NO170936B - ANALOGY PROCEDURE FOR THE PREPARATION OF THERAPEUTIC ACTIVE, SUBSTITUTED VINYL Cephalosporins - Google Patents

Abstract

Vinylcephalosporins of the formula (I) <IMAGE> in which the symbols R<1>, R<2>, R<3>, R<4> and R<5> have the meaning given in the description.

Description

The present invention relates to an analogous method for the production of therapeutically active β-lactam compounds of formula I

and pharmaceutically acceptable salts thereof.

It is known that different representatives of 7-a-amino-acylcephalosporins with different substituents in the 3-position of the molecule, thus e.g. cephalexin [7-(D-α-phenylglycylamido)-3-methyl-3-cephem-4-carboxylic acid, comp. DE-OS 24 32 485], cefaclor [7-(D-α-phenylglycylamido)-3-chloro-3-cephem-4-carboxylic acid, comp. DE-OS 24 08 698 and 27 28 578] and cefadroxil [7-(D-a-p-hydroxyphenylglycylamido)-3-methyl-3-cephem-4-carboxylic acid, comp. DE-OS 27 18 741] has a good antibiotic effect.

Furthermore, 3-alkenyl-substituted cephalosporins are mentioned as orally active compounds in DE-OS 34 02 642 and US-PS 4 619 925.

Benzothiazolylglycylamido-substituted vinyl cephalosporins are known from DE-OS 35 08 258. The present invention relates to a selection of the substances mentioned in DE-OS 35 08 258.

Analogous process for the preparation of therapeutically active β-lactam compound of formula I and pharmaceutically acceptable salts thereof, characterized by

[A] substituted cephalosporin compounds of formula

(II)

there

X means a group of formula

while

R13 and R<14> are the same or different and mean alkyl,

phenyl or tolyl

and

zØ means a halide anion, preferably chloride, bromide or iodide,

reacted in inert solvents in the presence of bases with aldehydes of formula (III)

or that

[B] carboxylic acids of the general formula (VI)

in which

R<3>' means an amlnoprotecting group,

after activation of the carboxyl group by transfer to a mixed anhydride, for example with chloroformate ethyl ester or chloroformate isobutyl ester or methanesulfonic acid chloride or by transfer to the acid halide or by transfer to an activated ester, for example with N-hydroxybenztriazole and dicyclohexylcarbodiimide (DCC),

reacted with vinylcephalosporin amines of the general formula

(VIII)

then any protective groups are removed and the desired pharmaceutically acceptable salts or from the salts the free acids are prepared in a known manner.

Amino-protecting groups within the framework of the above-mentioned definition usually mean one of the common protecting groups in g<->lactam chemistry from the series: benzyl, tert-butoxycarbonyl, benzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, fluorenyl-( 9)-methoxycarbonyl, N-diphenyl-methoxycarbonyl, acetoacetyl, 2-nitrobenzoyl, 2-nitrophenyl-sulphenyl, phthaloyl, trityl, vinyloxycarbonyl, formyl, benzyloyl, allyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 2-methylthio-ethoxycarbonyl, 1 ,3-dithian-2-ylmethoxycarbonyl (Dmoc), trimethyl-, triethyl, triphenylsilyl, tert-butyl-dimethylsilyl, tert-butyl-diphenylsilyl, l-methyl-2-benzoyl-vinyl, l-methyl-2- methoxycarbonyl-vinyl, l-methyl-2-ethoxycarbonyl-vinyl, l-methyl-2-(2,6-dimethoxybenzoyl)-vinyl, 4-methoxy-benzyloxycarbonyl (see E. Wilnsch, Methoden der Organischen Chemie, Houben-Weyl, Volume 15/1 (1974)).

The compound produced according to the invention can be used to treat bacterial infectious diseases.

Suitable inert solvents are the usual organic solvents, which do not change under the reaction conditions. These preferably include ethers such as diethyl ether, butyl methyl ether, dioxane or tetrahydrofuran, or hydrocarbons such as benzene, toluene, xylene or cyclohexane, or amides such as dimethylformamide or hexamethylphosphoric acid triamide, or alcohols such as methanol, ethanol, propanol or isopropanol, or chlorinated hydrocarbons such as methylene chloride , chloroform or carbon tetrachloride, or acetone, acetonitrile or acetate. Likewise, it is possible to use mixtures of the mentioned solvents.

The usual basic compounds are suitable as bases. These preferably include alkali or alkaline earth hydroxides, such as sodium hydroxide, potassium hydroxide or barium hydroxide, or alkali carbonates such as sodium carbonate, sodium hydrogen carbonate or potassium carbonate, or alkali alkali alcohols such as sodium methanolate, sodium ethanolate, potassium ethanolate, potassium ethanolate or potassium tert-butylate.

The reaction is usually carried out in a temperature range of -30°C to +80°C, preferably from 0°C to 30°C.

The turnover can be carried out at normal, elevated or reduced pressure (e.g. in a range of 0.5 to 5 bar). Usually you work at normal pressure.

The compound produced according to the invention is effective against a very broad spectrum of microorganisms. With their help, gram-negative and gram-positive bacteria and bacteria-like microorganisms can be fought, and the diseases caused by their producers can be prevented, improved and/or cured.

The compound produced according to the invention is particularly effective against bacteria and bacteria-like microorganisms. They are therefore particularly well suited for prophylaxis and chemotherapy of local and systemic infections in human and veterinary medicine and which are produced by these producers.

For example, local and/or systemic diseases caused by the following agents or by mixtures of the following agents can be treated and/or prevented: Gram-positive cocci, e.g. staphylococci (Staph. aureus, Staph. epidermidis) and streptococci (Strept. agalactiae, Strept. faecalis, Strept. pneumoniae, Strept. pyogenes); gram-negative cocci (Neisseria gonorrhoeae) as well as gram-negative rods such as enterobacteriaceae, e.g. Escherichia coli, Hamophilus influenzae, Citrobacter (Citrob. freundii, Citrob. divernis), Salmonella and Shigella; further klebsiels (Klebs. pneumoniae, Klebs. oxytoca), enterobacters (Ent. aerogenes, Ent. agglomerans), Hafnia, Serratia (Serr. marcescens), Proteus (Pr. mirabilis, Pr. rettgeri, Pr. vulgaris), Providencia, Yersinia , as well as the genus Acinetobacter. In addition, the antibacterial spectrum includes the genus Pseudomonas (Ps. aeruginosa, Ps. maltophilia) as well as strictly anaerobic bacteria such as e.g. Bacteroides fragilis, representatives of the genus Peptococcus, Peptostreptococcus and the genus Clostridium; further mycoplasmas (M. pneumoniae, M. hominis, M. urealyticum) as well as mycobacteria, e.g. Mycobacterium tuberculosis. In particular, the compound produced according to the invention works against staphylococci, streptococci, enterococci and Hemophilus influenzae. When administered parenterally or especially orally, the new compounds are very effective against microorganisms such as staphylococci, streptococci, enterobacteriases, Escherichia coli, Klebsiella, Salmonella, Shigella and Proteus.

The above list of producers is only exemplary and should not be construed as limiting in any way. As diseases, which are caused by the aforementioned agents or mixed infections and can be prevented, improved or cured by the compound produced according to the invention, the following should be mentioned, for example: Infectious diseases in humans, such as Otitis, Pharyngitis, Pneumonia, Peritonitis, Pyelonephritis, Cystitis, Endocarditis, Systemic infections, Brochitis (acute, chronic), septic infections, diseases of the upper respiratory tract, diffuse Panbronchiolitis, pulmonary emphysema, dysentery, Enteritis, liver abscesses, Urethritis, Prostatitis, Epididymitis, gastrointestinal infections, bone and joint infections, cystic Fibrosis, skin infections, postoperative -tive wound infections, abscesses, Phlegmons, wound infections, infected burns, burns, infections in the mouth area, infections after dental surgery, Osteomyelitis, septic Arthritis, Cholecystitis, Peritonitis with Appendicitis, Cholangitis, intra-abdominal abscesses, Pancreatitis, Sinusitis, Mastoiditis, Mastitis, Tonsillitis, Typhoid , Meni ngitis and infections of the nervous system, Salpingitis, Endometritis, genital infections, Pelveoperitonitis and eye infections.

