FI57111B - EXAMINATION OF ANTIBACTERIAL 7- (O-AMINOMETHYLPHENYL-ACETAMIDO-OCH-PROPIONAMIDO) -3- (TETRAZOLO (4,5-B) PYRIDAZIN-6-YLTIOMETHYL) -3-CEFEM-4-CARBOXY - Google Patents

Description

RSSF ^ l [B] (11) ADVERTISEMENT -

JWA lJ 1} UTLÄGGININCSSKRIFT b / 1 I I

C Patent cyännrtty 10 C6 1900 (45) Patent meldc lat ^ ^ (51) Kv.ik. * / Ta * »ci.e C 07 D 501/36 FINLAND — FINLAND (21) PttMttlhakwMM - PttMKaM & kning 2670/73 (22) H «k * mlip« lYl - AmMmingtdag 27.08.73 '' (23) Alkupiivt — GUtlghutadig 27.08.73

(41) Tullut | ulklMl («l - BIMt offtntllg 01.03.7U

Patent and Registration Office ........- " .__. . .....

· (44) Date of Nihtiviktlpuwn et al. -

Patents and registrations Amekan utiagd och uti. * Krtft * n puMkarad 29.02.80 '· (32) (33) (31) Pyj ^ Atty atuoikaui — Begird prlorkvt 30.08.72 Ο6.Ο9.72 USA (US) 28U792, 286793 '(Tl) Bristol-Myers Company, 3 ^ 5 Park Avenue, New York, NY, USA (72) Takayuki Naito, Tokyo, Jun Okumura, Yokohama, Ha jime Kamachi,

Yokohama, Japan-Japan (JP) (7M Oy Kolster Ab (5U) Method for antibacterial 7- (o-aminomethylphenylacetamido and propionamine) -3 - (t et r at Solo A »5-b / pyrid dat for the preparation of 6-yl-isomethyl-3-chemo-U-carboxylic acids - For the preparation of antibacterial compounds with 7- (o-aminomethylphenyl-acetamido-och-propionamido) -3- (tetrazolo / k, 5 This invention relates to the process for 7- (o-aminomethylphenylacetamido) -3- (tetrazo-1,5-b] pyridazin-6-ylthiomethyl) - For the preparation of 3-cephem-U-carboxylic acid and 7- (o-aminomethylphenylpropionamido) -3- (tetrazolo [u, 5-b] pyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid of the formula _.ch2nh2 C00H \ = / wherein n is 1 or 2 and for the preparation of dimethanesulfonate derivatives of these acids and their non-toxic pharmaceutically acceptable salts.

The novel compounds of the invention have been found to be valuable bactericidal agents, dietary supplements for animal feed, mastitis treatment agents for cattle and therapeutic agents for poultry and other animals, including humans, in the treatment of infectious diseases caused by many gram-positive and gram-negative bacteria.

2 57111

There is a wealth of literature dealing with cephalosporin antibiotics. The main prior art data in this field are presented below: 7-Phenylacetamidocephalosporanic acid, also referred to as the N-phenylacetyl derivative of 7-ACA, cephaloram, PACA, and apparently also phenazporine are well known in the scientific literature. The publications on the preparation and / or properties of this compound with or without substituents on the benzene ring and of the corresponding compounds in which the 3-acetoxymethyl group has been replaced by methyl, hydroxymethyl and / or pyridinium methyl are as follows:

Chauvette. 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, 84 5401-4302 (1962). Chauvette. R.R., et al. “Structure-Activity of Relationships Among 7-Acylami-docephalosporanic Acids”, Antimicrobial Agents and Chemotherapy -1962, 687-694.

Cocker. J.D. et al., "Cephalosporanic Acids, Part II. Displacement of the Acetoxy Group by Nucleophiles", Journal of the Chemical Society, 5015-5031 (1965).

Cocker. J.L., et al., ”Cephalosporanic Acids. Part IV. 7-acylamidoceph-2-em-4-carboxylic Acids, Journal of the Chemical Society, 1142-1151 (1966).

Culp. H.W., et al., "Metabolism and Absorption of the 7- (Phenylacetamido-1-14, C; -cephalosporanic Acid", Antimicrobial Agents and Chemotherapy - 1963, 243-246.

Jago, M., "Antibacterial Activity of Some Derivatives of 7-Aminocephalosphoric Acid Against Staphylococcus Auereus and Synergism Between These and Other Antibiotics," Britt. J. Pharmacol., 22, 22r23 (1964).

Loder. B., et al., "The Cephalosporin C Nucleus (7-aminocephalosporanic Acid) and some of its Derivatives", Biochemical Journal, 79, 408-416 (1996). Nishida, M., et al., "Studies on Microbial Degradation of Cephalosporin C Derivatives II ", The Journal of Antibiotics, 21, 375-478 (li68).

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 *

Stedman. R.J. et al., "7-aminodesacetocycephalosporanic Acid and its Derivatives", J. Med. Chem., 7 (1), U7-U9 (1964).

5 57111

Sullivan. H.R., et al., "Metabolism of Oral Cephalothin and Rexlated 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 Hygiene, Epidemiology, Microbiology and Immunology, 10, 180-189 (1966).

Many other 7-acyl derivatives of 7-aminocephalosporanic acid have been reported in the patent literature, including 7- (4- (α-aminoalkyl) phenylacetamido) cephalosporanic acids (U.S. Patent No. 3,382,241).

7 - ((p-aminophenyl) acetamido) cephalosporanic acid (U.S. Patent No. 3,422, 100), 7- (halophenylthioacetamido) cephalosporanic acids (U.S. Patent No. 3 * 335 * 136) and an almost unlimited number of variations of the compounds included to the general formula (and which is often not otherwise described) disclosed in patents such as Dutch Patent 69/02013 (Farmdoc 39,172). 7- (p-Aminophenylacetamido) cephalosporanic acid is disclosed in U.S. Patent No. 3 * 422,103 as well as the corresponding N-trityl derivative (see also Japanese Patent No. 2712/67) (Farmdoc 25,406).

U.S. Patent No. 3,219,662 claims compounds having the structure R-CHg-CO-ACA, wherein R is a phenyl, nitrophenyl (especially para-nitro), chlorophenyl, alkylphenyl and alkoxyphenyl group, and the like. phenoxy and substituted compounds and all the corresponding compounds in which the 3-acetoxymethyl group has been replaced by a 3-pyridinium methyl group. A broader group of such compounds, including a series in which R is a phenylthio group, and also compounds in which R is a benzyl group (i.e., 7- (f3-phenylpropionamido) cephalosporanic acid), an alkoxybenzyl, alkanoyloxybenzyl, aminobenzyl group, etc. are disclosed for use at least in general. as starting materials in English Patent Nos. 1,012,943 * and 1,153,421 (Farmdoc 23,984) »see also English Patent No. 1,001,478 and U.S. Patent No. 3,280,118. Other 7-phenylacetamidocephalosporanic acids having substituents on the benzene ring, such as hydroxy and amino groups, are disclosed as starting materials in British Patent Nos. 1,082,943 and 1,082,962.

U.S. Patent No. 3 * 341,531 describes 7- (o-, m- and p-carboxyamidomethylphenylacetamido) cephalosporanic acids and their betaines.

A number of 7- (halo-, dihalo-, nitro-, and halonitrophenylacetamido) cef? * Losporanic acids are mentioned as starting materials for the reaction with certain nucleophiles in 4,511,111 U.S. Patent No. 3 * 431 * 259 (Parmdoc 27,715) · Other 7- (phenylacetamido) Cephalosporanic acids having various substituents on the benzene ring are disclosed in Japanese Patent No. 2712/67 (Parmdoc 25 * 406), Japanese Patent No. 26105/69 (Parmdoc 40,860), English Patent No. 1,178,471 (Parmdoc 27,715). 67/00906) and Japanese Patent No. 25785/69 (Parmdoc 40,847).

The replacement of the 3-acetoxy group of a cephalosporin with various heterocyclic thiols is described in a) South African patent 70/2290 (see also Dutch patent 70/05519 (Parmdoc 80,188 R)), in which the side chains were e.g.

