NO148375B - ANALOGUE PROCEDURE FOR THE PREPARATION OF THERAPEUTIC ACTIVITY 3-CHLORO-7-ALFA-AMINO-ACYLCEPHALOSPORINE - Google Patents

Description

The present invention relates to the production of new therapeutically effective 3-chloro-7-a-aminoacylcephalosporin compounds with the formula:

where R is phenyl, 3- or 4-hydroxyphenyl or 4-chlorophenyl, as well as pharmaceutically acceptable, non-toxic salts thereof.

Several 7-α-aminoacylcephalosporin antibiotics are known

with different substituents in the 3-position in the molecule. Examples include the well-known antibiotic cephalexin, 7-(D-α-phenylglycylamino)-3-methyl-3-cephem-4-carboxylic acid, which is described in US patent no. 3,507,861.

The chloro-cephalosporin compounds provided by the present invention are structurally unique compounds in the cephalosporin group in that the chlorine atom is directly linked to the carbon atom in the 3-position of the dihydrothiazine ring.

The compounds of formula I are prepared according to the present invention by a 3-chloro-3-cephem ester of the formula: where is benzyl, p-methoxybenzyl, p-nitrobenzyl, diphenylmethyl, 2,2,2-trichloroethyl, trimethylsilyl or t-butyl , is reacted with an acylating agent of the formula: where R has the above meaning, and is t-butyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, trichloroethoxycarbonyl or a trityl group, or enamines formed with methyl acetoacetate and acetylacetone, or a reactive derivative thereof, after which removing the carboxylic acid protecting and amino protecting groups, and, if desired, converting a compound thus obtained into a pharmaceutically acceptable, non-toxic salt thereof.

The ester groups indicated for R^ in formula II are all known ester groups of the type usually used in connection with cephalosporins to protect the C^-carboxyl group in the cephalosporin molecule when carrying out reactions involving other reactive positions in the molecule. The preparation of esters of formula II is carried out by the methods indicated below.

By the term "pharmaceutically acceptable, non-toxic salts" are meant both the salts of the C 1 -carboxylic acid function and acid addition salts of the α-amino group in the 7-glycylamino side chain. Pharmaceutically acceptable salts containing the C 1 -carboxylic acid function include salts which. are formed with inorganic bases such as sodium, potassium and calcium salts, and these can be prepared with sodium bicarbonate, potassium carbonate, calcium hydroxide or sodium hydroxide. Pharmaceutically acceptable amine salts can also be prepared with organic amines

.such as dicyclohexylamine, benzylamine, 2-aminoethanol, diethanolamine or diisopropylamine. Acid addition salts of the α-amino group include salts formed with mineral acids such as

hydrochlorides, hydrobromides and sulphates, as well as salts formed with organic sulphonic acids such as p-toluenesulphonate.

The compounds of formula I can exist in zwitterionic form which occurs via intramolecular salt formation, when R 1 is converted to hydrogen.

Due to having an asymmetric carbon atom in the α-amino-acyl group, 3-chloro-cephalosporins of formula I can include D, L and DL forms. The D form is the preferred isomeric form.

Compounds of formula I or pharmaceutically acceptable, non-toxic salts of such compounds are valuable antibiotics that can be used to combat infections in warm-blooded mammals caused by gram-positive and gram-negative microorganisms. They are effective when administered parenterally, subcutaneously, or intramuscularly, as well as orally. The compounds of formula I are orally acceptable and more potent than existing orally acceptable cephalosporins, i.e. cephalexin and cephradine.

Norwegian application 792681 deals with compounds that are useful as intermediates for the production of compounds of formula I; these intermediates have the formula:

where R 1 is hydrogen, benzyl, p-methoxybenzyl, p-nitrobenzyl, diphenylmethyl, 2,2,2-trichloroethyl or t-butyl; and R~ is hydrogen, t-butyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, trichloroethoxycarbonyl or trityl, or enamines formed with methyl acetoacetate and acetylacetone; provided that R.| and R 2 are not both hydrogen.

The compounds of formula I have a broad antibacterial spectrum as illustrated by the in vitro spectrum of 7-(D-phenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid (cefaclor) as shown in subsequent tables.

Table I indicates minimum inhibitory concentrations (MIC) in micrograms per milliliters (µg/ml) obtained with this compound was a standard agar dilution sample.

Table II below indicates the diameter in millimeters of the growth inhibition zone of the indicated microorganism in a standard agar plate sample, and which was obtained with 7-(D-phenyl-glycylamino)-3-chloro-3-cephem-4-carboxylic acid.

Table III shows MIC values obtained with 7-(D-phenyl-glycylamino)-3-chloro-3-cephem-4-carboxylic acid against a variety of gram-positive and gram-negative microorganisms in a standard agar dilution test.

In a standard agar dilution test, 7-(D-phenyl-glycylamino)-3-chloro-3-cephem-4-carboxylic acid showed activity against Hemophilus influenzae with MIC values ranging from 1-4 mg/ml for

a number of different strains.