Besides in humans, bacterial infections can also be treated in other species. For example, the following should be mentioned: Pigs: Coll diarrhoea, Enterotoxemia, Sepsis, Dysentery, Salmonellosis, Metritis-Mastitis-Agalactiae syndrome, Mastitis; Ruminants (cattle, sheep, shepherds): diarrhoea, Sepsis, Bronchopneumonia, Salmonellosis, Pasteurellosis, Mycoplasmosis, Genital infections;

Horse: Bronchopneumonia, foal paralysis, puerperal and post-puerperal infections, Salmonellosis;

Dog and cat: Bronchopneumonia, diarrhoea, Dermatitis, Otitis, urinary tract infections, Prostatitis;

Poultry (hen, turkey, quail, pigeon, ornamental birds and others): Mycoplasmosis, E. coli infections, chronic respiratory diseases, Salmonellosis, Pasteurellosis, Psittacosis.

Likewise, bacterial diseases can be treated when breeding and keeping commercial and ornamental fish. As the antibacterial spectrum beyond the above-mentioned pathogens is extended to further pathogens, such as Pasteurella, Brucella, Campylobacter, Listeria, Erysipelo-thrix, Corynbacteria, Borellia, Treponema, Nocardia, Rickettsien, Yersinia.

In the following tables, the minimum inhibitory concentration (MHK values, pg/ml) for example 6 compared to Cefaclor [M. Gorman et al., Cefaclor, Chronicles of Drug Discovery, Vol. 2, 49, J. Wiley & Sons (1983)]. The MHK values were determined by agar dilution test using a Multipoint inoculator, the reading taking place after 18 to 24 hours of cultivation at 37<*>C. Isotenside test agar is used as growth medium. Generally, in both human and veterinary medicine, it has proven beneficial to administer the active substance(s) in total amounts of approx. 0.5 to approx. 500, preferably 5 to 100 mg/kg body weight every 24 hours, possibly in the form of several single administrations to achieve the desired results. A single administration contains the active substance produced according to the invention, preferably in amounts of approx. 1 to 80, especially 3 to 20 mg/kg body weight. However, it may be necessary to deviate from the aforementioned dosages, namely depending on the type and body weight of the object to be treated, the type and severity of the disease, the type of preparation and application of the medicine as well as the time period or the interval within which the administration takes place.

Thus, in some cases it may be sufficient to come out with less than the above-mentioned amount of active substance, while in other cases the above-mentioned amount of active substance must be exceeded. Determination of the resp. the necessary optimal dosage and type of application of the active substance can easily be carried out by any professional due to his professional knowledge.

The new compound can be had in the usual concentrations and preparations together with for resp. with preparations or with drinking water. Thereby, an infection can be prevented, improved and/or cured due to gram-negative or gram-positive bacteria, thereby achieving a promotion of growth and an improvement in utilization of the forage.

The compound produced according to the invention can be combined with other antimicrobial active substances and lactamasein inhibitors, e.g. with penicillins, which are particularly penicillin-linasefast and clavulanic acid. Such a combination would e.g. be the one with oxacillin or dicloxacillin.

The compound produced according to the invention can also be combined with aminoglycoside antibiotics, such as e.g. gen-tamicin, sisomycin, kanmicin, amikacin or tobramicin.

Progress examples

Example 1 (Intermediate)

7-amino-3-chloromethyl-3-cephem-4-carboxylic acid benzhydryl ester

To a suspension of 50 g (0.0972 mol) 7-phenylacetamido-3-hydroxymethyl-3-cephem-4-carboxylic acid benzhydryl ester in 500 ml methylene chloride, 19.64 ml (0.242 mol) pyridine is added at room temperature. After cooling to -20°C, 40.48 g (0.0972 mol) of phosphorus pentachloride are added and stirred for 5 minutes at -20°C. With an ice bath, heat to 0°C and stir for 10 minutes, then heat with a water bath for 15 °C and stirred for 1 hour. Then the mixture is cooled to -70 °C and 720 ml of cold methanol is quickly added. Then it is stirred for 5 minutes at -70 °C, 10 minutes at 0 °C and 25 minutes at 15 °C. Then the solution is evaporated strongly in vacuum and mixed with 1,400 ml of saturated sodium bicarbonate solution. This solution is extracted 3 times with methylene chloride, the organic phase is dried with sodium sulfate and evaporated in vacuum. The crude product is chromatographed on 500 g of silica gel 60 (0.04-0.063 mm) with methylene chloride.

Yield: 29.0 g (72% of the theoretical)

C21H19C1N2°3S (414.9)

NMR (CDCl 3 ) δ = 2.06 (s, 2H); 3.45 (d, 1H), 3.62 (d, 1H);

4.25-4.41 (q, 2H); 4.75 (d, 1H); 4.93 8d, 1H); 6.97 (s, 1H); 7.25-7.46 (m, 10H) ppm.

Example 2 (Intermediate)

D-7 - [2 - (t-butoxycarbonylamino)-2-(2-aminobenzothiazol-6-yl)-gly cylam i do]-3-chloromethyl-3-cephem-4-carboxylic acid benzhydryl ester

To a mixture of 28.3 g (0.0875 mol) of D-α-t-butoxycarbonyl-amino-α-(2-aminobenzothiazol-6-yl)acetic acid and 24.1 g (0.058 mol) of 7-amino-3-chloromethyl-3 -cephem-4-carboxylic acid benzhydryl ester (Example 1) in 136 ml of tetrahydrofuran and 77 ml of dimethylformamide, 18.07 g (0.0875 mol) of N,N'-dicyclohexylcarbodiimide (DCC) dissolved in 150 ml of tetrahydrofuran at 0°C are added , then stir for 2 hours at room temperature and evaporate to dryness. The residue is suspended in 1,200 ml of vinegar, stirred for 10 minutes and thus undissolved components are separated by suction. After distillation from ethyl acetate, the residue is chromatographed on silica gel 60 (0.04-0.063 mm) with toluene/ethyl acetate (1:1).

Yield: 17.6 g (42SÉ of the theoretical)

C35H34C1N5S206 (720.3)

NMR (DMSO): S = 1.37 (s, 9H); 3.46 (d, 1H); 3.64 (d, 1H);

4.32-4.43 (q, 2H); 5.11 (d, 5Hz, 1H); 5.31 (d, 1H); 5.8-5.86 (d, 1H); 6.98 (s, 1H);

7.15-7.5 (mm, 14H); 7.67 (s, 1H) ppm.

Example 3 (Intermediate)

D-7-[2-( t -butoxycarbonylamino )-2-( 2-aminobenzothiazol-6-yl)glycylamido]-3-iodomethyl-3-cephem-4-carboxylic acid benzhydryl ester

A mixture of 20.3 g (0.0282 mol) of D-7-[2-(t-butoxycarbonylamino)-2-(2-aminobenzothiazol-6-yl9-glycylamido]-3-chloromethyl-3-cephem-4- Carboxylic acid benzhydryl ester (Example 2) and 12.68 g (0.0846 mol) of sodium iodide in 300 ml of acetone are stirred for 2 hours at room temperature and evaporated to dryness. The residue is taken up in 500 ml of acetic acid, washed with an aqueous sodium thiosulphate solution, water and sodium chloride solution. After drying over sodium sulphate is distilled off from the solvent and the residue is digested in ether.

Yield: 22 g

The connection is used directly in the next step.

Example 4 (Intermediate)

D-7 -[2-(t-butoxycarbonylamino)-2-(2-aminobenzothiazo1-6-yl)glycylamido]-3-(triphenylphosphonio)methyl-3-cephem-4-carboxylic acid benzhydryl ester iodide

A mixture of 22 g (0.0271 mol) of D-7-[2-(t-butoxycarbonylamino)-2-(2-aminobenzothiazol-6-yl)glycylamido]-3-iodomethyl-3-cephem-4-carboxylic acid- benzhydryl ester (example 3) and 21.32 g (0.0813 mol) of triphenylphosphine in 500 ml of acetic acid are stirred for 1 hour at room temperature. The product falls out after 30 minutes. The mixture is evaporated under reduced pressure to approx. 150 ml and mixes the concentrate with 500 ml of ether. The formed precipitate is filtered off and washed with ether.