7-or-aminophenylacetamido groups and typical heterocyclic thiols included 2-methyl-1,3,4-thiadiazole-5-thiol and 1-methyl-1,2,3,4-tetratolol-5-thiol; and b) in U.S. Patent 3 * 516,997 »wherein the 7-position side chains had a structure such as R 1 - (alk) m -CO-NH- and R 1 -S- (alk) m-CO-NH-, wherein R 1 - was one of many aromatic heterocyclic groups, and the numerous heterocyclic thiols in the 3 ** position were, e.g., 1-methyltetrazole-5-thiol and 2-methyl-1,3,4-thiadiazole-5-thiol, and c) in U.S. Pat. 3,563,983.

Various cephalosporins, including cephalosporin C by chance, have been reacted with nucleophilic, aromatic mercaptans to produce compounds having the structure / s \.

Acyl-NH-CH-CH CH_

II I

O-C-N 2 -O-CH 2 -S-Ar 2 f

COOH

In U.S. Patent No. 3,278,531, Ar is a phenyl group or a certain substituted phenyl group or a particular aromatic heterocyclic ring, designated e.g. in English patent 1,101,422, all published in the name of Glaxo * A similar representation can be found in English patent published in the name of Ciba 1,109,525 e.g.

5 57111 from the heading "h". Additional nucleophiles of this type were disclosed by Fujisawa in Belgian Patent 714,518 (Farmdoc 35,307 »Dutch Patent 68/06129 and South African Patent 2695/68), Canadian Patent 818,501 (Farmdoc 38,845), English Patent 1,187,323 and Farmdoc 31,936 in particular U.S. Pat. No. 3,516,997 (Farmdoc 34,328; Dutch Pat. Agents and Chemotherapy-1969, American Society for Microbiology, Bethesda, Maryland, pp. 236-243 and J. Antibiotics (Japan) 23 (3), 131-148 (ΐ97θ).

More recently, the replacement of the 3-acetoxy group of a cephalosporin with various heterocyclic thiols has been disclosed in U.S. Patent 3,563,983 and Dutch Patent 70/03519 (Farmdoc 80,188 R) in which the side chains were e.g. 7-α-aminophenylacetamido groups and typical heterocyclic ol 3,4-thiadiazole-5-thiol and 1-methyl-1,2,3,4-tetrazole-3-thiol; the latter corresponds to U.S. Patent 3,641,021 published February 8, 1972 on the basis of an application filed April 18, 1969. Other similar disclosures can be found in U.S. Patent No. 3,563,983, Belgian Patent No. 771,189 (Farmdoc 12,817 T), Japanese Patent No. 72/0555 ° (Farmdoc 12,921 T) and Japanese Patent 72/0551 (Farmdoc 12,922 T).

Various cephalosporins with structure

Acyl-NH-CH-CIT CH0

I 1 I

0-C-N. C-CH2-S-alkyl ^ C '

COOH

wherein the acyl represents various side chains including the α-aminophenylacetyl group is described in some of the above patents and in Glaxo's Belgian Patent 734,532 (Farmdoc 41,619) and Belgian Patent 734,533 (Farmdoc 41,620) and U.S. Patent 3,668,203.

Cephalosporins having the structure 6 57111 / s \

Acyl-NH-CH-CH CH2

I I I

O-C-N 2 -C-CH 2 -X

c 0 ° H 0 wherein X comprises the groups -S-C- and -S-C- are disclosed in some of the above patents and U.S. Patent 3,239,515 # 3,239,516; 3,243,435 # 3,258,461 # 3,431,259 and 3,431,259 and 3,446,803.

Related publications in the scientific literature are J. Med. Chem. 8, 174-181 (1965) and J.Chem.Soc. (London) 1595-1605 (1965), 5015-5031 (1965) and 1959-1963 (1967).

6-Mercaptotetrazolo (4,5-b) pyridazine was described by B. Stanovnik et al., J. Org. Chem. 35 (4), 1138-1141 (1970) as compound 8 (R-H) under the name 6-mercaptotetrazolo (1,5-h) pyridazine ·

There are various presentations in the literature on methanesulfonates of antibiotics. Accordingly, Belgian Patent 742,720 (Farmdoc 41466 R) describes ampicillin monomethanesulfonate. U.S. Patents 3,268,508 and 3,295,246 describe the conversion of one or more of kanamycin from four free amino acids to a methanesulfonate derivative. Methanesulfonate sodium betrazine is disclosed in U.S. Patent 3,205,137. And for neomycin, see J. Antibiotics (Japan) 12, 114-115 (1959). The injectable antibiotic colistimethane (marketed by Varner-Chilcott as "Coly-Mycin") is the sodium salt of colistin methanesulfonate, exemplified by Koyama et al., Japan 4898 (1957), which is described in detail in pages 315-320 of TT.S. Dispens. 26th Edition, JB Lippincott Company, Philadelphia, Penns. It is described as the pentanodium salt of the pent (methanesulfonic acid) derivative of Colist Form A on pages 621-622 in U.S. Patent No. XVII. also English Patents 874,028, 902,992 and 896,774 and Japanese Patent 15948/61.

T 57111

The invention relates to a process for the preparation of antibacterial 7- (o-aminomethylphenylacetamido and propionamido) -3- (tetrazolo [5,5-b] pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acids of the formula ch2nh2 w

COOH

- wherein n is 1 or 2 and for the preparation of non-toxic pharmaceutically acceptable salts thereof.

Non-toxic pharmaceutically acceptable salts referred to above include non-toxic carboxylic acid salts, e.g. non-toxic metal salts such as sodium, potassium, calcium and aluminum salts, ammonium salt and substituted ammonium salts of progeny amine, e.g. , dibenzylamine, N-benzylbeta-phenethylamine, 1-ephenamine, N, N'-dibenzylethylenediamine, dehydroabietylamine, N, N'-bis-dehydroabiethylethylenediamine, N- (lower) alkylpiperidine, such as N-ethylpiperidine, such as N-ethylpiperidine, such as N-ethylpiperidine has been used to form salts with benzylpenicillin; and non-toxic acid addition salts thereof (i.e., amine salts) including mineral acid addition salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate and phosphate, and organic acid addition salts such as maleate, acetate, citrate, buccinate, succinate, oxalate, succinate, oxalate, , ascorbate, etc.

"Particularly preferred salts of the compounds of formula I are the sodium salts of dimethanesulfonate. These salts, when soluble in water, are particularly useful in the preparation of injectable preparations.

Intermediates or metabolic precursors of the compounds of formula I are those in which the amino group is "protected" by substituents such as t-butoxycarbonyl, carbobenzyloxy, formyl, o-nitrophenylsulphenyl, η 6, β, β- trichloroethoxycarbonyl, U-oxo-2-pentenyl-2-, 1-carboethoxy-1-propenyl-2, etc. Such protecting groups include, in particular, ketones (especially acetone) and aldehydes (especially formaldehyde and acetaldehyde) which are disclosed, e.g., in U.S. Patents 3,198,80U and 3,3 ^ 7,851 and β 57,111 /? ketoesters and Δ-diketones disclosed, e.g., in U.S. Patent 3,325-1 + 79 and β-ketoamides · 2U71M

The process according to the invention is characterized in that the compound of formula

. S

H H 1 r Λ / 2 '1 i "w J-N CH2 ~ S V = ^ N 11

COOH

or a salt thereof is reacted with a reactive acid derivative of the formula ch2nhb

F> V- (CH2) n-C00H HI

wherein B is an amino protecting group and n is the same as above to prepare a compound of formula

CH2NHB

N - (CH2) n-C-NH-N, N-W / N-N

J-n

IV COOH

wherein B and n are the same as above or to prepare a salt thereof, and then the amino-protecting group B is removed to prepare the desired compound of formula I or a non-toxic pharmaceutically acceptable salt thereof.