Table IV shows the oral absorption of 7-(D-phenylglycyl-amino)-3-chloro-3-cephem-4-carboxylic acid at blood and urine levels obtained in mice. In this test, Mc-Allister Swiss mice weighing 11-13 g were used, and they were fasted overnight and then orally administered 20 mg/kg of 7-(D-phenylglycyl-amino)-3-chloro-3-cephem- 4-carboxylic acid. Blood and urine samples were taken at the indicated times, and the concentration of the antibiotic in each sample was determined by a microbiological test using Sarcina lutea in a pH 6.0 agar in a standard agar plate sample.

The effective dose (EDj-q) for 7-(D-phenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid which is illustrative of the effective dose for compounds of formula I is 0.74 mg/kg x 2 orally and 0.48 mg/kg x 2 subcutaneously against Streptococcus pyogenes, and 5.5 mg/kg x 2 orally against Escherichia coli and 17.6 mg/kg x 2 orally against Diplococcus pneumoniae as determined in mice.

Table V below indicates minimum inhibitory concentrations (MIC) against a number of representative gram-negative bacteria for 3-chloro-3-cephem compounds of formula I.

The inhibitory concentrations were determined by the gradient plate method as essentially described by Brijson and Szybalski, Science 116, 45 (1952). In Table V, R<1> refers to the structural formula.

In the subsequent Table VI, the activity of 3-chloro-3-cephem compounds against clinical isolates of penicillin-resistant staphylococci is indicated. The activity is indicated as minimum inhibitory concentrations for the test compound. This concentration was determined by the gradient plate method.

In the tables VII and VIII below, tests have been carried out with compounds produced according to the invention, namely 7-(D-phenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid (cefaclor) and 7-[D-(p-hydroxyphenyl) )glycylamino]-3-chloro-3-cephem-4-carboxylic acid (p-hydroxycefaclor) as well as the previously known compound 7-(D-phenylglycylamino)-3-methyl-3-cephem-4-carboxylic acid (cephalexin); and these tests confirm the advantageous properties possessed by the compounds produced according to the invention compared to the known compound. Compounds of formula I are prepared by N-acylation of the benzyl, p-methoxybenzyl, p-nitrobenzyl, diphenylmethyl, 2,2,2-trichloroethyl, trimethylsilyl or t-butyl ester of 7-amino-3-chloro- 3-cephem-4-carboxylic acid with a phenyl-substituted glycine of the formula:

or a derivative thereof, where R and R2 have the same meaning as indicated in connection with formulas I and III. Activated derivatives of the carboxyl group in the substituted glycine may be acid halides such as acid chlorides, the activated esters such as those formed with pentachlorophenol, the azide or a mixed anhydride formed with the glycine and methyl chloroformate and iso-butyl chloroformate. Furthermore, amino-protected glycine can be used directly in the acylation of the desired 3-chloro core ester by using a condensing agent, e.g. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Thus, e.g. p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate is reacted with N-(t-butyloxycarbonyl)-D-phenyl-glycine in a dry, inert solvent, e.g. tetrahydrofuran, whereby p-nitrobenzyl-7-(D-phenylglycylamino)-3-chloro-3-cephem-4-carboxylate is formed.

In general, any known amide coupling method can be used for the acylation of 7-amino-3-chloro-3-cephem-4-carboxyl-

the acid esters. When the activated derivative is an acid halide, the acylation can be carried out in the presence of a hydrogen halide acceptor such as sodium bicarbonate, pyridine, sodium bisulfite or an alkylene oxide such as propylene oxide. When using a mixed anhydride in the acylation, the mixed anhydride can be formed using N-ethoxycarbonyl-2-ethoxy-1,2-dihydro-quinoline (EEDQ). The acylation can also be carried out in the presence of a condensing agent such as N,N'-dicyclohexylcarbodiimide.

Illustrative examples of substituted glycines which can be used in the preparation of compounds of formula I are pentachlorophenyl-D-phenyl-N-(t-butyloxycarbonyl)glycinate, N-(t-butoxycarbonyl)-D-phenylglycine, or N-(1-carbomethoxy -2-propenyl)-D-phenylglycine.

The acylation is preferably carried out in an inert solvent, e.g. in acetone, acetonitrile, dimethylformamide or methylene chloride, combustible at temperatures between -20 and 20°C.

Illustrative examples of 7-amino-3-chloro-3-cephem-4-carboxylic acid esters which can be used as starting compounds for the preparation of compounds of formula I are p-nitrobenzyl-7-amino-3-chloro-3-cephem- 4-carboxylate or diphenylmethyl-7-amino-3-chloro-3-cephem-4-carboxylate.

