylacetamido-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid

Into a 1 liter three-necked round-bottomed flask fitted with a drying tube, stirrer and thermometer, place 2U, 0 g (0.08 mol) of finely divided potassium o- / 1-carbomethoxypropen-2-ylaminomethyl / phenylacetate in 350 ml of dry tetrahydrofuran containing k drops of N, N-dimethylbenzyl broth a. The suspension is stirred vigorously and cooled to -35 to 10 ° C in an acetone-dry ice bath. 13 * 6 g (0.06 mol) of isobutyl chloroformate are added to the suspension in one portion and the temperature is kept below -35 ° C at all times. After 2.5 minutes, the acetone-dry ice bath is removed and a solution of 0.036 mol of 7-amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid and 5.0 g (0.050 mol) of ) n-methylmorpholine in 250 ml of water. The reaction mixture is stirred until its temperature reaches room temperature (1-2 hours). The tetrahydrofuran is removed under reduced pressure. The aqueous solution is layered with 100 ml of ethyl acetate and the pH of the mixture is lowered to 1.7-2.0 with concentrated hydrochloric acid. The mixture is treated with 3.5 g of activated carbon ("Darco KB") and filtered. The layers are separated and the aqueous phase is extracted with ethyl acetate (2 x 100 mL). The aqueous phase is filtered through filter paper and the pH is adjusted to U, 0. After scraping, the solution is left overnight at 5 ~ 7 ° C to crystallize. The product is filtered off, washed with a small amount of water and dried in a vacuum desiccator over P2O2. The yield of 7-O-aminomethylphenylacetamido-3 ”(3-hydroxypyridazin-6-ylthicomethyl) -3” cephem-1-carboxylic acid is about 30%.

Example 26

A suspension of 7- (o-aminomethylphenylacetamido) -3- (3-hydroxy-pyridazin-6-ylthiomethyl) -3'-cephem-U-carboxylic acid (0.361 g) in 3 ml of methanol is cooled in ice and a few drops of concentrated hydrochloric acid until a clear solution is formed. 7- (o-Aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid hydrochloride precipitates as a light brown solid when ether is added and the precipitate is collected and dried in vacuo over P 2 O 3.

Example 27

To a stirred suspension of 0.361 g of 7- (o-aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid zwitterion is added 1N aqueous sodium hydroxide solution at room temperature until a clear solution (pH 10.6). Immediate freeze-drying of this solution gives crude solid sodium 7- (o-aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylmethyl) -3-cephem-U-carboxylate.

36 5 7420

Preparation of dimethanesulfonate Example 2d CH 2 OH.

J.

(,) - CH -C-NH-CH-CH CH2 i2

y - ya2-s- \ y — 0H

0 ^ (Γ X = l /

COOH

Ϊ2 ”5 + HOCHgSO4 Na + CH3 (CH2) 3CH-C00Na (30% SEH acetone solution) 1. Heat in water 2. Treat with activated carbon and filter ^ 3. Suo do s is added to the anhydrous alcohol CH_N (CH.SO.Na).

Ö2 2 3 2

-CH 2 -C-NH-CH-dT "^ CH 6 N_N

C -] L .C-CH - S - // V-OH

✓ \ = / 0 | COONa + HgO + N'-ethylhexyl acid 57,57420

Procedure:

2.0 mol (about 980 g calculated on the anhydrous basis) of 7 '(o-aminomethyl-phenylacetamido) -3' (3 "lyyroxypyridazin-6-ylthiomethyl) -3-cephem-1-carboxylic acid, 5.0 g of sodium formaldehyde bisulphite ( U, 03 moles), 3000 ml of water and 2700 ml (U, 87 moles) of a 30% acetone solution of SEH (sodium 2-ethylhexanoate) in a suitable tank and the mixture is heated to Jt-1> 5 ° C with stirring. After heating the reaction mixture for 15 minutes at 1 * 0-U5 ° C, 50 g of activated carbon ("Darco KB") are added and the solution is stirred for a further 15 minutes at 1 * 0-U5 ° C when the reaction mixture is stirred for a total of 30 minutes at 50 DEG C., it is filtered through diatomaceous earth ("Dicalite") and the carbon layer is washed with 2000 ml of a 50% ethanol-water mixture.