The amphoteric compounds of this invention are prepared by coupling a 7-amino-3- (tetrazolo [5,5-b] pyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid as defined in formula II with an acid of formula III or a compound having the same reactivity as a primary amino group acylating agent. After coupling, the amino protecting group B is removed to provide the desired product. Formula III B shows a protecting group of the type used either in peptide syntheses or in one of the numerous syntheses of ampicillin or cephaloglycine or cefalexin starting from 2-phenylglycine. Particularly valuable protecting groups are protons, such as in the compound of formula 9 57111 ch2nh2.hci ^ y ^ (CE2) nC-Cl or β-diketone or β-ketoester, as in British Patent 1,123,333 and U.S. Patents 3,325-1 + 79, and 3,316 21 + 7, e.g. methyl acetoacetate or (β-ketoamide as in Japanese Patent 71/21 + 711 + (Farmdoc 1 + 7 321 S), in which case the acid containing the protected amino group is preferably converted to a mixture of anhydrides with e.g. ethyl chloroformate before administration react with compound II or a salt thereof to form the desired product I after removal of the protecting group.

Following the above representation of the protecting groups used in the free amino group of the side chain acid during its incorporation into compound II, the protecting group is then removed to form the products of this invention, e.g., the t-butoxycarbonyl group can be removed by treatment with formic acid or trifluoroacetic acid, carbobenzyloxy the hydroxy-1-naphthycarbonyl group can be removed by acid hydrolysis, the trichloroethoxycarbonyl group can be removed by treatment with zinc dust in glacial acetic acid, and the 1-carboethoxy-1-propenyl-2 group is preferably removed by treatment with formic acid. It will be appreciated that other amino protecting groups having the same reactivity may be used and such groups are considered to be within the scope of this invention.

Thus, with respect to the acid of formula III to be used for incorporation into compound II, compounds of similar reactivity are the corresponding acid anhydrides, including mixed anhydrides and especially those mixed anhydrides} prepared from stronger acids such as carboxylic acid or alkyl and alkyl and esters of acidic sulfonic acids and steric acids. , such as lower aliphatic monoesters of diphenylacetic acid. In addition, an acid azide or an active ester or thioester (e.g. with p-nitrophenyl, 2,1 + -dinitrophenol, thiophenol or thioacetic acid) may be used or the free acid itself may be added to compound II after said free acid has been reacted with Ν, Ν'-dimethyl with chloroforminium chloride (cf. English Patent 1,008,170 and Novak and Weichet, Experientia XXI, 6, 360 (1965)) or using enzymes or N, N'-carbonyldiimidazole or N, Nf-carbonylditriazole (cf. South African Patent 63 / 2681 +) or a carbodiimide reagent (especially Ν, Ν'-dicyclohexylcarbodiimide, Ν, Ν'-diisopropylcarbodiimide or N-cyclohexyl-N '- (2-morpholinoethyl) -carbodiimide see Sheehan and Hess J. Amer. Chem. Soc., 77, 1067 (1955)) or an alkylamine amine reagent (cf. R. Buijle and HG Viehe, Angew, Chem. International Edition 3, 582, (196M)) or isoxazolium salt reagent. (cf. RB Woodward, RA Olofson and H. Mayer, J. Amer. Chem. Soc., 83, 1010 (1961)) or ketene imine reagent (cf. C. L. Stevens and M.E. Mond, J. Amer. Chem. Soc., 80 (I + O65)).

Another compound corresponding to the acid chloride is the corresponding azolide, i. an amide of the corresponding acid, the amide nitrogen of which is a member of a semi-aromatic five-membered ring containing at least two nitrogen atoms, i. imidazole, pyrazole, triazoles, benzimidazole, benzotriazole and their substituted derivatives. As an example of a general process for the preparation of an azolid, N, N'-carbonyldiimidazole is reacted with a carboxylic acid in equimolar ratios at room temperature in tetrahydrofuran, chloroform in dimethylformamide or a similar inert solvent to liberate the carboxylic acid imidazolidide substantially to give the carboxylic acid imidazolidide. Diimidazolide is obtained from dicarboxylic acids. Page 57111 tucrte imidazole precipitates and can be separated and imidazolide isolated, but this is not necessary.

Methods for carrying out these reactions to prepare a cephalosporin and for isolating the cephalosporin thus obtained are well known in the art *

The use of enzymes to incorporate the free acid with its euated amino groups into compound II was mentioned above. Such methods include the use of this free acid ester, e.g., a methyl ester, with enzymes produced by various microorganisms, such as those described by T. Takahashi et al., 'J. Amer. Chem. Soc., 94 (II), 4035-4037 (1972) and T.

Hara et al., J. Antibiotics (Japan) 24 (5) · 321-323 (1971) ·

Upon completion of the coupling and deprotection reactions described above, zwitterionic products of formula CHgNHg ©

P y (CH2) n - c - NH - | -p J N-N N

\ = / o ^) ~ N

° If desired to convert dimethanesulfonate to the sodium salt of formula CH2N (CH2SO3Na) 2 Ö-? COONa

The conversion of zwitterionic cephalosporanic acid to dimethanesulfonate derivatives can be performed by methods known in the art. In a preferred method, the zwitterion is reacted 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. The above mixture is warmed above room temperature, e.g. above about 40 ° C and preferably between 40-45 ° C for a short time until a solution of the desired product is formed. The product is recovered as a solid by precipitation, e.g. by the addition of a water-soluble, substantially anhydrous solvent, preferably an alcohol such as ethanol or isopropanol. Alternatively, binary products can be converted to non-toxic, pharmaceutically acceptable salts by methods known in the art.

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 the desired 7-side chain acid or a substance having the same reactivity and then replacing the 3-acetoxy group with 6-mercaptotetrazolo. to obtain the product. _

The starting 7-amino-3- (tetrazolo [5-b] pyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid of formula II

g H N-CH-CH 2 XCH 5 N -

* 11 I * // \ __ I

C — N C-CH0-S- \> = N II

o '' 'C * 2 \ - /

COOH

or a salt thereof can be prepared by reacting 7-aminocephalosporanic acid or a salt thereof with a thiol of formula

N-N

The replacement of an ester group of a 7-aminocephalosporanic acid, e.g. a 3-acetoxy group with a thiol group, of HS 2 = -li or a salt thereof, preferably a sodium or potassium salt, is a well-known reaction and can be carried out in aqueous solution in the presence of a dilute base such as sodium bicarbonate. The reaction is preferably carried out preferably at about 50-100 ° C.

7-Amino-3- (tetrazolo [1,5-b] pyridazin-6-ylthiomethyl) -3-cephem-1-carboxylic acid is a useful intermediate in the preparation of compounds of formula I.

In the treatment of bacterial infections in humans, the compounds of the invention are administered parenterally in about 5-200 mg / kg / day, preferably about 5-20 mg / kg / day in divided doses, e.g. three or four times a day. They are administered in dosage units containing, for example, 125-250 or 500 mg of active ingredient with suitable physiologically acceptable carriers or diluents. Dosage units are in the form of liquid preparations such as solutions or suspensions.

13 571 1 1

The following examples illustrate the method of the invention. All temperatures are in degrees Celsius. 7-Amino-cephalosporanic acid is abbreviated as 7-ACA; -ACA- represents a radical having the structure -NH-CH-Clr CHg / -N. > CH -

COOH

0

II

and thus the 7-ACA compound can be represented by the formula H-ACA-Q-C-CH 2 ·

Example 1

OO "™" Βα000 "3 -> QQ“ ™ 0C

A Ji (ί ^ Υ ^ ΝΗ-ΒΟΟ f ~ \ | I // \ H-ACA-S-f) = - N K— '' \ = / rr woman ^^

NH-BOC

kX / co-T-rA

Qi-N \ ^ ”CVS · / CFgCOOH ^ j8 co2h \ = / rr ^

0 J — n cVs ~ y y = ^ R

co2h 20 57111 14 7-Amino-3- (tetrateolo- (4,5-b) pyrazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid HN._ / Ν ^ κ

n2Nl J N — N I

o J — ch2-o-coch5 -h ^

COgH

7-ACA

"2 · Η -! ^ S '' j n— / ^ 1}

1c02H

ϋ 7-Amino-3- (tetrataolo- (4,5-b) pyrazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (14) i) In a hot solution (50-60 ° C) containing 9? 56 g (0f062 moles) of product 13 and 10.42 g (0.124 moles) of sodium bicarbonate in 300 ml of water, 16.86 g (0.062 moles) of 7-ACA were carefully added and the mixture was kept at 80-83%. min. About 4 g of sodium bicarbonate was added to the reaction mixture to dissolve the insoluble matter. The solution was treated with charcoal, filtered and the euodoe acidified to pH 3 with dilute hydrochloric acid. The precipitate was collected by filtration, washed with water, air dried and finally in vacuo with PgO 2 to give 14.47 β (64 en) of product 14 ·

Sp. 248-250 ° C (decomposes).

ii) A stirred solution of 16.8 g (0.11 moles) of product 13 and 18.48 g (0.22 moles) of NaHCO 3 in 1 L of 0.1 M phosphate buffer (pH 6.4), was maintained at 50 ° C and 30 g (0.11 moles) of 7-ACA was added portionwise to the solution. The mixture was kept at 80 ° C for 2.5 hours, during which time insoluble matter was retained. The reaction mixture was cooled to room temperature and the stirred product 14 was collected by filtration, washed thoroughly with 200 ml of water and air dried.