7-amino-3-chloro-3-cephem-4-carboxylic acid and esters thereof can be prepared in the following way. A 7-acylamino-cephalosporanic acid or an ester thereof is converted to a 7-acylamino-3-exomethylene cepham-4-carboxylic acid or ester. An ester of the 3-exomethylene cepham compound can then be oxidized with ozone to form an intermediate ozonide as the 3-exomethylene group is converted, and this ozonide will give 7-acylamino-3-hydroxy-3-cephem-4-carboxylic acid upon decomposition. The 3-hydroxy-3-cephem ester can then be chlorinated, whereby the corresponding 3-chloro-3-cephem is obtained. The 7-acyl group in the 3-chloro-cephem can then be removed by a well-known side chain cleavage reaction by using phosphorus pentachloride in pyridine, whereby one obtains the imino chloride derivative in the side chain, and this can be reacted with an alcohol such as methanol, whereby one obtains the iminoether, and this again can easily is hydrolysed so that the 7-amino-3-chloro-3-cephem-4-carboxylic acid ester is obtained.

Thus, e.g. a 7-acylaminocephalosporanic acid such as 7-phenoxyacetamidocephalosporanic acid is reacted with a sulphur-containing nucleophilic compound using known methods, whereby a nucleophilic displacement of the acetoxy group in the cephalosporanic acid is obtained and a 7-acylamino-3-thio-substituted-methyl is obtained -3-cephem-4-carboxylic acid. This compound can then be reduced with zinc/formic acid in the presence of dimethylformamide (DMF) or with Raney nickel in the presence of hydrogen, whereby a 7-acylamino-3-exomethylene cepham-4-carboxylic acid is obtained. Thus, e.g. 7-phenoxyacetamido-3-acetoxymethyl-3-cephem-4-carboxylic acid is reacted with potassium ethyl xanthate, whereby 7-phenoxyacetamido-3-ethoxythionocarbonylthiomethyl-3-cephem-4-carboxylic acid is obtained. A reduction of the latter compound with zinc and formic acid in the presence of DMF gives 7-phenoxyacetamido-3-exomethylene-cene-4-carboxylic acid. After the reductive displacement reaction described above, the 3-exomethylene cepham-4-carboxylic acid produced is esterified, e.g. with p-nitrobenzyl bromide, p-methoxybenzyl bromide, diphenyldiazomethane or 2,2,2-trichloroethyl chloroformate or other ester-forming compounds, after which the 3-exomethylene cephem ester is reacted with ozone to form the 3-hydroxy-3-cephem ester. The ozonolysis reaction of a 3-exomethylene encephamester is carried out in an inert solvent at temperatures between -80 and 0°C, preferably between -80 and -50°C to form an intermediate ozonide. The ozonide is then decomposed in situ in the cold, whereby the corresponding 3-hydroxy-3-cephem ester is obtained, which is illustrated in the subsequent reaction core.

In the above formula, R" is equal to the remainder of an acylamino group, for example R" can be benzyl, phenoxymethyl, methyl, 2-thienylmethyl or 2-furylmethyl, and R1 is as defined above.

The ozonolysis of a 3-ethoxymethylene encephamester is carried out by bubbling ozone into a solution of the ester in an inert solvent until ozonide formation is complete. Inert solvents that can be used here are those in which the 3-exo-methylene cep amester is partially soluble and which does not react with the ozone under the described conditions. Satisfactory results will usually be obtained with solvents such as methanol, ethanol, ethyl acetate, methyl acetate, iso-amyl acetate and methylene chloride.

The ozone gas is produced using an ozone generator of the type usually used in synthetic and analytical chemical work. Such generators produce ozone by the action of an electrical discharge in oxygen. The ozone is developed in a stream of oxygen which is then fed directly into the reaction vessel. The percentage content of ozone in the oxygen stream can vary as desired, e.g. by varying the speed of the oxygen flowing through the generator as well as by varying the intensity of the electrical discharge.

The concentration of the aforementioned 3-exomethylene encephamester i

the inert solvent is not critical, but it is preferred to use a sufficient volume of the solvent to form a complete solution.

When the ozonide formation is complete, any excess of ozone in the reaction mixture is removed from it by bubbling through nitrogen, oxygen or another inert gas such as argon. After removing an excess of the ozone, the ozonide is decomposed by adding a reducing agent selected from the group consisting of sodium bisulfite, sulfur dioxide and trimethylphosphite, whereby the 3-hydroxy-3-cephem-4-carboxylic acid ester is obtained. The decomposition is carried out by adding an excess of the decomposing reagent and then stirring the reaction mixture until it is negative in the potassium iodide starch test. A preferred agent for decomposing the ozonide is gaseous sulfur dioxide.

The 3-hydroxy-3-cephem esters are recovered from the reaction mixture by evaporation of the volatile solvents, and in this way a residue is obtained from which the reaction product is crystallized.

7-acylamino-3-hydroxy-3-cephem-4-carboxylic acid esters prepared in that way are then chlorinated, whereby the desired 7-acylamino-3-chloro-3-cephem-carboxylic acid ester is obtained.