Yield: 19.6 g (67% of the theoretical)

<C>53H4glN506PS2 (1,074.1)

NMR (DMS0): δ = 1.35 (s, 9H); 3.32-3.42 (dd, 2H); 4.81-4.93

(t, 2E); 5.2 (d, 1H); 5.33 (d, 1H); 5.72-5.79 (q, 1H); 6.24 (s, 1H); 7.2-7.49 (mm, 15H); 7.6-7.79 (m, 15H) ppm.

Example 5

D-7-[2-(t-butoxycarbonylamino)-2-(2-aminobenzothiazol-6-yl)glycylamido]-3-[(Z)-1-propen-1-yl]-3-cephem-4-carboxyl -acid benzhydryl ester

For a cold solution of 6.08 g (0.07 mol) lithium bromide in 50 ml dimethylformamide and 150 ml methylene chloride, at -5°C you have 7.04 ml (0.126 mol) acetaldehyde and 7.6 g (0.007 mol) D -7-[2-(t-butoxycarbonylamino)-2-(2-aminobenzothiazol-6-yl)-glycylamin do]-3-(triphenylphosphonio)methyl-3-cephem-4-carboxylic acid benzylhydryl ester -iodide (Example 4). The mixture is stirred for 20 hours at -5°C and then for 5 hours at room temperature. The solution is evaporated in vacuo to approx. 50 ml and distributed in a solvent mixture of 200 ml vinegar and 200 ml water. The upper layer is separated and washed once with aqueous sodium chloride solution. After drying over sodium sulfate and distilling off the solvent, the residue is taken up in toluene and applied to a column packed with silica gel (0.04-0.063 mm). First elute with toluene and then with the solution mixture toluene/acetic ester (5:1) and toluene/acetic ester (1:1).

Yield: 2.9 g ($58 of the theoretical)

C37H37N506S2 (711.9)

NMR (CDCl 3 ): δ = 1.35 (dd, 3H); 1.43 (s, 9H); 3.15 (d, 1H);

3.31 (d, 1H); 4.97 (d, 1H); 5.3 (s, 1H);

5.46-5.55 (m, 1H); 5.71 (width s, 2H);

5.78-5.85 (q, 1H); 6.03 (d, J = Hz, 1H);

6.87 (s, 1H); 7.2-7.4 (mm, 12H); 7.5 (s, 1H) ppm.

Example 6

D-7-[(2-aminobenzothiazol-6-yl)glycylamido]-3-[(Z)-1-propen-1-yl]-3-cephem-4-carboxylic acid, cis isomer

2.9 g (4.1 mmol) D-7-[2-(t-butoxycarbonylamino)~2-(2-aminobenzothiazol-6-yl)glycylamido]-3-[(Z)-1-propen-1- yl]-3-cephem-4-carboxylic acid benzhydryl ester (example 5) is dissolved in 20 ml of methylene chloride, mixed with 40 ml of trifluoroacetic acid (TFA) and stirred magnetically for 60 minutes at room temperature. Methylene chloride and trifluoroacetic acid are removed in vacuo, the remaining red oil is triturated in ether, filtered off with suction and washed with ether. The pale yellow trifluoroacetate is dried in a vacuum, then suspended in 100 ml of water, filtered off from insoluble yellow flecks over silica gel and washed with 30 ml of water. The still slightly cloudy solution is again filtered over a membrane filter (Millipore, 0.45 pm). The filtrate is pumped onto an RP 18 column (Hibar 250-25, Merck). The column is first eluted with 200 ml of water (fraction 1), then with 400 ml of 55^-mg methanol (fraction 2) and finally with 105^-mg of methanol, as the resp. collect 300 ml fractions (fractions 3 to 12). The fractions are examined using analytical HPLC and fractions 6 to 10, which contain the desired peaks, are combined, methanol is distilled off in vacuum and lyophilized.

Yield: 400 mg

C19H19N5°4S2 (445.5)

NMR (DCOOD): S = 1.67 (dd, 3H); 3.41 (d, 1H); 3.55 8d, 1H);

5.3 (dd, 1E); 5.78 (s, 1H); 5.81-5.91 (q and m, 2H); 6.25 (d, J = 11.6 Hz, 1H); 7.81-7.9

(q, 2H); 8.18 (s, 1H) ppm.

HPLC analytical: Hibar 250-4, RP-8, 10 pm, 254 nm Eluent: 1000 ml CH3CN - 30 ml acetic acid, 870 ml water

Flow: 4 ml/min., concentration: 1 mg/ml Retention: 3.22 (content: 97.356).

Example 7

D-7-[(2-aminobenzothiazol-6-yl)glycylamldo]-3-[(E)-1-propen-1-yl]-3-cephem-4-carboxylic acid, trans isomer

From the preparative high pressure liquid chromatography of D-7-[(2-aminobenzothiazol-6-yl)glycylamido]-3-[(Z)-l-propen-l-yl]-3-cephem-4-carboxylic acid ( cis-isomer; example 6) the E-isomeric compound is prepared by further elution with 30% methanol.

Yield: 137 mg

C19H19N5°4S2 (445.5)

NMR (CDOOD): S = 1.9 (d, 3H); 3.61 (s, 2H); 5.25 (d, 1H);

5.72 (s, 1H); 5.87 (d, 1H); 6.23-6.46 (m, 1H); 7.04 (d, J = 15.8 Hz, 1H); 7.8-7.88

(q, 2H); 8.18 (s, 1H) ppm.

HPLC analytical: Hibar 250-4, RP-8 10 pm, 254 nm Eluent: 100 ml CH3CN - 30 ml acetic acid - 870 ml water

Flow: 4 ml/min., concentration: 1 mg/ml

Retention: 5.20 (content: 67.2%)

Example 8 (Intermediate)

7-phenylacetamido-3-(triphenylphosphonium)methyl-3-cephem-4-carboxylic acid benzhydryl ester iodide

32.5 g (0.0609 mol) of 7-phenylacetamldo-3-chloromethyl-3-cephem-4-carboxylic acid benzhydryl ester are dissolved in 330 ml of acetone and mixed sequentially with stirring with 10.1 g (0.0674 mol) of Nal and 17.6 g (0.0671 mol) of triphenyl osf in. After stirring for 1.5 hours at room temperature, the insoluble material is separated by suction and the clear mother liquor is stirred into 1000 ether. The precipitated white fluffy material is suctioned off, washed with 300 ml of ether and dried in a vacuum.

Yield: 51 g ($94 of the theoretical)

C47H40IN2O4PS (886.8)

NMR (DMSO): S = 3.51-3.61 (q, 4H); 4.93-5.05 (t, 1H); 5.22-5.33 (d and t, 2H); 5.7-5.76 (q, 1H); 6.26 (s, 1H); 7.21-7.46 (mm, 15H); 7.68-7.79 (m, 15H); 9.14 (d, 1H) ppm.

Example 9 (Intermediate)

1. 7-phenylacetamido-3-[(Z)-propen-1-yl]-3-cephem-4-carboxylic acid benzhydryl ester

15.9 g (17.9 mmol) of 7-phenylacetamido-3-(triphenylphosphonio)-methyl-3-cephem-4-carboxylic acid benzhydryl ester iodide (Example 8) is placed in a 250 ml three-necked flask in 100 ml of methylene chloride and 12.8 ml (229.6 mmol) of acetaldehyde. It is cooled to 10<e>C and 100 ml of water is added. 16.3 ml of 1N NaOH is then allowed to be added drop by drop over the course of 4 hours while keeping the pH value constant at 8.6. The reaction solution is diluted with methylene chloride, the organic phase is separated, washed once with water and then dried over sodium sulphate. After separation of the drying agent, the methylene chloride solution is mixed again with 13 ml (233 mmol) of acetaldehyde and stirred overnight. The reaction solution is then evaporated to dryness, dissolved again in a little methylene chloride and applied to a column filled with 500 ml of silica gel (0.04-0.063 mm). 400 ml fractions are collected and with the help of analytical HPLC, all fractions are examined for the cis-isomeric compound.