Additional product 14 was obtained from the filtrate and washings by acidification and pH 5 with dilute HCl. Total catch 52.9 g ($ 85) · M.p. 245-250 ° C (decomposes).

IR: γ0 * 1800, 1615 * 1558, 1560 cm -1, max UV: r 1 (HalClO 2 J 7 (190000)), 275 nm (ε 12000), 510 nm (sh) (δ 5700).

max HMR: δ DgO + KgCO 3 5.55 (1H, d, 18 Hz, 2-H), 5.76, (II, d, 18 Hz, 2-H), ppm 4.00 (1H, d, 10 Hz, 5-CH 2), 4.48 (1H, d, 10 Hz, 5-0¾). 4.95 (1H, d, 4 Hz, 6-H), 5.32 (1H, d, 4 Hz, 7-H), 7.46 (1H, df 10 Hz, pyridazine-H), 8.18 (1H , d, 10 Hz, pyridazine-H).

Calculating from the formula: ^ 2 ^ 11 ^ 7 ^ 5 ^ 2: C * ^ 9.44; H, 5.05; N, 26.85; S, 17.55 Experimental: C, 59.19; H, 2.71; H, 26.84; S, 17.55 · t-butoxycarbonyl azide (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 for 40 min. while maintaining the temperature at 10-15 ° C. At the end of the addition, stirring was continued for another 50 min. at the same temperature. To the mixture was added 100 ml of water, and the separated oil was extracted with five 100 ml portions of methylene chloride. The combined organic extracts were washed with 100 ml of 10 L of sodium bicarbonate solution and 100 ml of water successively and dried over anhydrous sodium sulfate. The methylene chloride was removed under reduced pressure with a water bath maintained at 40-45 ° C. The remaining azide was distilled off and recovered at 45 ° C and 20 mm Hg. It weighed 92.7 (84 lbs) · o- (t-butoxycarbonylaminomethyl) phenyl acetic acid (16)

To a solution of 70 g (0.55 moles) of o-aminomethylphenylacetic acid hydrochloride (4) and 116 g (1.15 moles) of triethylamine (TXi) in 400 ml of water was added dropwise a solution of 64 g (0 moles) of .45 moles) of t-butoylcarbonyl azide (15) in 300 ml of tetrahydrofuran (THF) with stirring at 0 ° C. At the end of the addition, the temperature was allowed to rise to room temperature and stirring was continued for 20 hours. Tetrahydrofuran was distilled off below 40 ° C and the aqueous solution was washed with 200 ml of ether, over which 200 mL was deposited.

57111 16 ethyl acetate and acidified with dilute hydrochloric acid to pH 3 with cooling to 0 ° C. The organic layer was separated and the Yes layer was extracted with four 200 mL portions of ethyl acetate. The combined ethyl acetate solution was washed with 200 mL of water, dried over anhydrous sodium sulfate and concentrated in vacuo. The concentrate was treated with $ 00 mL of n-hexane to give 87 x 9 g (95 50 products as 16 colorless needles) melting at 114-116 ° C.

2,4'-Nitrophenyl p-t-butoxycarbonylaminomethylphenyl acetate (17)

Dicyclohexylcarbodiimide (17 * 72 g »0> 086 moles) (DCC) was added in one portion to a mixture of o- (t-butoxycarbonylaminomethyl) phenylacetic acid (16) (22.73 g, 0.086 moles) and 2.4 -dinitrophenol (13 * 82 g, 0.086 mol) (2,4-DHP) in 230 ml of equal THF. The reaction mixture was stirred for 2 hours at room temperature. The precipitated dicyclohexylurea was filtered off and washed with 100 ml of TH 2. The filtrate and washings were combined and concentrated under reduced pressure below 30 ° C to give a viscous yellow oil which was triturated in n-hexane (130 mL) to give the product 17 as yellow needles. Yield 34.9 (94 50. M.p. 76-77 ° C.

YS 3340 * 1785 »1685» 1610 · 1540 * 1530 »1500» 1340 o * "1 ·

Calculated from C, 55.68 »H, 4.91 * I, 9.74

Experimental: C, 55.70 * H, 5.05 * H, 9.93.

7- (α-Butoxycarbonylaminomethylphenylacetamido) -3- (tetrazolo- (4,5-b) -pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (18) To a solution of 20.26 g (0.047 mol) product 17 in 130 ml of tetrahydrofuran, a solution of 14.40 g (0.039 mol) of thiote 14 and 19.19 g (0.19 mol) of? E4 in 130 ml of 30 ml of an aqueous solution of tetrahydrofuran was added in one portion. At 5 ° C · The reaction mixture was stirred for 18 hours and concentrated under reduced pressure to remove tetrahydrofuran below 30 ° C. The aqueous concentrate was washed with two 200 mL portions of ether, acidified to pH 2 with dilute hydrochloric acid and extracted with five 200 mL portions of ether, acidified to pH 2 with dilute hydrochloric acid and extracted with five 200 mL portions of ethyl acetate. The combined extracts were washed with two 100 ml portions of water, dried over anhydrous sodium sulfate, treated with activated carbon and filtered. Evaporation of the solvent gave a pale yellow oil which was triturated with ether to give 13 * 89 g (58 JO of product 18). Mp 166-173 ° C (dec).

17 5 71 1 1 γΞϊ 177 ° »171 °» ι69 ° »1535» 157 ° »1255» 1170 ο® "1 ·

Analysis by calculating from the formula 1 ° »49.51? Η »4.79 ^ Η, 17.77? S, 10.17.

Experienced: C, 49.58? 49.65 # H, 4.15? 4.59? V, 17.41? 17.66 * S, 10.14.

7- (o-Aminomostylphenylacetamido) -3- (atetrazolo (4,5-6) -pyridazin-6-ylthiomethyl) -5-cephem-4-carboxylic acid (20) To 20 ml of trifluoroacetic acid was added in one portion 15, 80 g (0.022 mol) of product 18 and the mixture were stirred for 45 min. 0.10 ° C sea. To the Eeaktb mixture was added 500 mL of ether to give the product 20 as its trifluoroacetate, which was collected by filtration and washed with ether. The trifluoroacetate was dissolved in 20 ml of water and the pH was adjusted to 5 with ammonium hydroxide to give the zwitterion as a gum, which was separated by decantation and triturated with water. The solid product was collected by filtration, washed with 20 ml of water and 200 ml of acetonitrile successively and dried in vacuo over phosphorus pentoxide to give 5.10 g (45%) of product 20, which was fairly pure but amorphous.

Recrystallization of product 20 - an amorphous powder (5.50 g) was dissolved in 400 ml of a 50% aqueous solution of tetrahydrofuran with heating to 60-70 ° C with vigorous stirring. The solution was treated with a small amount of activated carbon. The filtrate was cooled, the walls of the vessel were scraped with a glass rod and the solution was allowed to stand overnight in the refrigerator to give 2.27 g of product as 20 fine needles melting at 190-195 ° C (decomposes).

Analysis: Calculating from the formula C21H20 ^ 8 ° 4S2 * ^ »5H2O5 c» 46.76? H, 4.50? N, 20.77? S, 11.89.

Experimental: C, 47.18? 47.57? H, 4.08? 5.88? H, 20.95? 20.25? S, 12.05.