The 3-chloro-3-cephemester starting materials can be prepared by reacting a 7-acylamino-3-hydroxy-3-cephemester or a 3-l 3 1~ _ O -P^ty, _V -; ^ -v^ c 4- ^ v- i PlMP \ Tn^rl i^n

reactive chlorine compound which with said DMF forms chlorodimethyliminium chloride as indicated by the following formula

The reactive iminium chloride with the above formula is formed in situ and is a highly reactive chlorination intermediate. Chlorine compounds which form the above iminium chlorides include commonly used chlorinating agents such as phosgene (carbonyl chloride), oxalyl chloride, thionyl chloride and phosphorus chlorides, e.g. phosphorus trichloride and phosphorus oxychloride (phosphoryl chloride). Phosphorus pentachloride can be used in the preparation of 3-chloro-3-cephem compounds, but this reagent reacts simultaneously with the 7-acylamino side chain in the starting material to form the imino chloride, the reactive intermediate in the well-known cephalosporin side chain cleavage reaction. It is therefore preferred to use one of the other specified chlorinating agents.

The chlorination can be carried out by adding the chlorination reagent to a solution of the 3-hydroxy-3-cephemester in dry DMF at temperatures of 5-15°C, after which the reaction mixture is allowed to stand at room temperature for 4-8 hours or longer. The reaction is initially exothermic and the reaction vessel should therefore initially be cooled in an ice-water bath and then kept at approx.

25°C during the rest of the reaction. Said DMF is preferably dried before use using a molecular sieve. Although the reaction can be carried out in DMF as solvent, a co-solvent can also be used which can be used together with said DMF. Such a co-solvent is e.g. tetrahydrofuran, dioxane, methylene chloride, dimethylacetamide or dimethylsulfoxide which can be used together with DMF.

The desired 3-chloro-3-cephem esters can be recovered from the reaction mixture by pouring it into a water-ethyl acetate mixture and separating the organic phase containing the product. The organic phase can be washed, dried and then evaporated, whereby the 3-chloro-3-cephemester is obtained as an •amorphous residue. The compound can be obtained, in crystalline form, by treating the residue with ether or n-hexane.

After the preparation of the stated 7-acylamino-3-chloro-3-cephem-4-carboxylic acid esters as described above, the respective 7-amino-3-chloro-3-cephem-4-carboxylic acid esters can be prepared by carrying out the well-known N-deacylerin gene of the 7-acylamino side chain. Thus, the 7-acylamino-3-chloro-cephalosporin ester can be reacted with phosphorus pentachloride in methylene chloride in the presence of pyridine, whereby the imino chloride intermediate is formed. This can then be reacted with an alcoholic solvent such as methanol or isobutanol, whereby the corresponding iminoether is obtained. This can then be hydrolysed, whereby the 7-amino-3-chloro-3-cephem-4-carboxylic acid ester is obtained as a hydrochloride salt.

In a specific embodiment of the preparation of starting compounds and compounds of formula I, 7-phenoxyacetamidocephalosporanic acid is reacted with thiourea to produce the isothiouronium salt by a displacement of the acetoxy group in the 3-position of the cephalosporanic acid dihydrothiazine ring. The isothiouronium salt is then reacted with zinc and an excess of 90% formic acid in the presence of dimethylformamide at temperatures of approx. 25°C, whereby the 7-phenoxy-acetamido-3-exomethylene cepham-4-carboxylic acid is produced. This acid can then be esterified with p-nitrobenzyl bromide in the presence of a hydrogen-halide acceptor, whereby the 3-exomethylene encepham-4-carboxylic acid p-nitrobenzyl ester is obtained. This ester can then be ozonized in methylene chloride at temperatures of approx. -70°C, and the ozonolysis mixture is treated with sulfur dioxide to decompose the intermediate ozonide, yielding p-nitrobenzyl-7-phenoxyacetamido-3-hydroxy-3-cephem-4-carboxylate. The 3-hydroxy ester is then reacted with phosphorus trichloride in dry DMF, which gives p-nitrobenzyl-7-phenoxy-acetamido-3-chloro-3-cephem-4-carboxylate. The 3-chloroester is then reacted in methylene chloride with phosphorus pentachloride in the presence of pyridine, which in situ gives the imino chloride intermediate which is then reacted with methanol to produce the corresponding imino ether intermediate. When water is added to the reaction mixture, the iminoether will decompose to p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate. The 7-amino-3-chloro-3-cephem-4-carboxylic acid ester can then be acylated as previously described, with an amino-protected D-phenyl-glycine derivative, thereby obtaining an N-protected derivative of 7-(.-D-f enylglycylamino )-3-

chloro-3-cephem-4-carboxylic acid-p-nitrobenzyl.es t-

is a. After removal of the α-amino protecting group and the C4~carboxylic ester group, 7-(D-phenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid is obtained. It is understood from the preceding description of the preparation of starting compounds that a number of known 7-acylamino-cephalosporanic acids can be used for the preparation of the 7-amino-3-chloro-3-cephem-4-carboxylic acids described here.

7-(D-ct-phenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid is a preferred compound because it is of great commercial value•

The following examples illustrate the production of starting materials and final connections.

Example 1

p-nitrobenzyl-7-amino-3-methylene cepham-4-carboxylate hydrochloride.