HPLC analytical: Hibar 250-4, Lichrosorb Si 60, 5 pm, 254 nm. Eluent: 100 ml methylene chloride - 3 ml methanol Flow: 2 ml/min., concentration: 1 mg/ml

Retention: 5.80 (content 73.1$)

Yield: 4.75 g (5156 of the theoretical)

C31H28N2°4S (524.6)

NMR (CDCl 3 ): S = 1.4 (dd, 3H); 3.22 (d, 1H); 3.41 (d, 1H);

3.65 (q, 2H); 5.0 (d, 1H); 5.48-5.6 (m, 1H); 6.07 (d, 1H); 6.92 (s, 1H); 7.21-7.4 (m, 15H) ppm.

II. 7-phenylacetamido-3-[(Z)-propen-1-yl]-3-cephem-4-carboxylic acid benzhydryl ester (other method)

177.2 g (0.2 mol) of 7-phenylacetamido-3-(triphenylphosphonio)methyl-3-cephem-4-carboxylic acid benzhydryl ester iodide is dissolved almost completely in 800 ml of CH2Cl2 and 80 ml of CH3OH, cooled to 5' C, and mixed with 168.8 ml (3.0 mol) of acetaldehyde, while the temperature should not rise above 20°C.

26.4 g (0.25 mol) of sodium carbonate dissolved in 200 ml are then slowly added over 15 minutes at 14°C. The ice bath is then removed and the solution is stirred for 2.5 hours at room temperature.

The course of the reaction is checked with thin-layer chromatography in acetone:trile:water = 9:1 and toluene:acetic ester = 8:2. After the Wittig reaction has ended, the organic phase is separated and the aqueous phase is washed again with CH2CI2. The combined CH2Cl2 phases are filtered over 150 g of silica gel (Merck, 0.04-0.063 mm) and washed with CH2Cl2 (approx. 1,000 ml) until the filtrate is colourless.

The CH2Cl2 ~ filtrate is dried over Na2SO4, then evaporated to an oily residue and stirred with 800 ml of ethanol. The ethanolic solution is allowed to stir on a rotary evaporator, as the product gradually crystallizes out. Ethanol is further distilled off and the formed crystal slurry is stirred with approx. 90 ml ether/100 ml n-pentane, aspirated off and washed with 60 ml n-penta. The product is dried in vacuum over P40^q overnight.

Yield: 41.2 g (39.256 of the theoretical)

HPLC analytical: Hibar 250-4; Lichrosorb Si 60, 5 pm, 254 nm.

Eluent: 850 ml toluene - 150 acetic acid

Flow. 2 ml min-<1>

Retention: 4.73 (80.256; Z-isomer)

4.00 (17.456; E-isomer)

Example 10 (Intermediate)

I. 7-Amino-3-[(Z)-1-propen-1-yl]-3-cephem-4-carboxylic acid benzhydryl ester

6.4 g (12.2 mmol) of 7-phenylacetamido-3-[(Z)-propen-1-yl]-3-cephem-4-carboxylic acid benzhydryl ester (Example 9) is dissolved in 64 ml of methylene chloride and cooled with a dry ice bath to - 40°C and 2.47 ml (30.5 mmol) pyridine and 2.54 g (12.2 mmol) phosphorus pentachloride are added in order. After 5 minutes it is allowed to warm up to -20°C, then the temperature must rise within 20 minutes to -10°C and finally to +10°C.

The solution is now stirred for 1 hour at +10°C to +15°C. The mixture is then cooled to -40°C, 100 ml of methanol (-30°C) is added and stirred for a further 30 minutes at 10°C. The reaction solution is evaporated gently, the precipitated oil is dissolved in 600 ml of methylene chloride, stirred into 800 ml of sodium bicarbonate solution and stirred for 10 minutes. The methylene chloride phase is separated, washed once with water and dried over sodium sulfate. The methylene chloride filtrate is chromatographed on 400 ml of silica gel (0.04-0.063 mm) , eluting first with methylene chloride and then with methylene chloride while adding methanol (gradient up to 10%). The eluate is examined by means of analytical HPLC and DC (thin layer chromatography)

(methylene chloride/methanol = 100:1).

Yield: 4.2 g

C23H22N203S (406.5)

NMR (CDCl 3 ): S 1.4 (dd, j = 2 Hz and 7 Hz, 3H); 3.3 (d, J = 17 Hz, 1H); 3.48 (d, J = 17 Hz, 1H); 4.75

(d, J = 4.5 Hz, 1H); 4.98 (d, J = 4.5 Hz, 1H); 5.45-5.55 (d and q, J = 10 Hz, 1H);

6.96 (s, 1H); 7.23-7.42 (m, 10H); 8.6 (d,

2H) ppm.

HPLC analytical: Hibar 250-4, Merck, Lichrosorb Si 60, 5 pm, 254 nm.

Eluent: 1000 ml methylene chloride - 5 ml methanol Flow rate: 2 ml/min, concentration: 1 mg/ml Retention: 12.75 (content: 70.4$)

II. 7-amino-3-[(Z)-1-propen-1-yl)]-3-cephem-4-carboxylic acid benzhydryl ester hydrochloride (another method)

187.05 g (0.8976 mol; 1.57 equivalents) of phosphorus pentachloride is placed at room temperature in 3,300 ml CH2CI2 in a 4 liter three-necked flask and added dropwise over 5-10 minutes 66.42 ml (0.821 mol; 1.44 equivalents ) pyridln dissolved in 330 ml of CH2CI2. As the temperature rises to 24-27°C, a clear, colorless solution is formed. The solution is cooled to -2°C and 300 (0.572 mol) of 7-phenylacetamido-3-[(Z)-propen-1-yl]-3-cephem-4-carboxylic acid benzhydryl ester is added, the temperature should not rise above 2°C. The cooling bath is then removed and stirred for 40 minutes, as the temperature of the reaction solution rises to 10-12°C (imino chloride solution). 1 In a 6-liter three-necked flask, 255 ml (2.843 mol, 4.99 equivalents) of 1,3-butanediol dissolved in 1,650 ml of CH2Cl2 are cooled to -20°C to -25°C (acetone/dry ice) and the imino chloride solution introduced therein using a vacuum within 5-10 minutes. The temperature should therefore not rise above -20°C. The cooling bath is then removed and the reaction solution is stirred for 2 hours, the temperature rising to 10°C.

The reaction solution is now washed with 1,200 ml of ice water, with

1200 ml 2N HC1 and 1200 ml saturated saline solution. The CH2Cl2 phase is briefly dried over Na2SO4, separated from the drying agent and the filtrate is evaporated to dryness. The precipitated crystalline material is stirred with 1,200 ml to 2,000 ml of acetic acid, then suctioned off and washed with ether. The product is dried overnight in a high vacuum:

Yield: 173.9 g (68.756 of the theoretical)

C23H23CIN2O3S (442.96)

NMR (DMSO): S = 1.48 (dd, 3H); 3.56 (d, 1H); 3.79 (d, 1H);

5.2 (d, 1H); 5.31 (d, 1H); 5.56-5.72 (m, 1H); 6.21 (weak d, 1H); 6.28 (weak d, 1H);

6.91 (s, 1H); 7.25-7.47 (m, 10H) ppm.

In addition to the Z-isomer, the product contains some E-isomer (Z/E = 91:9).