19 57111

Example 2 CR-CCCHpCCCEt _ fH3 ^ r ^ V ^ ^ - o = cH-cocEt ^^ N-X'COOH CgHjONa ^^ N ^ COCKa k 23 N — N ^ ^ 3 CICCCEt H - /. C / -S \ = -ύ r ^ | ^ ~ N.'HC = CH-COOEt -> -—-> v ^^ T-r8 ") nm ^>

Nvj ^ 04-S-LCH2

^ co2h ^ HCCCH> CC-H ^ 3 co2h 20.

Sodium o- (1-ethocycarbonyl-1-propen-2-ylaminomethyl) phenylacetate (19)

To an alcoholic solution of sodium ethoxide (sodium metal 9.75 g (0.25 g atom) and absolute ethanol 500 ml) was added 41.26 g (0.25 mol) of o-aminomethylphenylacetic acid (obtained by neutralizing the hydrochloride with 4 aqueous ammonia) and 52.5 g (0.25 mol). moles) of ethyl acetoacetate sequentially. The mixture was refluxed for 6 hours and treated with activated carbon and filtered through diatomaceous earth ("Dicalite"). The filter pad was washed with 200 mL of hot ethanol. The combined filtrate and washings were evaporated to near dryness and cooled to 0 ° C to give product 19 as colorless needles which were collected by filtration, washed with 200 ml of ethanol and dried in vacuo over PgO 2. Solid product 19 weighed 44.51 g * Additional product 19 was obtained by concentrating the mother liquor. Total yield 56.51 g (79 #).

Sp. 251-252 ° C (decomposes).

if 5,7111 7- (o-Aminomethylphenylacetamido) -3- (tetrazolo (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (20)

Ethyl formate (6.87 g, 0.0063 mol) was added in one portion to a stirred suspension of 17 * 44 g (0.037 mol) of sodium O- (1-ethocycarbonyl-1-propen-2-ylaminomethyl) phenylacetate (19) 200 ml of dry THF containing 1 ml of Ν, Ν-dimethylbenzylamine at -13 ° C * Stirring was stopped and a cooled solution of 20.60 g (0.037 mol) of product 14 and 9 * 60 g (0.093 mol) ) triethylamine in 200 mL of a 30% aqueous solution of THP was added slowly along the wall. The mixture was stirred vigorously for 30 min. At 0-13 ° C, treated with activated carbon and filtered. Through "Dicalite" diatomaceous earth. The layer was washed with 30 mL of 30 x aqueous THF containing 8 mL of triethylamine. Formic acid (3 mL) was added to a combined solution of the filtrate and washings to precipitate unreacted 7-ACA (2.3 g), which was filtered off. The filtrate was mixed with 200 ml of ether followed by 13 ml of formic acid. The mixture was stirred for 10-13 min. at room temperature and the resulting precipitate was collected by filtration, washed with 100 ml of ether and 300 ml of water successively and dried in vacuo over P 2 O 4 to give 23.03 g (79 10 of product 20, mp 180-186 ° C ( dec).

Recrystallization of product 20 - The amorphous product described above (12.93 g) was dissolved in 1.2 L of THF at 30-60 ° C with vigorous stirring, treated with 3 g of activated carbon and filtered. The filtrate was seeded and stored in the refrigerator overnight to give 7.80 g of product as 20 fine needles. Sp. 186-189 ° C (decomposes).

Example 3 A suspension of 7- (o-aminomethylphenylacetamido) -3- (tetrazolo (4,3-b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid zwitterionic form (0.36 l g) in 3 ml netanol is cooled with ice. and treated with a few drops of concentrated hydrochloric acid until a clear solution is obtained. 7- (o-Aminomethylphenylacetamido) -3- (tetrazolo (4,5) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid hydrochloride precipitates as a light brown solid upon addition of ether and is collected by filtration and dried in vacuo over PgO. Using ^ xn.

Example 4 To a stirred suspension of the zwitterionic form (0.361 g) of 7- (o-aminomethylphenylacetamido) -3- (tetrateolo (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid is added an aqueous solution of 1 N sodium hydroxide at room temperature. until a clear solution (pH 10.8) is obtained. This solution is freeze-dried immediately to give the crude solid sodium 7- (o-aminoethylphenylacetamido) -3- (tetrazolo (4,5-fc) pyrazin-6-ylthiomethyl) -3'-cephem. -4-carboxylate.

Method for the preparation of dimethanesulfonate Example 3 / = \ S / s \ / »%„

C00H

? 2 »5 4- HOCHgSCyia - {- CH5 (CH2) 3CH-C00Na (30 ^ SEH in acetone) 1 · Heat in water 2« Treat with charcoal and filter 2 * Add the filtrate to anhydrous alcohol.

Ψ.

CH 2 N (CH2S03Na) 2

/ V “X * \ / S

Solid C V-CHg-C-NH-CH CH CHg tf j w / -1 / ^ c * COONa h2o _J_ EHA.

ai 57111

Procedure:

Place 2.0 moles (about 1025 g calculated on the anhydrous basis) of 7- (o-aminomethylphenylacetamido) -3- (tetrazolo- (4,5-b) -pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid, 540 g of sodium formaldehyde bisulfite (4.03 moles), 3000 ml of water and 2700 ml (4 * 87 moles) of $ 30 in SEH (sodium 2-ethylhexanoate) dissolved in acetone in a suitable tank and heat the mixture with stirring to 40-45 ° C . The mixture dissolves in about 10 minutes to a yellow solution.

After heating for 15 minutes, add 50 g of decolorizing carbon ("Parko KB") to the solution and stir for a further 15 minutes at 40-45 ° C.

Filter through tin ("Dicalite") after maintaining the reaction mixture at 40-45 ° C for a total of 30 min.

Wash the carbon cake with 2000 ml of 50 Of which aqueous ethanol. Combine the extracts, adjust the temperature to 25 ° C and add the solution at 25 ° C to 112 liters of rapidly stirred 100 l of ethyl alcohol. A fine, white, amorphous precipitate is formed which is sodium di- (o-aminomethylphenylacetamido) -3- (tetrat-Eolo (4,5-b) pyridazin-6-ylthioethyl) -3-cephem-4-carboxylate -methane sulfonate) ·

Stir the suspension for about 10 min. and then filter and wash the cake with 15 L of 100 of ethyl alcohol.

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

The yield is about 1200-1400 g of amorphous, white, solid sodium 7- (o-aminomethylphenylacetamido) -3- (tetrazolo (4,5-b) pyridatein-6-ylthiomethyl) -3-cephem-4-carboxylate di (natrtummetaan! sulphonate). The product usually contains a few percent water and possibly a small amount of ethanol.

This product may also be called sodium 7- (oH, N-bis (sodium sulfomethyl) aminomethylphenylacetamido) -3- (tetrazolo (4,5-b) pyridatin-6-ylthiomethyl) -3-cephem-4- carboxylate.

Example 6

The following slurry is prepared: 2.19 g of sodium formaldehyde bisulfite (2 equivalents) 3.5 g of 7- (o-aminomethylphenylacetamido) -3- (tetrazolo (4,5-b) pyridazin-6-yl] methyl) -3-cephem-4- carboxylic acid zwitterion (100-200 mesh).

25 ml of water (volume can be varied).

14 ml of 30 SEH isopropanol solution 22 57111

An almost finished solution is obtained after rapid stirring for about 0.5 hours at 24 ° C. The temperature of the mixture is rapidly raised to 40-43 ° C. This is maintained for about 2 minutes and then rapidly cooled to 20-23 ° C.

The solution is filtered to remove small insolubles (total dissolution time should not exceed 2 hours).

A solution with a pH of 7.3 is added for 5 min. during 600 any very rapidly stirred absolute ethanol (other alcohols such as anhydrous isopropanol may be used). An amorphous precipitate of sodium 7- (o-aminomethylphenylacetamido) -3- (tetrazolo (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate di (sodium methanesulfonate) is formed. The mixture is stirred for 5 min. The precipitate is collected by filtration, washed with 60 ml of ethanol (or isopropanol) and dried in vacuo at 50 ° C for 24 hours. The yield is about 4.3 g.