A solution of 965 mg (2 mmol) of p-nitrobenzyl-7-phenoxy-acetamido-3-methylene cepham-4-carboxylate in 10 ml of methylene chloride was added to 175 mg of dry pyridine and 460 mg of phosphorus pentachloride and the mixture was stirred at room temperature for 6 hours.

1 ml of isobutanol was added to the mixture which was then stored at

0°C overnight. The reaction product, p-nitrobenzyl-7-amino-3-methylene cepham-4-carboxylate hydrochloride in response formed as a crystalline precipitate was filtered off to give 430 mg

(58% yield).

Elemental analysis for C^^H-^gN^O^SCl

Theoretical: C 46.69, H 4.18, N 10.89

Found: C 46.40, H 4.20, N 10.62

I.R. (Nujol)

Carbonyl absorption at 5.65 (.-3-lactam) and 5.75 (ester) ym. NMR (DMSO dg) signals at 6.35 (2d, 2H, C2~ H2), 4.98 (d, 1H,

Cg-H), 4.7-4.4(m, 6H, C4~H, ester CH2, C4~CH2 and C?-H), and 2.4-1.6 (m, 4H, aromatic H) rope.

Example 2

p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride.

A solution of 4 g of p-nitrobenzyl-7-amino-3-methylene-cepham-4-carboxylate hydrochloride in 620 ml of methanol was cooled in a dry ice-acetone bath, and ozone was bubbled through the cold solution for approx. 20 minutes. The reaction mixture was purified from residual ozone by passing nitrogen through the solution, after which 10 g of sodium bisulphite was added.

The reaction mixture was stirred for 1 hour at ice bath temperature and the mixture gave a negative potassium iodide starch test at this time.

The mixture was evaporated in vacuo, whereby the reaction product was obtained as an amorphous yellow residue. This was crystallized in acetone to give 3.4 g of p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride as a crystalline acetone solvate.

I.R. (Nujol)

Carbonyl absorption bands at 5.60 (3-lactam) and 6.04'

(ester carbonyl hydrogen bonded to 3-hydroxy) etc. N.M.R. (DMSO db,) signals at 7.92 (s, 3H, 0.5 mol acetone),

6.22 (2d, 2H, C2-H2), 5.07 (d, 1H, CgH), 4.8-4.5 (m, 3H, ester CH^ and C7H), 2.4-1.6 (m, aromatic II) Lau.

Example 3

p-nitrobenzyl 7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate

A solution of 1.55 g of p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride in 30 ml of acetone containing 364 mg (0.5 ml, 3.6 mmol) of triethylamine was added 962 mg urea. While stirring at room temperature, a solution of 730 mg (4.4 mmol) of 2-thiopheneacetyl chloride in 20 ml of acetone was added dropwise. After 2.5 hours, the reaction mixture was filtered and evaporated. The residue was dissolved in ethyl acetate, and the solution was washed successively with water, a 5% solution of sodium bicarbonate, 5% hydrochloric acid and a saturated solution of sodium chloride. The washed solution was dried and concentrated by evaporation in vacuo, and this gave 1.2 g of reaction product as a crystalline residue. The product was recrystallized from ethyl acetate to give pure p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate with the following spectral properties.

I.R. (Nujol)

Absorption peaks at 3.0 (amide NH), 5.68 (α-lactam carbonyl) and 6.1 (amide and ester hydrogen bonded to

3 OH) etc.

N.M.R. (CDC13 d):

Signals at 6.54 (2d, 2H, C2H2), 6.16 (s, 2H, side-chain CH2), 4.90 (d, 1H, CgH), 4.60 (d, 2H, ester CH2), 4.43 (q, 1H, C7H), 3.1-1.6 (m, 7H, aromatic H) and 1.30 (d, 1H, amide NH) tau.

Example 4

Diphenylmethyl 7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate

a) A solution of 34 g (100 mmol) of 7-[2-(2-thienyl)-acetamido]-3-methylenecepham-4-carboxylic acid in 500 ml of methylene chloride was

added 21.4 g (110 mmol) of diphenyldiazomethane, and the resulting mixture was stirred for 2 hours at room temperature. The solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate. The ethyl acetate solution was washed with a 5% solution of sodium bicarbonate, then with water and then dried over magnesium sulfate. The dry solution was concentrated to a small volume. On standing, 40 g of diphenylmethyl-7-[2-(2-thienyl)acetamido]-3-methylene cepham-4-carboxylate with a melting point of approx. 132-133°C.

IR (chloroform):

Absorption peaks at 2.9 (amide N-H), 5.65, 5.75 and 5.93 (|3-lactam, ester and amide carbonyl groups respectively) and 6.62 (amide II) ym.