The filtrates are combined, the temperature is adjusted to 25 ° C and the solution is added at 25 ° C to 112 liters of effectively stirred 100% ethanol. There is obtained as a fine, white, amorphous precipitate of sodium 7- (o-aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylate di (sodium methanesulfonate). The suspension is stirred for about 10 minutes, after which it is filtered and the precipitate is washed with 15 liters of 100% ethanol. The precipitate is first dried in an oven at 50-55 ° C with circulating air for 2 hours and then under vacuum (U-6 mm) for 2 hours. About 1200-100 g of amorphous white solid sodium 7 '(o-aminomethylphenylacetamido) -3' (3'-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylate di (sodium methanesulfonate) are obtained. The product usually contains several percent water and possibly some ethanol. The product is also called sodium 7- [6-bis (sodium sulfomethyl) aminomethylphenylacetamide] -3- (3-hydroxy-pyridazin-6-ylthiomethyl) -3'-cephem-U-carboxylate.

Example 29

Prepare a slurry of 2.19 g of sodium formaldehyde bisulfite (2 equivalents), 3.5 g of 7 "(o-aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid zwitterion (100- 200 mesh), 25 ml of water (volume can be changed) and 10 ml of 30% SEH isopropanol solution.

When stirred vigorously for 0.5 hours at 2 ° C, dissolution occurs almost completely. The temperature of the mixture is rapidly raised to 1 → 0-1 + 3 ° C. The temperature is maintained here for about 2 minutes, after which it is rapidly cooled to 20-23 ° C. The insoluble matter is removed from the solution by filtration (the total time in solution should not exceed two hours). The solution (pH 7.3) is added over 5 minutes to 600 ml of very efficiently stirred absolute ethanol (other alcohols such as isopropanol can be used). Sodium 7- (o-aminomethylphenyl-38,57420 acetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylate di (sodium methanesulfonate) is formed as an amorphous precipitate. 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 2 hours. The yield is about U, 3 g. The solubility of the product in water at pH 7 is at least 200 mg / ml. This solution stays at room temperature for at least 2 hours, more dilute solutions are more stable. The product has the same bacterial spectrum as the corresponding zwitterion and is fully biologically active whether it is hydrolyzed back to the zwitterion or not.

Example 30

Sodium 7- [6-N, N-bis (sodium sulfomethyl) aminomethylphenylacetamide] -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylate. Manufacture from hydroxymethanesulphonate.

A mixture of 1 g (2.05 mmol) of the zwitterion of 7- (o-aminoacetylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylate, 1.56 g (10 mmol) of sodium hydroxymethanesulfonate monohydrate .6 ml (6 mmol) of a 1 M solution of SEH in ethyl acetate, 10 ml of isopropanol and 10 ml of water were stirred at room temperature for 3.5 hours. The resulting solution was treated with 1 g of activated carbon and stirred in 300 ml of absolute ethanol and the resulting mixture was stirred at room temperature for 30 minutes to give a crystalline product which was collected by filtration, washed with absolute ethanol (3 x 50 ml) and dried over PgO 2 + (U5 -52 ° C / 1 mm) in 20 hours to give 1.6 U g of sodium 7- [O-N, Libis (sodium sulfomethyl) aminomethylphenylacetamido] -3- (3-hydroxypyridate-6-ylthiomethyl) -3-cephem- 4-carboxylate, which is highly water-soluble (tl g / ml), m.p. 7,270 ° C.

IR: λmax 1760 '1680-1620 »158 °» lU00, 1200, lOUO cnf1 LIV: λmax 2U9 nm (£ 10000), 272 nm (£ 10900, sh), 320 nm (£ 2100, sh).

NMR: D 2 O 3.30-U.0 (en, m S-CH, O -CH -CO and O-CH-N or ppm 3-CH 2 -S), 4.0¾ (2H, s, N- CH 2 -O or 3-CH 2 -S), 1 +, 30 (1 »H, s, H-CH 2 -SO 2 a) U, 89 (1H, d, UHz, 6-H), 5.U0 (1H , d, kHz, 7-H), 6.78 (1H, d, 9.5 Hz, pyridazine-H), 7.1 ', 0.7 (UH, phenyl-H), 7.32 (1H, d , 9.5 Hz, pyridazine-H).