The product is soluble in water at a pH of about 7 in an amount of at least 200 mg / ml. Such a solution is stable for at least 2 hours at room temperature; dilute solutions are stable for longer. The product exhibits the same bactericidal properties as the preceding two ions and is biologically fully active whether or not it is hydrolyzed back to a bis ion.

Example 7

Sodium 7- (o-N, H-bis- (sodium sulfoinethyl) aninomethylphenylacetamido) -3- (tetrateolo (4,5-b) pyridatein-6-ylthiomethyl) -3-cephem-4-carboxylate A. Preparation using hydroxymethanesulfonate

A mixture of 1 g (1.95 mmol) of 7- (o-aminomethylphenylacetamido-3- (tetrateolo (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 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, and the resulting solution was treated with 1 g of activated carbon and poured into with stirring to 300 nl of absolute ethanol and the mixture was stirred at room temperature for 30 min to give a crystalline product which was collected by filtration, washed with three 50 ml portions of absolute ethanol and dried at 45-52 ° C under 1 mm pressure for 20 hours to give 1.51 g of sodium 7- (oN, N-bis (sodium sulfomethyl) aminomethylphenylacetamido) -3- (tetrazole- (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem-4 carboxylate which was readily soluble in water (> 1 g / ml), mp> 270 ° C.

25 5 71 1 1 ΙΕ: γ * 5 * 1760, 1660-1620, 1605, 1540, 1400, 1200, 1040 cm-1.

DftX

UV: λmax 242 nm (ε 18,000), 271 nm (ε 11,600 sh), 312 nm (ε 5,100, sh), max NMR: δ D2 3.34 (1H, d, 18 Hz, S-CH 2), δ , 76 (1H, d, 18 Hz, S-CH2), 3.85 ppm (2H, s, CO-CH2-ii), 4.04 (4H, H-CH2-jrf & 3-CH2-S), 4.30 (4H, b, N-CH 2 -SO 2 Ha), 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, pyridateine-H), 8.11 (1H, d, 10 Hz, pyridazine ni-H).

B. To be prepared using formalin and sodium bisulphite a) To a solution of 1 ml (10 mmol) of 30% formalin and 1 g of sodium bisulphite in 10 ml of water was added successively 1.026 g (2 mmol) of 7- (o-aminomethylphenylacetamido) - 3- (Tetrazolo (4,5-b) pyridatein-6-ylthiomethyl) -3-cephem-4-carboxylic acid, 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 obtained sodium 7- (oN, N-bis (sodium sulfoethyl) aminomethylphenylacetamido) -3- (tetrazolo (4,5-t)) pyridazin-6-ylthiomethyl) -3-phephem-4-carboxylate was collected by filtration, washed with three 50 ml portions of ethanol and dried in vacuo. Yield 1.61 g.

b) To a mixture of 1.026 g (2 mmol) of 7- (o-aminomethylphenylacetamido) -3- (tetrazolo (4,5-b) pyridatein-6-ylthiomethyl) -3-cephem-4-carboxylic acid, 6 ml of a 1 M solution of SEH in ethyl acetate, 10 ml * of isopropanol and 10 ml of water, 1 ml (10 mmol) of formalin were added. The mixture was stirred for 2 hours at room temperature to give a clear solution of a small amount of oily precipitate. After 1 g of sodium bisulfite was added to the solution, it was stirred for another 2 hours, during which time an oily precipitate dissolved in the solution. The reaction mixture was poured into 300 ml of ethanol with vigorous stirring to give 1.61 g of sodium 7- (oH, H-bie (sodium sulfomethyl) aminomethyl-phenyl) ethamido) -3- (tetrazolo (4,5-b) pyridazin-6- ylthiomethyl) -3-cephem-4-carboxylate, which was collected by filtration, washed with three 50 ml portions of ethanol and dried in vacuo.

Example 8 A. 7- (O- (α-Butoxycarbonylaminomethylphenyl) propionamido) -3- (tetrateolo (4,5-b) pyridatein-6-ylthiomethyl) -3-cephea-4-carboxylic acid S4 57111 Η, ΙΡ- Diethylcyclohexylcarbodiimide (0.41 g, 2 moles) was added to a mixture of N- (tert-butoxycarbonylaminomethylphenyl) propionic acid (0.56 g, 2 moles) and 2,4-dinitrophenyl (0.37 g , 2 moles) in 3 x 1 THF and the mixture was stirred for 1 hour at room temperature. The precipitated urea was filtered off. A solution of 7-amino-3- (tetrat Eolo (4,5-b) pyridin-6-ylmethyl) -3-cephem-4-carboxylic acid (0.75 gf) was added to the filtrate. 2 moles) and triethylamine (0.81 g, 8 mmol) in 10 mL of 30 aqueous THP and the mixture was stirred for 18 h at room temperature. * The reaction mixture was washed with ether (2 x 20 mL) and the aqueous layer was acidified to pH 2 with dilute ECl: and extracted with ethyl acetate (3 x 50 mL). The combined extracts were washed with water (30 ml), treated with activated carbon, dried over anhydrous Ha 2 SO 4 and evaporated in vacuo to an oil, the oil triturated with ether to give 7- (8- (α-butoxycarbonylaninomethylphenyl) propionamido) -3- (tetrateolo (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid as a colorless solid which was collected by filtration and dried in vacuo over PgO 2.

Hall 0.33 (£ 26). Sp. 110-120 ° C (decomposes).

IR. 1780, 1710, 1690, 1550, 1370, 1250 cm-1.

i * UV: Ai.? ΔHCO 3 240 nm (ε 19,400), 270 nm (sh) (ε 12,500), 310 nm (sh) “(ε 5000).

M: δ DEG 1.37 (9H, s, t-Bu-H), 4.96 (1H, d, 4Hs, 6-H), 5.55 (1H, dd, 4 4 8 Ha , 7-H), 6.99 (4H, e, phenyl-H), 7.57 (1H, d, 10 Hz, pyridazine-H), 8.38 (1H, d, 10 Hz, pyridazine-H) , 8.69 (1H, d, 8 Hz, CONH).

Analysis calculated by the formula: C 9 H 18 N 2 O 2 • Sg 1 / H 2 O: C, 51.02; H, 4.91; H, 17.63. Experimental: C, 51.74; H, 4.83; N, 17.88.

B. 7- (P- (o-Aminomethylphenyl) propionamido) -3- (tetrazolo (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid Trifluoroacetic acid (0.5 ml) and 7- (trans- (o-tert-butocycarbonylaminomethylphenyl) propionamido) -3 '(tetrazolo (4,5-b) pyridathin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (0.28 g , 0.46 mol) was stirred with cooling to 0 ° C and stirred for 30 min. Setter (50 mL) was added to the mixture to give 7- (8 '(o-aminomethylphenyl) propionamido) -3- (tetrazolo- (4,5-t) -pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid. trifluoroacetate, which was separated by decantation, washed with ether, dissolved in water (1 mL) and the pH was adjusted to 6 with dilute NH 4 OH. The product 7- (8- (O-4 aminomethylphenyl) propionamido) -3- (tetrateolo (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid was collected by filtration and dried. ▼ V in vacuo with PgO 2. Yield 0.10 g (42, mp 190-197 ° C (ha)).

IR: γ * ® * 1760, 1665, 1600, 1535 »cm’1.

211 flUL

Analysis calculated for C 22 H 22 O 8 O 4 S 2 * 1 · 2 H 2 O: C * * 9.33; H »4» 33f N, 20.92; 8, 12.1Θ.

Experimental: C, 49.04; H, 4.26; N, 20.17; S, 11.96.