Example 11

D-7-[2-(t-butoxycarbonylamino)-2-(2-aminobenzothiazol-6-yl)glycylamido]-3-[ (Z )-1-propen-l-yl]-3-cephem-4- carboxylic acid benzhydryl ester

3.4 g (10.5 mmol) D-a-t-butyloxycarbonylamino-ot-(2-aminobenzothiazol-6-yl)acetic acid and 4.0 g (7 mmol) 7-amino-3-[(Z)-1- propen-1-yl]-3-cephem-4-carboxylic acid benzhydryl ester (Example 10) is dissolved in 100 ml of tetrahydrofuran. The clear yellow solution is mixed with 2.2 g (10.5 mmol) of DCC and then stirred for 2 hours at room temperature. After stirring for 2 hours, the mixture is evaporated to dryness, the residue is suspended in 150 ml of vinegar, stirred magnetically for 10 minutes and the insoluble components are separated by suction. The acetate filtrate is evaporated to dryness, the residue is dissolved in 50 ml of methylene chloride and chromatographed on 200 ml of silica gel (0.04-0.063 mm). The elution takes place with methylene chloride and methylene chloride/methanol mixtures in the following order: 1) methylene chloride (fractions 1 to 6): 200 mg, discarded 2) methylene chloride - 2% methanol (fractions 7, 8): 200 mg, discarded

3) methylene chloride - 3% to 4* methanol (fraction 9, 10)

4) methylene chloride - 4# to 5% methanol (fractions 11 to 13) Yield of fractions 9 to 13: 3.1 g 5) methylene chloride - 556 to 1056 methanol (fractions 14, 15): 1.9 g 6) methylene chloride - methanol (1:1, fraction 16): 0.7 g, discard

According to analytical HPLC examination, fractions 9 to 13 contain the desired compound.

Yield: 3.1 g (4456 of the theoretical)

C37H37N5O6S2 (711.9)

NMR (CDCl 3 ): S 1.35 (dd, 3H); 1.43 (s, 9H); 3.15 (d, 1H);

3.31 (d, 1H); 4.97 (d, 1H); 5.3 (p, 1E);

5.46-5.55 (m, 1E); 5.71 (broad s, 2E); 5.78-5.85 (q, 1E); 6.03 (broad d, J - 11 Ez, 1E);

6.87 (s, 1H); 7.2-7.4 (mm, 12E); 7.5 (s, 1E) ppm.

Example 12

D-7-[(2-aminobenzothiazol-6-yl)glycylamido]-3-[(Z )-1-propen-1-yl]-3-cephem-4-carboxylic acid trifluoroacetate

3.1 g (4.35 mmol) D-7-[2-(t-butyloxycarbonylamino)-2-(2-aminobenzothiazol-6-yl)glycylamido]-3-[(Z)-1-propen-1 -yl]-3-cephem-4-carboxylic acid diphenyl methyl ester (Example 11) is dissolved in 20 ml of methylene chloride, mixed with 50 ml of trifluoroacetic acid and 5 ml of anisole and stirred for 60 minutes at room temperature. The mixture is evaporated in vacuo, 200 ml of ether is added to the concentrate, the precipitated solid is filtered, and washed with 100 ml of ether. The pale yellow trifluoroacetate is dried in a vacuum.

Yield: 2.7 g

C21H21F3N5°6S2 (560.6)

HPLC analytical: Hibar 250-4, Merck RP-8, 10 pm, 254 nm Eluent: 250 ml CH3CN - 75 ml glacial acetic acid - 2,175 ml water

Flow rate: 4 ml/min, concentration: 1 mg/ml Retention: 3.33 (content: 76.4#)

Example 13

D-7-[(2-aminobenzothiazol-6-yl)glycylamido]-3-[(Z)-1-propen-1-yl]-3-cephem-4-carboxylic acid

2.6 g (4.64 mmol) D-7-[(2-aminobenzythiazol-6-yl)glycylamido]-3-[(Z )-1-propen-1-yl]-3-cephem-4-carboxylic acid -trifluoroacetate (Example 12) is suspended in 100 ml of water, separated from yellow insoluble particles over diatomaceous earth and washed with 30 ml of water.

The still slightly cloudy solution is filtered over a membrane filter (Millipore, 0.45 pm) and the 3-propenylcephalosporin is isolated from the filtrate analogously to example 6 by means of preparative HPLC.

Yield: 500 mg

C19H19N5°4S2 (445.5)

NMR (DCOOD): S = 1.67 (dd, 3H); 3.41 (d, 1H); 3.55 (d, 1H);

5.3 (d, 1H); 5.78 (s, 1H); 5.81-5.91 (q and m, 2H); 6.25 (broad d, J = 11.6 Mz, 1H);

7.81-7.9 (q, 2H); 8.18 (s, 1H) ppm.

Example 14 (Intermediate)

The sodium salt of D-oc-[(1-methyl-2-methoxycarbonyl<y>1-vinyl )-amino]-(2-aminobenzothiazol-6-yl)acetic acid

100 g [equivalent to 97.2 g (0.0435 mol)] D-2-aminobenzythiazol-6-yl)glycyl (content = 97.256, ee = 89.756) is introduced into methanolic caustic soda prepared from 18.6 g (0.465 mol, i.e. .756 excess) NaOH and 1000 ml methanol. During reflux and stirring, a clear solution forms, which is mixed with 64 ml (0.59 mol, approx. 4056 excess) of acetoacetic acid methyl ester, dissolved in 100 ml of methanol, over the course of 40 minutes (after completion of instillation pH = 10.3, sample/ water = 1:1). The solution is then heated at reflux for 1 hour and then stirred for several hours without heating. The crystallized material is suctioned off and washed with toluene (2 times 200 ml). The filter residue is heated in 1,000 toluene for 30-40 minutes until boiling, then 300 ml of toluene is distilled off and washed by adding fresh toluene drop by drop and dried overnight in a fresh air cabinet at 70°C.

Yield: 81.9 g ( 52 , 156 of the theoretical)

c14H14N3°4SNa H2° (361»4)

Calculated: C 46.53 H 4.46 N 11.62 S 8.87 Na 6.36 Found: C 46.2 H4.4 N 11.9 S8.4 Na 5.6 Br 0.1 Cl 0.5 NMR (DMSO): S = 1.63 (s, 3H); 3.49 (s, 3H); 4.25 (s, 1H);

4.7 (d, J = 7.5 Hz, 1H); 7.12 (dd, 1H);

7.21 (d, 1H); 7.34 (s, 2H); 7.48 (s, 1H);

9.55 (d, 1H) ppm.

ee = 10056

Example 15 (Intermediate)

7-amino-3-[(Z)-1-propen-1-yl]-3-cephem-4-carboxylic acid (7-ÅPCÅ)

54.5 g (0.123 mol) of 7-amino-3-[(Z)-1-propene-1- yl]-3-cephem-4-carboxylic acid benzhydryl ester hydrochloride (Example 10). The mixture is stirred for 1 hour at room temperature, then evaporated at 30°C in a vacuum and the oily residue is stirred with 600 ml of ether for 1 hour. The precipitate is suctioned off, washed with 300 to 400 ml of ether and the filter residue is dried for 3 hours in a vacuum. The trifluoroacetate is suspended in 300 ml of water, the slurry is cooled to 5°C and adjusted with 12N HCl to a pH value of 0.2-0.4. The clear solution formed is cooled to 5°C and stirred with 4 g of activated charcoal for 10 minutes. One vacuums over diatomaceous earth and then washes with approx. 50 ml of 0.1N HCl. The filtrate is adjusted at 5°C with 2056 ig NaOH to pH 2.1 and the precipitated product is left in a refrigerator for 1 hour to complete crystallization. The crystal slurry is suctioned off, washed with 100 ml of water and 300 ml of acetone and dried in a vacuum.

Yield: 16.4 g (55.456 of the theoretical)

C10<H>12N2°3S (240.3)

NMR (DCOOD): S = 1.8 (dd, 3H); 3.61 (d, 1H); 3.77 (d, 1H);

5.39 (d, 1H); 5.52 (d, 1H); 5.91-6.06 (q,

1H); 6.5 (d, 1H) ppm.