Example 31

Sodium 7 '> N-bis (sodium ulfomethyl) aminomethylphenylacetamido-S- (3-hydroxypyridazin-6-ylthiomethyl) -3 "cephem-U-carboxylate. Preparation using formalin and sodium bisulphite.

39 5 7420 a) To a solution of ml (10 mmol) of 30% formalin and 1 g of sodium bisulfite in 10 ml of water was successively added 0.975 g (2 mmol) of 7- (O-aminomethylphenylacetamido) -3- (3- hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid zwitterion, 6 ml of 1 M SEH solution and 10 ml of isopropanol.

The mixture was stirred for 2.5 hours at room temperature and poured into 300 ml of ethanol. The resulting sodium 7- [6-N, Ν-bis (sodium sulfomethyl) aminomethylphenylacetamide] -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-1 * carboxylate was collected by filtration, washed with ethanol (3 x 50 mL). ) and dried in vacuo.

The yield was 1.6 g.

b) To a mixture of 0.975 g (2 mmol) of 7- (o-aminomethylphenylacetamido-3- (3-hydroxypyrazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid zwitterion, 6 ml of 1M SEH in ethyl acetate, 10 ml isopropanol and 10 ml of water, 1 ml (10 mmol) of 30% formalin were added, and after stirring for 2 hours at room temperature, a clear solution of a small amount of oily precipitate was formed, and when 1 g of sodium bisulfite was added and the solution was stirred for another 2 hours, the oily precipitate dissolved. When the reaction mixture was poured into 300 ml of ethanol with vigorous stirring, 1.8 U g of sodium 7- [3-N, N-bis (sodium sulfomethyl) aminomethylphenylacetamide] -3- (3-hydroxypyridazin-6-ylthiomethyl) -3- cefem-14-carboxylate, which was collected by filtration, washed with ethanol (3 x 50 ml) and dried in vacuo.

Claims (2)

1.0 57420 Patents: Process for the preparation of antibacterial 7-substituted 3- (3-hydroxypyridazin-6-yl-thiomethyl) -3-cephem-N-carboxylic acids of the formula CH NH / 22 - / V X- (,} - S -CH-C-NH-CH-CH CHO \\ // m 2 I 2 Nj IN -N \ CN C - CH0 - S ('--- OH / \ c ^ · 2 \ _ / C00H I where X is H or OH and m is 0 or 1, provided that m is 0, when X is OH, and for the preparation of these pharmaceutically acceptable salts, characterized in that a compound of formula S κ \ HN - CH-CH CH d | * N -N C - ^ - C-CH-S— \ Λ- OH i COOH II or a salt thereof for the reaction with an acylation derivative of the formula / ch2nhb X- (. \ - (S) - CH - COOH> / “2 \ -ym wherein B represents an amino protecting group and m and X are the same as above, wherein the product is a compound of the formula ^ 57420 CHgNHB x Γ ^ \ S \ / m 2 I 2 N - N // ^ „C -N. C-CH -S— (f x -> ------- - OH 2 \ 7 7 IV COOH wherein B, X and m are the same as above, or a salt thereof, and then the amino protecting group is removed to give the desired compound of formula I or a non-toxic, pharmaceutically acceptable salt thereof. For the preparation of the antibacterial 7-substituent 3- (3-hydroxypyridazine-6-ylthiom ethyl) -3-cephem-1-carboxylic acid with the formula ch2nh2 / = \ s X \ X | | i2 C- N C-CH - S - // X \ _0H ° / wy COOH color X är H eller OH och m är 0 eller 1, under förutsättning, att när X är OH, är m O, och giftfria, farmaceutiskt godtagbara salter därav, kännne -tecknat därav, att man omsätter en förening med formula S / \ HN - CH --- CH CH CI e N - N. C - N. C-CH -S— (f '-) oh // \ c ^ 2 \ / 0 I COOH or another site of the acyl derivative derived from the formula CH NHB _ / X - /) - (S) - CH_ - COOH Y_X m III color B is an amino acid group of the och m och X har ovannämnda betydelse , for the purposes of this Regulation