Example 9 A. Sodium β- (o- (1-ethoxycarbonyl-1-propen-2-ylaminomethyl) -phenyl) propionate

To an alcoholic solution of sodium ethoxide (metallic sodium 3.73 δ (0.23 g atom) and absolute ethanol 500 ml) is added 0.23 mol of β- (o-aminomethylphenyl) propionic acid (obtained by neutralizing its hydrochloride with aqueous ammonia) and 32.3 g (0.25 moles) of ethyl acetoacetate in succession. The mixture is refluxed for 6 hours and treated with activated carbon and filtered through diatomaceous earth ("Oicalite"). The filter pad is washed with 200 ml of hot ethanol. The combined filtrate and washings are evaporated to near dryness and cooled to 0 ° C to give sodium β- (o- (1-ethoxycarbonyl-1-propen-2-ylaminomethyl) phenyl) propionate as colorless needles which are taken by filtration, washed with 200 ml of ethanol and dried in vacuo over PoOc. Additional product is obtained by concentrating «3 mother liquors. Total catch about 50 8 · B. 7- (8- (o-Aminomethylphenyl) proplonamido) -3- (tetrazolo (4,5-b) -pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid

Ethyl chloroformate (6.87 g, 0.0063 mol) is added in one portion to a stirred suspension of 0.037 mol of sodium 8- (o- (1-ethoxycarbonyl-1-propen-2-ylaminomethyl) phenyl) propionate in 200 ml: in dry THF containing 1 ml of Ν, Ν-dimethylbenzylamine at -13 ° C. Stirring is stopped and a cooled solution of 20.80 g (0.037 moles) of 7-amino-3- (tetrazolo (4,5 ”b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid and 9, 60 g (0.093 mol) of triethylamine in 200 ml of 3 ° aqueous THF are slowly added along the wall. The mixture is stirred vigorously for 30 min. At 0-13 ° C, treated with charcoal and filtered through nDicalite "diatomaceous earth. The layer is washed with 50 ml of 50 .mu.l of an aqueous solution of THF containing 8 ml of triethylamine. Formic acid (3 ml) is added to the filtrate and washings. to a combined solution to precipitate unreacted 7-ACA (2.3 g) which is filtered off, 200 ml of ether 57111 u and then 15 ml of formic acid are stirred in. The mixture is stirred for 10-15 minutes at room temperature and the precipitate obtained is collected by filtration, Wash with 100 ml of ether and 500 ml of water successively and dry in vacuo over PgO to give 7- (P- (o-aminomethylphenyl) propionamido) -3- (tetrazolo (4 ', 5-fc) - pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid about 20 g.

Recrystallization of 7- (β- (o-aminomethylphenyl) propionamino) -3- (tetrazolo (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid - as described above the amorphous product (13 g) is dissolved in 1.2 l of a 50% aqueous solution of THF at 50-60 ° C with vigorous stirring, treated with 5 g of activated carbon and filtered. The filtrate is seeded and stored in the refrigerator overnight to give about 8 g of product as fine needles.

Example 10 A suspension of 7- (β- (o-aminomethylphenyl) propionamido) -3- (tetrazolo (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid zwitterionic form (0.361 g) in 3 ml In methanol, the mixture is cooled with ice and treated with a few drops of concentrated hydrochloric acid until a clear solution is obtained. 7- (P- (o-Aminomethylphenyl) propionamido) -3- (tetrateolo (4,5-h) pyridatein-6-ylthiomethyl) -3-cephem-4-carboxylic acid hydrochloride precipitates as a light brown solid upon addition of ether and recovered and dried in vacuo over PgO 2.

Example 11 To a stirred suspension of the zwitterionic form (0.361 g) of 7- (β- (o-aminomethylphenyl) propionamido) -3- (tetrazolo (4,5-b) pyridatin-6-ylthiomethyl) -3-cephem-4-carboxylic acid is added. Aqueous 1 N sodium hydroxide solution at room temperature until a clear solution (pH 10.8) is obtained. This solution is freeze-dried immediately to give crude solid sodium 7- (β- (o-aminomethylphenyl) propionamido) -3- (tetrazolo (4'-b) pyridazin-6-yl-thiomethyl) -3-cephem-4 carboxylate instead.

27 57111

Methods for preparing dimethanesulfonate Example 12 ch2nh2 • 1 O 3 · <T ^ ch2_ch2-c-nh-ch-c / \ h2 J — fy / WXc ^ c ~ CHg ~ s \ _ /

COOH

? 2 "5“ H HOCHgSO 2 Na - {- CH 2 CH 2 ^ CH-COONa · (30 ^ SEH in acetone) 1. Heat in water 2. Treat with charcoal and filter 5. Add the filtrate to anhydrous alcohol Ψ · CH 2 N (CH 2 SO 3 Na) 2 / r \ 8 / s \ / ** 5,

Solid (/ yCH2 -CH2-C-NH-CH CH CHO N— / / C · COONa - |. H2o_j_eha.

29 57111

Procedure:

Place 2.0 moles (about 1033 g calculated on the anhydrous basis) of 7- (β- (o-aminomethylphenyl) prepionido) -3- (tetrazolo (4,5-b) pyridatein-6-ylthiomethyl) -3-cephem -4-carboxylic acid, 540 g of sodium formaldehyde bisulfite (4.03 moles), 3000 ml of water and 2700 ml (4.87 moles) of a solution of 30 [mu] l of SEH (sodium 2-ethylhexanoate) in acetone in a suitable tank and heat the mixture with stirring. -45 ° C. The mixture dissolves in about 10 minutes to a yellow solution.

After heating for 15 minutes, add 50 g of decolorizing carbon ("Darco KB") to the solution and stir for a further 15 minutes. 40-45 ° C esa. Filter through diatomaceous earth (Dicalite) after maintaining the reaction mixture at 40-45 ° C for a total of 50 min.

Wash the carbon cake with 2000 ml of 50 l of aqueous ethanol. Combine the filtrates, adjust the temperature to 25 ° C and add the solution at 25 ° C to 112 L of rapidly stirred 100% ethyl alcohol. A fine, white, amorphous precipitate is formed which is sodium 7- (O- (o-aminonethylphenyl) -propionamido) -5- (tetrahydro (4,5-t)) pyridin-6-ylthiomethyl) -3-cephem-4-carboxylate di- (sodium methanesulfonate),

Stir the suspension for about 10 min. and then filter and wash the cake with 15 1 of 100 of ethyl alcohol.

Dry the cake at 50-55 ° C ** ea in a convection oven for about 2 hours and then under vacuum at 4-6 mm for 24 hours.

The yield is about 1200-1400 g of amorphous white solid sodium 7- (O- (o-aminomethylphenyl) propionamido) -3- (tetrazolo- (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem- 4-carboxylate di (sodium methanesulfonate).

The product usually contains a few percent water and possibly a small amount of ethanol.

This product is also known as sodium 7- (O- (oN, N-bis- (sodium sulfomethyl) aminomethylphenyl) propionamido) -3- (tetrazolo (4,5-b) -pyridazin-6-ylthiomethyl) -3-cephem- 4-karbokaylaatti.

Example 13

The following slurry is prepared: 2.19 g of sodium formaldehyde bisulfite (2 equivalents) 3.5 g of 7- (O- (o-aminomethylphenylpropionamido) -3- (tetrat8olo (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem 4-Carboxylic acid crude ion (100-200 mesh) in 25 ml of water (volume can be varied) in 14 ml of 30 isopropanol solution of SEH.

An almost finished solution is obtained with rapid stirring at 29 ° C for about 0.5 hours at 24 ° C. The temperature of the mixture is rapidly raised to 40-43 ° C. This is maintained for about 2 minutes and then rapidly cooled to 20-23 ° C.

The solution is filtered to remove small insolubles (the total dissolution time el must not exceed two hours).

A solution with a pH of 7 »3» is added for 5 min. to 600 ml of very rapidly stirred absolute ethanol (other alcohols such as anhydrous iropropanol may be used). Formed amorphous sodium 7- (β- (o-aminomethylphenyl) propionamido) -3- (tetrazolo (4,3-b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate di (sodium methanesulfonate) sediment. The mixture is stirred for 3 min. The precipitate is collected by filtration, washed with 60 ml of ethanol (or isopropanol) and dried in vacuo at 50 ° C for 24 hours.

The catch is about 4 g.

The product is soluble in water at a pH of about 7 in an amount of at least 200 mg / ml. Such a solution is stable for at least 2 hours at room temperature; dilute solutions are stable for longer. The product exhibits the same bactericidal properties as its preceding zwitterion and is biologically fully active whether or not hydrolyzed back to the zwitterion.