HPLC analytical: Hibar 250-4, Merck RP-8, 10 pm, 254 nm. Eluent: 100 ml CH3CN - 30 ml glacial acetic acid - 870 ml water Flow rate: 2 ml/min, concentration: 0.25 mg/ml Retention: 2.39 (85.756; Z-isomer)

3.16 (11.356; E-isomer)

Example 16

I. D-7-[(2-aminobenzothiazol-6-yl)glycyl-amido]-3-[(Z)-1-propen-1-yl]-3-cephem-4-carboxylic acid

a) Activation of the precursor acid:

12.09 g [equivalent to 11.48 g (33.4 mmol)] sodium D-cx-[(1-methyl-2-methoxycarbonyl-vinyl)-amino]-(2-aminobenzothiazol-6-yl)acetate ( content = 9756, example 14) is dissolved in 57 ml of dimethylformamide, then 23 ml of acetonitrile is added. The solution is cooled to -70°C and 115 µl of 3-dimethylaminopropanol and 3.30 ml (34.4 mmol) ethyl chloroformate are added in sequence and stirred for 20 minutes at -70°C.

b) Preparation of the cephalosporin component (7-APC)

9.61 g (40 mmol) of 7-amino-3-[(Z )-1-propen-1-yl]-3-cephem-4-carboxylic acid (Example 15) are suspended in 57 ml of dimethylformamide and 23 ml of acetonitrile and converted by adding 1N caustic soda (36.8 ml) at room temperature until pH 8.5 to a clear solution. The solution is cooled to

-20°C to -30°C.

c) Coupling, unblocking and isolation of the crude betaine

The cooled 7-APCA solution (-20°C to -30°C) acc

point b) is allowed to slowly drip into the solution of the mixed anhydride of the precursor acid according to point a) at -70°C and then stirred for 10 minutes at -70°C. The temperature of the solution is thereby allowed to rise within 45 minutes

to 0'C (without cooling) and stir with 1.2 g of activated carbon and 1.2 g of diatomaceous earth for a further 10 minutes. The reaction mixture is filtered over a Seitz filter, washed with a little dimethylformamide and the filtrate is mixed with 6.9 ml of concentrated hydrochloric acid. The volume of the solution is concentrated to 115 ml, the precipitated salts being separated. The filtrate is set over magnetic stirring with 2556 NH3 solution at pH 4.0 and mixed with 800 ml of acetone, the crude betaine falling out. The precipitate is stirred for 10 minutes, aspirated off and washed with acetone and the material is dried in a vacuum.

Yield: 12.65 g

HPLC analytical: Hibar 250-4, Merck RP-8, 10 pm, 254 nm Eluent: 100 ml CH3CN - 30 ml glacial acetic acid - 870 ml water

Flow rate: 2 ml/min, concentration: 1 mg/ml Retention: 7.06 (73.856; Z-isomer)

11.18 (11.556; E-isomer)

The crude betaine is suspended in water, dissolved with semi-concentrated hydrochloric acid at pH 1.2 and stirred for 15 minutes with 1.2 g of activated charcoal. The mixture is sucked off over a silica gel ring, washed with 20 ml of 0.1N hydrochloric acid and the filtrate is pumped onto an RP 18 column (Hibar 250-25, Merck). The column is first eluted with water, then with 556 µg methanol. The fraction is examined by means of analytical HPLC and the fractions, which contain the Z-isomer derivative, are combined, methanol is distilled off in vacuum and the aqueous solution is lyophilized.

Yield: 5.2 g (32.356 of the theoretical)

C19H19N5°4S2' 2H2° (481.56)

II. D-7-[(2-aminobenzothiazol-6-yl)glycyl-amido]-3-[(Z)-1-propen-1-yl]-3-cephem-4-carboxylic acid (another method)

a) Activation of the precursor acid:

100 g [equivalent to 95 g (0.277 mol) pure material, 1.2

equivalents] sodium D-α-[(1-methyl-2-methoxycarbonyl-vinyl)-amino]-(2-aminobenzothiazol-6-yl)acetate (content 9556, Example 14) is clearly dissolved in 500 ml of dimethylformamide and 200 ml of acetonitrile. The solution is cooled to -60°C and 1 ml of 3-dimethylamino-1-propanol and 27.7 ml (0.281 mol) ethyl chloroformate are added in sequence and stirred for 30 minutes at -60°C.

b) Preparation of cephalosporin components:

102 g (0.2304 mol) of 7-amino-3-[(Z)-1-propen-1-yl]-3-cephem-4-carboxylic acid benzhydryl ester hydrochloride (Example 10/11) are dissolved clearly in 500 ml of dimethylformamide and 100 ml acetonitrile, is mixed at room temperature with 31 ml of water and 32.2 ml (0.2289 mol) of triethylamine and stirred for 5 minutes under ice cooling.

c) Coupling:

The cephalosporin solution (b) cooled to 0°C sets

slowly to the solution of the mixed anhydride (a) at -60°C, the temperature increasing from -60°C to -30°C. The mixture is stirred for a total of 30 minutes and the temperature of the reaction solution is allowed to reach 0°C. 57 ml of concentrated hydrochloric acid are then added and the solution is stirred for 15 minutes at 0°C.

d) Isolation of the trifluoroacetate salt:

Acetonitrile is distilled off from the reaction solution and

adjust with 25$ NH3 solution under ice-cooling at pH 7.5. The solution is shaken in 5 liters of vinegar and 3 liters of 10#-ig NaHC03 solution, which contains common salt. The mixture is stirred intensively for 5 minutes and aspirated

then over a Seitz filter. The acetic ester phase is separated, washed once with 4 liters of saturated NaHC03 solution and twice with 4 liters of water. The acetic ester phase is then dried over NaSO4, sucked off the desiccant and finally evaporated in a vacuum to dryness. The resin foam formed is dried for 30 minutes in a high vacuum. Yield: 170 g.

e) Unblocking

The hard foam is dissolved in 1,600 ml CltøC^t cooled to 0'C

and mixed with a mixture of 750 ml of trifluoroacetic acid and 4 ml of anisole. The solution is then stirred for 45 minutes

at room temperature, is then evaporated to an oil and the oily residue is digested with 6 liters of ether. The crystallized material is suctioned off, washed with ether and dried overnight in a vacuum.

Yield: 125 g (9756 of the theoretical)

C19H19N5°4S2' CF3 C00E (559.55)

HPLC analytical: Hibar 250-4, Merck RP-8, 254 nm Eluent: 1000 ml CH3CN - 30 ml glacial acetic acid - 870 ml water

Flow rate: 4 ml/min.

Retention: 2.25 (90.156; Z-isomer)

3.77 (8.356; E-isomer)

f) Purification by h. gel of adsorber resin chromatograph in: 141 g D-7-[(2-aminobenzothiazol-6-yl)glycyl-amido]-3-[(Z )-1-propen-1-yl]-3- cephem-4-carboxylic acid trifluoroacetate [wet material = 128.8 g (10056 of the theoretical) in

1,000 ml of water is stirred intensively for 15 minutes, then

Insoluble material is sucked off and washed with water. The filtrate (pH 1.3) is applied to a column filled with 8 liters of adsorber resin LGP 4429 ("Lewatit" OC 1062, BAYER AG). The column is first eluted with 5 liters of water and then with resp. 2 liter portions of water, to which it converts acetone with an increasing part from 256 to 1056 . A total of 15 fractions with a volume of resp. 2000 ml:

The fractions from 3 to 10, which contain the desired product in pure form, are distilled from acetone in vacuum and lyophilized.

Yield: 47.7 g (43.056 of the theoretical)

C19H19N5°4S2' 2H2° (481.56)

g) Methanol solvate formation 168.3 g of D-7-[2-aminobenzothiazol-6-yl)-glycyl-amido]-3-cephem-4-carboxylic acid as lyophilisate (f) is stirred in 1,700 ml of methanol for 90 minutes, aspirated and washed with 500 ml of methanol on the Nutschen. The remaining nuts are stirred in again

1,000 ml of methanol for 45 minutes, aspirated off and washed

with 500 ml of methanol on Nutschen. The product is dried overnight in a tight vacuum.