NMR (CDCl 3 ):

Signals at 6.72 (ABq, 2H, C2~H2), 6.21 (s, 2H, a-CH2), 4.83-4.65 (m, 4H, C4~H, Cg-H and C3~ CH2), 4.39 (q, 1H, C7-H), 3.4-2.65 (m, 15H, C?-NH, ester CH and aromatic H) tau.

b) A solution of 8.1 g (16 mmol) of the above-mentioned ester i

80 ml of methylene chloride was added to 1.57 g (1.6 ml, 19.6 mmol)

dry pyridine and 3.8 g (18.1 mmol) phosphorus pentachloride. The reaction mixture was stirred for 2 hours at room temperature and then cooled in an ice-water bath. The cold mixture was treated with 8 ml of isobutanol with stirring. Stirring was continued for 2 hours and during this time 3 g of diphenylmethyl-7-amino-3-methylene cepham-4-carboxylate hydrochloride formed as a crystalline precipitate. This product was filtered off and washed with methylene chloride and vacuum dried.

Elemental analysis (percent) for <C>21<H>21N203SC1: Theoretical: C 60.50, H 5.08, N 6.72, Cl 8.50 Found: C 60.70, H 5.02, N 6, 71, Cl 8.80

NMR (DMSO dg):

Signals at 6.45 (ABq, 2H, C2~H2), 5.00 (d, 1H, Cg-H), 4.68 (d, 1H, C?-H), 4.60 (s, 2H, 3-CH2), 4.44 (s, 1H, C4-H), 3.10 (s, 1H, ester CH), and 2.61 (s, 10H, aromatic H) tau.

c) The 7-amino-3-exomethylene encephamester hydrochloride salt 2.1 g (5 mmol), was dissolved in 200 ml of methanol and the solution was

cooled in an acetone-dry ice bath. Ozone was bubbled into the cold solution for 7 minutes to form the intermediate ozonide. The ozonide was decomposed by passing a stream of sulfur dioxide gas through the reaction mixture for 2 minutes. Then the mixture was evaporated and the residue

was treated with diethyl ether to give 1.6 g of diphenylmethyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride as a crystalline solid.

NMR (CDCl 3 ):

Signals at 6.4 (ABq, 2H, C2~H2), 5.0-4.5 (m, 2H, Cg-H and C7~H), 3.2-2.4 (m, 11H, ester CH and aromatic H) rope.

IR (chloroform):

Carbonyl absorption peaks at 5.57 and 5.70 (3-lactam and ester carbonyl respectively) ym.

UV (pH 7 buffer):

A max 275 my, e 7550.

Electrometric titration (60% aqueous DMF):

Titratable groups at 4.5 and 6.5.

d) A solution of 840 mg of diphenylmethyl-7-amino-3-hydroxy-3-cephem-4-carboxylate in 10 ml of water and 10 ml of acetone was

added 1 g of sodium bisulphite. The mixture was stirred and 800 mg of thiophene-2-acetyl chloride was added dropwise

10 ml of acetone. The mixture was stirred at room temperature i

4.5 hours and was then evaporated under reduced pressure. The residue was dissolved in a mixture of ethyl acetate and an aqueous 5% solution of sodium bicarbonate. The ethyl acetate layer was separated, washed with water and dried. The dried solution was evaporated and the residue treated with ether to give 500 mg of diphenyl-methyl-7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate.

NMR (CDCl 3 ):

Signals at 6.79 (s, 2H, C2"H2), 6.16 (s, 2H, a-CH2), 5.0 (d, 1H, Cg-H), 4.32 (q, 1H, C ?-H), 3.05-2.46 (m, 15H, C^-NH, ester CH and aromatic H) tau.

IR (chloroform):

Absorption peaks at 2.9 (amide NH), 5.6, 5.73 and 5.95 (3-lactam, ester and amide carbonyl respectively) and 6.65 (amine II) ym. e) To a solution of 4.2 g of diphenylmethyl-7-[2-(2-thienyl)-acet-amido]-3-hydroxy-3-cephem-4-carboxylate in 44 ml of dry dimethylformamide was added 865 mg of phosphorus trichloride. The mixture was stirred for 1.5 hours at room temperature and poured into a mixture of ethyl acetate and 5% aqueous hydrochloric acid. The ethyl acetate layer was evaporated, washed with 5% hydrochloric acid, water and dried. The dried solution was concentrated in vacuo, and a product crystallized out: the 3-chloroester was filtered off, washed with cold ethyl acetate and dried, whereby 2.2 g were obtained.

Elemental analysis (%) for C2gH21<N>2<0>4<S>2<Cl>:

Theoretical: C 59.48, H 4.03, N 5.34, Cl 6.75 Found: C 59.77, H 4.25, N 5.40, Cl 6.91

NMR (CDCl 3 ) :

Signals at 6.49 (ABq, 2H, C2~H2), 6.22 (s, 2H, a-CH2), 5.08 (d, 1H, Cg-H), 4.19 (q, 1H, C ?-H), 3.13-2.5 '(m, 15H, C7~NH, ester CH and aromatic H) tau.

IR (CHC13) :

Absorption peaks at 2.9 (amide NH), 5.55, 5.72 oy

5.90 (3-lactam, ester and amide carbonyl groups) and 6.60 (amide II) ym.