Example 14

Sodium 7- (β- (oN, N-bis (sodium sulfomethyl) aminomethylphenyl) propionamido) -3- (tetrazolo (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate Λ . Preparation using hydroxymethanesulfonate A mixture of 1.93 mmol of 7- (6- (o-aminomethylphenyl) propionamido) -3- (tetrazolo (4,5-b) pyridazin-6-ylthionethyl) -3-cephem-4- carboxylic acid, 1.32 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 hours. The resulting solution is treated with 1 g of activated carbon and poured with stirring into 300 ml of absolute ethanol, and the mixture is stirred at room temperature for 30 minutes. to give a crystalline product which is collected by filtration, washed with three 30 ml portions of absolute ethanol and dried over PoOc at 45-52 ° C under 1 mm pressure for 20 hours to give about 1.3 g of sodium 7- (β- (ο-N, N-bis (sodium sulfomethyl) aminomethylphenyl) propionamido) -3- (tetrasolo- (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate, which is readily soluble water (> 1 g / ml).

57111 50 B. Preparation using formalin and sodium bisulphite (a) To a solution of 1 ml (10 mmol) of $ 30 formalin and 1 g of sodium bisulphite in 10 ml of water are added successively 2 mmol of 7- (o-aminomethylphenyl) propionamido) - 3- (Tetrazolo (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid, 6 ml of 1 M SEC solution and 10 ml of isopropanol.

The mixture is stirred for 2.5 hours at room temperature and poured into 500 ml of ethanol. The resulting sodium 7- (3- (oN, N-bis (sodium sulfomethyl) aminomethylphenyl) propionamido) -3- (tetrazolo (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate is taken recovered by filtration, washed with three 50 ml portions of ethanol and dried in vacuo. Yield about 1.5 g · b) To a mixture of 2 mmol of 7- (3- (o-aminomethylphenyl) propionamido) -3 - »(tetrazolo (4,5 ** fc) P3nPlazin-6-ylthiomethyl) - 3-Cefene-4-carboxylic acid, 6 ml of a 1 M solution of SEH in ethyl acetate, 10 ml of isopropanol and 10 ml of water are added 1 ml (10 mmol) of 50 .mu.l of formalin. The mixture is stirred for 2 hours at room temperature to give a clear solution with a small amount of oily precipitate. After adding 1 g of sodium bisulfite to the solution, it is stirred for a further 2 hours, during which time the oily precipitate dissolves in the solution. The reaction mixture is poured into 300 ml of ethanol with vigorous stirring to give about 1.5 g of sodium 7- (β- (oN, N-bis (sodium sulfomethyl) aminomethylphenyl) propionamido) -3- (tetrazolo (4-5 (Fc) Pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate, which is collected by filtration, washed with three 50 ml portions of ethanol and dried in vacuo.

In vitro and In vivo studies 7 - ((o-aminomethyl) phenylpropionamido) -3- (tetrazolo (4,5-b) pyridazin-6-ylmethyl) -3-cephem-4-carboxylic acid is a novel, broad-spectrum a semi-synthetic cephalosporin having the structure 0 UUv-w '·' 'shown below,

cocP

It has been shown to have excellent in vitro and in vivo activities against a variety of gram-positive and gram-negative bacteria, kuuluvat * 5i 57111 including those that resist cephalothin and cephaloridine. Its dimethanesulfonate adduct having the structure / CH 2 -N (CH 2 SO 3 Na) 2 -S N-CO 2 Na is prepared as a water-soluble preparation useful for injection and in the study of absorption and excretion, acute toxicity and tendency to pain upon injection.

Initial studies of in vitro antibacterial activity by the tube dilution method or the agar dilution method using this new cephalosporin showed minimum inhibitory concentrations (MICs) of less than 1.0 μg / ml in all or almost all species tested, namely Staphylococcus aureoc, Biploceus and Streptococcus py values were usually less than 4 ug / ml and often less than 1.0 ug / ml against various Salmonella enteritidis and Enterobacter cloacae species.

The in vivo efficacy of this compound by subcutaneous injection was studied in mice with a test infection induced by a pathogenic, penicillinase-positive S. aureus species. The mean curative dose (CD, β) was about 1.6 mg / kgv 7 - ((o-aminomethyl) phenylacetamido) -3- (tetrazolo (4,5-b) pyridazin-6-ylthiomethyl) -3-cephem-4 -carboxylic acid is a novel broad-spectrum semi-synthetic cephalosporin having the structure shown below.

Θ ΟΚ, -Μ, UL-co-S - ^ \ 0X ~ * -yj- COO ^ 52 57111

It has been shown to have excellent in vitro and in vivo activities against a variety of gram-positive and gram-negative bacteria, including those that are resistant to cephalothin and cephaloridine. Its dimethane sulfonate adduct having the structure aCH_-N (CH 2 SO, Na) 2

H .S

CH-CO-U-r-f | N - N j COgNa was prepared as a water-soluble preparation »useful as an injection and in the study of absorption and excretion» acute toxicity and tendency to pain upon injection.

Initial studies of in vitro antibacterial activity by the tube dilution method or the agar dilution method using this new cephalosporin showed minimum inhibitory concentrations (MICs) of less than 1.0 μg / ml in all or almost all species tested, namely Staphylococcus aureus, Streptococcus pneumoniae, various Bacillus species such as Bacillus antharacis, mycoides and cereus, Klebsiella pneumoniae, Proteus rettgeri, Shigella and in particular Shigella flexneri, Salmonella enteritidis and Salmonella typhosa and MIC values were usually less than 4 ug / ml and often less than 1.0. μg / ml against different species of Escherichia coli, Klebsiella unspecified, Proteus vulgaris, Proteus morganii, Proteus mirabilis and Enterobacter Cloacae.

This series of gram-negative test organisms contained 14 cephalothin-resistant Enterobacteriacae (3 E. coli, 6 Proteus, 1 Enterobacter, 1 Shigella, and 3 Serratia) that were not inhibited by 100 μg / ml cephalothin and in many cases 100 μg / ml cephaloridine and cefapirin. nor did it prevent.

The compound of this invention was remarkably active against these organisms, inhibiting 12 of these 14 species at a concentration of 6.3 μg / ml or less.

The MICs of this compound did not significantly deteriorate in the presence of moderate concentrations of human blood, e.g., up to 30 #. The compound was orally stable in solution at 37 ° C in the pH range ranging from 4.6 to 8.4 with half-lives exceeding 24 hours.

The in vivo efficacy of this compound by subcutaneous injection was studied in mice with experimental infections caused by ten pathogenic bacteria, including penicillinase-positive 8. aureus and cephalosporinase-positive B. coli. The bacteria used were species S, aureus, S, pyogenes, Dpneumoniae, B.coli, K.pneumoninae, P.vulgaris and S.marcescens. The mean healing dose (CD_ „) never exceeded 20 mg / kg, only twice exceeded 10 mg / kg and in seven cases was 3.2 πφ / kg or less with a minimum value of 0.2 mg / kg.

Concentrations in mouse blood after subcutaneous injection of this compound at doses as low as 5 me / kg were determined and showed excellent absorption) the urine yield was good (about 60 #) and urinary paper chromatographic examination showed this compound to be the only bioactive substance present, indicating metabolic stability.

Acute toxicity studies in mice at subcutaneous doses as high as 1000 mg / kg showed no lethality. Two experiments designed to measure local tissue irritation after injection did not show irritation at concentrations of 12.3 μl or less.

Claims (1)

si. 57111 Claim: Process for the preparation of antibacterial 7- (o-aminomethylphenylacetamido and propionamido) -3- (tetrazolo [1,5-b] pyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acids having the formula _, ch2nh2 ow oj COOH wherein n is 1 or 2 and non-toxic pharmaceutically acceptable salts thereof; characterized in that the compound of formula ¥ T1 | 1 II oi-NN COOH or a salt thereof is reacted with a reactive acid derivative of the formula ch2nhb // X. (CH.) -COOH III \ Jr 2n wherein B is an amino protecting group and n is the same as above to prepare a compound, of the formula _JCHgNHB f ~ \ (CH2) nSm -, - IV COOH wherein B and n are the same as above or for the preparation of a salt thereof, and then the amino-protecting group B is removed to prepare the desired compound of formula I or a non-toxic pharmaceutically acceptable salt thereof .