Yield: 110.2 g (b5, 5% of theory) HPLC analytical: Hibar 250-4, Merck RP-8, 10 pm, 254 nm Eluent. lOOml CH3CN - 30 ml glacial acetic acid - 870 ml water

Flow rate: 4 ml/min.-<1>; concentration: 1 mg ml<-1>

Retention: 3.58 (98.456; Z-isomer)

6.54 (0.7956; E-isomer)

h) Eudrate formation 109.6 g D-7-[2-aminobenzothiazol-6-yl)glycyl-amido]-3-[(Z)-1-propen-1-yl-3-cephem-4-carboxylic acid methanol solvate ( g) Introduce while stirring in 1,100 ml of water (manufactured by Mi111-0 Watersystem, Millipore GmbH) and stir magnetically under vacuum for 2 hours. The product is suctioned off and washed 3 times with roughly equal portions of water (from the Milli-0 water system). The fabric is dried for 36 hours in a high vacuum without drying agent. Yield: 94.7 g (86.456 of the theoretical) C19H19N5°4S2' 2H2° (463.541)

HPLC analytical: Hibar 250-4, Merck RP-8, 10 pm, 254 nm Eluent: 100 ml CH3CN - 30 ml glacial acetic acid - 870 ml water

Flow rate: 4 ml min.-<1>; concentration: 1 mg ml<-1>

Retention: 3.56 (98.856)

Example 17

Sodium D-7-[(2-aminobenzothiazol-6-yl)glycyl-amido]-3-[(Z)-1-propen-1-yl]-3-cephem-4-carboxylate

15.0 g (0.0324 mol) D-7-[(2-aminobenzothiazol-6-yl)-glycyl-amido]-3-[(Z)-1-propen-1-yl]-3-cephem- 4-carboxylic acid (example 16/11) is suspended in 300 ml of water while stirring and

adjusted with IN caustic soda to pH 8.56 under pH-stat conditions (Memo-Titrator DL 40 RC). The light yellow solution formed is sucked off over a paper filter and the filtrate is lyophilized.

Yield: 15.0 g (92.156 of the theoretical)

c19H18N5Na04s2<*> 2H2° (503.54)

NMR (DCOOC): S = 1.64 (dd, 3H); 3.38 (d, 1H); 3.51 (d, 1H),

5.26 (d, 1H); 5.72 (s, 1H); 5.78-5.85 (m, 1H); 5.85 (d, 1H); 6.21 (d, 1H); 7.76-7.83

(q, 2H); 8.12 (s, 1H) ppm.

HPLC analytical: Hibar 250-4, RP-8, 10 pm, 254 nm Eluent: 100 ml CH3CN - 30 ml glacial acetic acid - 870 ml water Flow rate: 2 ml/min.

Concentration: 1 mg/ml

Retention: 4.36 (content: 98,756)

Example 18 (Intermediate)

D-α-t-butoxycarbonylamino-α-(benzothiazol-6-yl)acetic acid

To a solution of 20 g (0.0618 mol) of D-α-t-butyloxycarbonylamino-α-(2-aminobenzothiazol-6-yl)acetic acid in 160 ml of tetrahydrofuran is added dropwise 12.2 ml (0.102 mol) of tert-butyl nitrite dissolved in 25 ml of tetrahydrofuran within 30 minutes at 50 to 60°C. The mixture is then stirred for 30 minutes at 50 to 55°C, the solvent is distilled off and the residue is distributed in 300 ml of water and 300 ml of acetic acid. The mixture is acidified by ice-cooling with 2N HCl to pH 1.5, stirred for 5 minutes and then adjusted with 40# potassium carbonate solution to pH 8.0 to 8.5. The aqueous phase is separated, washed again with acetic acid and then acidified at 0°C with 2N HCl to pH 2.5. The acidic solution is extracted twice with vinegar, washed with sodium chloride solution and dried over sodium sulphate. The acetic ester phase is evaporated to 50 ml and stirred into petroleum ether.

Yield: 14.4 g (76# of the theoretical)

C14H16N2O4S (308.4)

[a]<20>589 = -130.3° (c = 1, methanol)

NMR (DMSO): S = 1.43 (s, 9H); 5.31 (d, 1H); 7.6 (dd, 1H);

7.73 (d, 1H); 8.08 (d, 1H); 8.2 (weak d, 1H); 9.4 (s, 1H) ppm.

Example 19 (Intermediate)

7-Phenylacetamido-3-[(Z)-propen-1-yl]-3-cephem-4-carboxylic acid p-methoxybenzyl ester

40.0 g (82.1 mmol) of 7-phenylacetamido-3-chloromethyl-3-cephem-4-carboxylic acid p-methoxybenzyl ester and 22.66 g (86.4 mmol) of triphenylphosphine are dissolved in 300 ml of dimethylformamide, mixed with 12 .95 g (86.4 mmol) of sodium iodide and stirred for 2 hours at room temperature. The reaction solution is then evaporated in high vacuum to dryness to an oil.

The oily residue of 4.2 g is taken up in 130 ml of methylene chloride (no completely clear solution), mixed in a 500 ml three-necked flask with 69.0 ml (1231.5 mmol) of acetaldehyde and then treated with 1N sodium hydroxide solution under pH-stat conditions using autotitration at pH 8.1. After 1 hour, 8.7 ml IN caustic soda has been consumed. Then, at pH 8.3, a further 72.5 ml of IN caustic soda is consumed within 20 hours. The aqueous phase is separated, the methylene chloride phase is washed twice with water and dried over sodium sulphate. The methylene chloride phase is then stirred again with 20 ml (358 mmol) of acetaldehyde for 2 hours at room temperature, the methylene chloride is distilled off and the remaining oil is obtained in toluene and concentrated on a column with 1 liter of silica gel (0.04-0.063 mm).

The elution takes place first with toluene (fractions 1 to 5) and then with toluene/acetic acid (5:1, fractions 6, 7), collecting 600 ml of fractions. Fractions 3 to 6 are combined, evaporated to dryness and the oil formed is expelled with 100 to 150 ml of ether. The precipitated white material is filtered off with suction and washed with ether (50 to 80 ml).

Yield: 12.1 g ($31 of the theoretical)

C26<H>26N2°5° (478.6)

NMR (CDCl 3 ): S = 1.52 (d, 3H); 3.23 (d, 1H), 3.41 (d, 1H);

3.61, (q, 2H); 3.78 (s, 3H); 4.95 (d, 1H);

5.13 (s, 2H); 5.59-5.69 (dq, 1H); 5.75-5.81 (q, 1E); 6.08 (broad d, 1H); 6.45 (d, 1H);

6.87 (d, 2H); 7.25-7.36 (m, 7E) ppm.

Fractions 7 to 11 are evaporated to a faint reddish oil, which cannot be assigned to the desired compound.

Example 20 (Intermediate)

7-amino-3-[(Z )-1-propen-1-yl]-3-cephem-4-carboxylic acid p-methoxybenzyl ester

12.1 g (25.28 mmol) of 7-phenylacetamldo-3-[(Z)-propen-l-yl]-3-cephem-4-carboxylic acid p-methoxybenzyl ester (Example 19) is dissolved in 1,133 ml of methylene chloride, cooled to - 50°C and 5.11 ml (63.2 mmol) pyridine and 5.26 g (25.28 mmol) phosphorus pentachloride are added in order. The temperature is then allowed to rise within 25 minutes to -10°C. After a further 20 minutes, the temperature of the solution is 0°C, then stirring is continued for 45 minutes to +15°C. The mixture is now cooled to -50°C, 200 methanol (approx. -30°C) is added at once and stirred without to cool for 30 minutes. The reaction solution is evaporated, the oily residue is dissolved in methylene chloride and applied to a column packed with 400 ml of silica gel (0.04-0.063 mm). The column is first eluted with methylene chloride, then in sequence with the solvent mixtures methylene chloride - 556 methanol and methylene chloride - 1056 methanol. The fractions, which are eluted with the mixture methylene chloride - 1056 methanol, are evaporated to dryness.

Yield: 10 g

The oil is taken up in 500 ml of ethanol, separated from an insoluble slime by decantation filtration, the filtrate is evaporated to dryness and the oil is dried in a vacuum.

Yield: 6.8 g (7556 of the theoretical)

C18H20N2°4S (360.4)

NMR (CDCl 3 ): S - 1.55 (dd, 3H); 1.95 (weak dd, 2E); 3.3 (d,

1E); 3.5 (d, 1E); 3.76 (p, 3E); 4.72 8d, 1E); 4.98 (d, 1E); 5.18 (s, 1H); 5.58-5.69 (dq, 1E); 6.1 (broad d, 1E); 6.88 (d, 2E);

7.31 (d, 2E); 8.61 (d, 1E) ppm.