UV (dioxane):

X max 275 my, e = 8700.

Example 5

p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate (via thionyl chloride).

A solution of 1.9 g (4 mmol) of p-nitrobenzyl-7-[2-(2-thienyl)-acetamido]-3-hydroxy-3-cephem-4-carboxylate in 10 ml of DMF (dried over a molecular sieve) was added 950 mg (0.58 ml, 8 mmol)

of freshly distilled thionyl chloride. The mixture was stirred at room temperature for 6.5 hours and then poured into 100 ml of ethyl acetate. The mixture was extracted three times with 30 ml portions of 5% hydrochloric acid and with a saturated solution of sodium chloride. The washed ethyl acetate solution was filtered and evaporated to dryness in vacuo. The residue was treated with ether, whereby 1.2 g of p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate was obtained as a brown crystalline solid, melting point approx. . 164-166°C.

Elemental analysis (%) for C__H,,No0,SoCl:

20 16 3 6 2

Theoretical: C 48.63, H 3.27, N 8.51, Cl 7.18

Found: C 48.47, H 3.29, N 8.78, Cl 6.96

IR (chloroform)

showed absorption bands at 2.9 (amide NH), 5.59 (β-lactam carbonyl), 5.75 (ester carbonyl) and 5.92 μm (amide carbonyl).

UV absorption spectrum (acetonitrile)

showed maxima at A max 235 my, e = 12,100 and

A max 268 my, e = 15,800.

The mass spectrum of the product showed a molecular ion at

4 93 m/e.

NMR (CDCl 3 )

showed signals at 6.39 (ABq, 2H, C2~H2), 6.17 (s, 2H,

a CH2), 4.99 (d, 1H, Cg-H), 4.64 (s, 2H, ester CH2),

4.19 (q, 1H, C?-H), 3.45 (d, 1H, Cy-NH), 3.1-1.67 (m, 7H, aromatic H) tau.

Example 6

p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate (via phosphorus trichloride).

A cooled solution of 439 mg (0.93 mmol) p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate in 4.4 ml DMF was slowly added 85 mg (0.05 ml, 0.63 mmol) phosphorus trichloride. The reaction mixture was allowed to stand for 4 hours

at room temperature, then the reaction mixture was worked up in the manner indicated in example 4, and a total of 374 mg of p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem- 4-carboxylate. The NMR spectrum of the product was in agreement with the expected product and corresponded to that found for the compound from Example 5.

Example 7

p-nitrobenzyl 7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate (via oxalyl chloride).

A solution of 439 mg (0.93 mmol) of p-nitrobenzyl-7-[2-(2-thien-nyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate in 4.4 ml of DMF cooled in a ice bath, 118 mg (0.07 ml, 0.93 mmol) of oxalyl chloride was added dropwise. The reaction mixture was left at room temperature for 4 hours and then poured into a mixture of aqueous 5% hydrochloric acid and ethyl acetate. The organic layer was separated and washed first with 5% hydrochloric acid, then with water and a saturated solution of sodium chloride. The washed layer was dried and evaporated to dryness to give the reaction product, p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate as an amorphous solid. The product was obtained in crystalline form by treating the amorphous residue with ether. Yield 360 mg. The infrared spectrum and the NMR spectrum of the crystalline product were consistent with a spectrum of authentic material.

Example 8

p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate hydrochloride.

A solution of 500 mg of p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate in 6 ml of methylene chloride was added

added 95 mg of dry pyridine and 237 mg of phosphorus pentachloride. The reaction mixture was stirred at room temperature for 1.5 hours, and then cooled in an ice bath to approx. 5°C, after which 0.6 ml of isobutyl alcohol was added. During continued cooling and stirring, the reaction product, p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate hydrochloride, was crystallized from the reaction mixture. The product was filtered off, washed with cold methylene chloride and dried, whereby 200 mg of crystalline product was obtained which, during decomposition, melted at approx. 168°C.

Percent elemental composition for C,.H,_C1N_0_S.HC1:

3 14 13 3 5

Theoretical: C 41.39, H 3.20, N 10.34, Cl 17.45

Found: C 41.14, H 3.31, N 10.44, Cl 17.29

IR (Nujol):

showed absorption bands at 5.55 (3-lactam carbonyl) and

at 5.78 (ester carbonyl) ym.

UV (pH 7 buffer):

showed absorption maximum X max 268 my (e = 13,800)

NMR (DMSO dg):

Signals at 5.97 (s, 2H, C2"H2), 4.8-4.5 (m, 4H,

C,-H and ester CH_) and 2.35-1.6 (q, 4H, aromatic H)

rope.

Example 9

Diphenylmethyl 7-amino-3-chloro-3-cephem-4-carboxylate.