Example 21

D-7-[2-( t-butyloxycarbonylamino )-2-(benzothiazol-6-yl)-glycylamido]-3-[(Z)-1-propen-1-yl]-3-cephem-4- carboxylic acid p-methoxybenzyl ester

To a solution of 5.83 g (18.9 mmol) of D-α-t-butyloxycarbonylamino-α-(benzothiazol-6-yl)acetic acid and 6.8 g (15.1 mmol) of 7-amino-3-[(Z )- 1-propen-1-yl]-3-cephem-4-carboxylic acid p-methoxybenzyl ester (Example 20) in 150 tetrahydrofuran, 3.90 g (18.9 mmol) of dicyclohexylcarbodiimide are placed under magnetic stirring at room temperature and then stirred for 2 hours. The formed dicyclourea is filtered off with suction, washed with tetrahydrofuran and the mother liquor is evaporated to dryness. The remaining oil is dissolved in 100 ml of methylene chloride and chromatographed on 600 ml of silica gel (0.04-0.063 mm), eluting first with methylene chloride (2 x 300 ml) and then with 556 µg of methanol in methylene chloride under control using thin-layer chromatography (methylene chloride:methanol = 100:5). It collects resp. 300 ml fractions. The desired fractions of the eluate (11 and 12) are combined with 556 µg of methanol and evaporated to dryness.

Yield: 8.8 g (8956 of the theoretical)

C32<H>34N4°7S2 (650.8)

NMR (CDCl 3 ): S = 1.4 (s, 9H); 1.5 (dd, 3H); 3.12 (d, 1H);

3.32 (d, 1H); 3.75 (s, 3H); 4.91 (d, 1H);

5.13 (s and d, 3H); 5.58-5.68 (m, 1H); 5.73-5.79 (q, 1H); 6.03 (broad d, 1H); 6.86 (d, 2H); 7.28 (d, 2H); 7.52 (d, 1H); 8.01 (p, 1E); 9.4 (s, 1H) ppm.

Example 22

D-7 - [ (benzothiazol-6-yl)glycylamido]-3-[(Z)-1-propen-1-yl]-3-cephem-4-carboxylic acid

A mixture of 8.8 g (13.4 mmol) of D-7-[2-(t-butyloxycarbonylamino)-2-(benzothiazol-6-yl)glycylamido]-3-[(Z)-1-propene-1 -yl]-3-cephem-4-carboxylic acid p-methoxybenzyl ester (Example 21), 3 ml of anisole and 100 ml of trifluoroacetic acid are stirred for 1 hour at room temperature. The mixture is evaporated in a vacuum, 40 ml of toluene is added to the pale pink, easily moving oil and evaporated again in a vacuum. The remaining oil is expelled with 400 ml of ether, the precipitated solid substance is sucked off, washed with ether and dried under vacuum. 6.3 g of trifluoroacetate is obtained, which is dissolved in 800 ml of water and separated from insoluble components by filtration over diatomaceous earth. The solution is passed over an HP-20 column (600 ml, Diaion adsorber resin, Mitsubishi), which is eluted with water and then with a mixture of water and methanol (gradient up to 5056). The eluate containing the desired compound is lyophilized.

Yield: 1.9 g

The lyophilisate is dissolved almost completely in water under magnetic stirring, 100 mg of activated carbon is added, stirred for 5 minutes, then suctioned off over diatomaceous earth and washed with 50 ml of water. The filtrate is filtered again with a syringe

using a Millipore membrane filter and then pumped onto a preparative column (Hibar 250-25, RP-18, Merck, flow rate: 10-15 ml min.-<1>). After sample collection, the column is eluted with the following elution systems in order:

1) 500 ml of water

2) 500 ml water with 1056 methanol

3) 1,000 ml of water with 1,056 to 4,056 methanol: The eluate is collected here in 50-100 ml fractions and then examined by means of analytical HPLC, determining that fractions 8 to 10 contain the cis-isomer derivative.

Yield: 468 mg

C19H18N4°4S2 (430.5)

NMR (DC00D): S = 1.61 (dd, 3H); 3.31 (d, 1H); 3.48 (d, 1H);

5.25 (d, 1H); 5.75-5.9 (m and q, 3H); 6.2 (broad d, 1H); 8.13 (dd, 1H); 8.48 (d, 1H);

8.67 (s, 1H); 10.33 (s, 1H) ppm.

HPLC analytical: Hibar 250-4, RP-8, 10 pm, 254 nm Eluent system: 790 ml water - 200 ml methanol - 10 ml buffer pH 7.0

Flow rate: 2 ml/min, concentration: 1 mg/ml Retention: 7.53 (content: 92 .456)

Example 23

D-7-[(2-aminobenzothiazol-6-yl)glycylamido]-3-vinyl-3-cephem-4-carboxylic acid

To a solution cooled to -50°C of 512.5 mg (1.585 mmol) of D-α-t-butyloxycarbonyl amino-α-(2-aminobenzothiazol-6-yl)acetic acid in 5 ml of dimethylformamide is injected slowly in sequence 276.1 µl (1.585 mmol ) ethylenediisopropylamine and 122.7 µl (1.585 mmol) mesyl chloride. It is stirred for 40 minutes at -50°C and a solution (-20°C) of 622 mg (1.585 mmol) of 7-amino-3-vinyl-3-cephem-4-carboxylic acid diphenylmethyl ester and 276.1 µl ( 1.585 mmol) of ethyldiisopropylamine in 5 ml of tetrahydrofuran and 3 ml of dimethylformamide. Stirring is continued for 5 minutes at -50°C and then a further 50 minutes without cooling. The reaction solution is then stirred into 40 ml of water and 120 ml of ethyl acetate, the ethyl acetate phase is separated, the aqueous layer is extracted again with 60 ml of ethyl acetate, the organic phases are combined, washed with 0.1N hydrochloric acid, sodium bicarbonate solution and table salt solution. After drying and distilling off the solvent, the residue is taken up in 20 ml of methylene chloride, 20 ml of trifluoroacetic acid with 1 drop of anisole are added and stirred for 45 minutes at room temperature. The trifluoroacetic acid/methylene chloride mixture is then distilled off in a vacuum and the residue is dissolved in 15 ml of 8056 ug acetic acid, pumped onto an RP-18 column (Hibar 250-25, Merck) and eluted with 3# acetic acid. The eluate, which contains the desired substance, is freeze-dried.

Yield: 800 mg

The lyophilisate is dissolved again in 10 ml of 3% acetic acid and rechromatographed on an RP-18 column (Hibar 250-25, Merck).

Yield: 165 mg

C18<H>17N5°4S2' 3H2° ' 1/2CH3C00H (505.5)

NMR (DCOOD): S = 3.61-3.76 (dd, 2H); 5.28 (d, 1H); 5.52 (d,

1H); 5.69 (d, 1H); 5.75 (s, 1H); 5.91 (d, 1H); 7.18-7.28 (q, 1H); 7.84 (m, 2H); 8.18 (s, 1H) ppm.

Claims (1)

Analogous process for the preparation of therapeutically active 3-lactam compound of formula I and pharmaceutically acceptable salts thereof, characterized in that [A] substituted cephalosporin compounds of formula (II) where X means a group of formula while R13 and R<14> are the same or different and mean alkyl, phenyl or tolyl and z<©> means a halide anion, preferably chloride, bromide or iodide, reacted in inert solvents in the presence of bases with aldehydes of formula (III) or that [B] carboxylic acids of the general formula (VI) in which R<3>' means an amino protecting group, after activation of the carboxyl group by transfer to a mixed anhydride, for example with chloroformic ethyl ester or chloroformate isobutyl ester or methanesulfonic acid chloride or by transfer to the acid halide or by transfer to an activated ester, for example with N-hydroxybenztriazole and dicyclohexylcarbodiimide (DCC), is reacted with vinyl cephalosporin amines of the general formula (VII), then any protective groups are removed and the desired pharmaceutically acceptable salts are prepared or from the salts the free acids, in a known manner.