To a solution of 525 mg of diphenylmethyl-7-[2-(2-thienyl)-acetamido]-3-chloro-3-cephem-4-carboxylate in 20 ml of methylene chloride was added 0.1 ml of dry pyridine and 237 mg of phosphorus pentachloride. The reaction mixture was stirred for 2 hours at room temperature and then cooled in an ice-water mixture. To the cold mixture was added 0.6 ml of isobutanol and after 30 minutes the mixture was evaporated. The residue was dissolved in ethyl acetate, and the solution was washed with a 5% sodium bicarbonate solution and then with water and then dried. The dried solution was evaporated to dryness and the residue was treated with ether to give 190 mg of the 3-chloro-nuclear ester, diphenylmethyl-7-amino-3-chloro-3-cephem-4-carboxylate.

IR (mill):

absorption peaks at 5.7 and 5.9 (3-lactam and ester carbonyl) etc.

NMR (CDCl 3 ):

Signals at 6.35 (ABq, 2H, C2~H2), 4.78 (2d, 2H, Cg-H and C?-H), 3.05 (s, 1H, ester CH) and 2.65 (s , 10H, aromatic H) .

Example 1 0

7-(D-a-phenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid

A suspension of 280 mg (1.2 mmol) of 7-amino-3-chloro-3-cephem-4-carboxylic acid in 14 ml of acetonitrile was stirred at

room temperature added 0.5 ml of N,0-bis-(trimethylsilyl)acetamide, whereby a soluble disilylmethyl derivative was obtained. The solution was cooled to 0°C and slowly added a solution of a mixed anhydride formed by reacting 408 mg (1.5 mmol) of methyl 3-α-carboxybenzylaminocrotonate sodium salt and 161 mg (1.7 mmol) of methyl chloroformate in the presence of 2 drops of dimethylbenzylamine in 7 ml of acetonitrile. The mixture was stirred at ice bath temperature for 2 hours, 1 ml of methanol was added and the mixture was filtered to remove insoluble impurities. 2 ml of water was added to the filtrate, and the pH was adjusted to 1.5, to remove the enamine block, and then to pH 4.5 with triethylamine. After stirring for a further 1 hour at ice bath temperature, the reaction product 7-(D-α-phenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid (zwitterion) was precipitated from the reaction mixture as a crystalline solid. The product was filtered, washed with acetonitrile and dried in vacuum, and a total of 200 mg was obtained.

The product had the following physical characteristics: Elemental analysis for C15H14N304SC1.0.5H20: Theoretical: C 47.80, H 4.01, N 11.15, Cl 9.40 Found: C 47.55, H 4.12, N 10, 98, Cl 9,21

IR spectrum

absorption peaks at 2.9 (amide NH), 5.70 (3-lactam carbonyl), 5.95 (amide carbonyl) and 6.28 (carboxylate)

■p.m.

NMR spectrum (D20/DC1):

signals at 6.5-6.7 (ABq, 2H, C2"H2), 4.84 (d, 1H, Cb ,-H), 4.26 (d, 1H, C_/-H) and 2.44 (p, 5H, aromatic

H) rope.

UV spectrum (pH 7 buffer):

X max 265 my (e = 6,800).

Example 11

To a solution of 500 mg (1.85 mmol) of methyl 3-α-carboxybenzyl-aminocrotonate sodium salt (formed with phenylglycine and methyl acetoacetate) in 20 mL of acetonitrile was added 4 drops of dimethylbenzylamine, and the solution was cooled in a dry ice-carbon tetrachloride mixture while stirring. To the cold solution was slowly added 184 mg (1.95 mmol) of methyl chloroformate to form a mixed anhydride. After 20 minutes, a pre-cooled solution of 750 mg (1.85 mmol) of p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate and 188 mg (1.85 mmol) of triethylamine in 40 ml of acetone was added . The addition took place over 3 minutes, and then the reaction mixture was stirred in the cold for 30 minutes and then at room temperature for 2 hours. The mixture was filtered to remove insoluble impurities and then evaporated in vacuo. The residue was dissolved in a mixture of ethyl acetate and water, and the pH of the solution was adjusted to 7. The organic layer was separated and washed with water. After drying over magnesium sulfate, the organic layer was concentrated in vacuo to a small volume. By adding n-hexane to the concentrate, 620 mg of p-nitrobenzyl-7-[N-(1-carbomethoxy-2-propenyl)-D-a-phenylglycylamino]-3-chloro-3-cephem-4-carboxylate were precipitated from the reaction mixture .

The product gave the following elemental analysis and had the following nuclear magnetic resonance spectrum:

Elemental analysis for C„-,H„^N .0oSCl:

J 27 26 4 8

Theoretical: C 53.87, H 4.35, N 9.31

Found: C 54.05, H 4.13, N 9.36

NMR (DMSO d, ):

b

Signals at 8.20 (s, 3H, enamine CH3), 6.60 (ABq, 2H, C2-H2), 6.45 (s, 3H, ester CH"3), 5.48 (s, 1H, enamine -vinyl H), 4.90-4.1 (m, 5H, Cg-H, C?-H, a-CH and ester CH2) and 3.10-1.5 (m, 9H, aromatic H) tau .

The product, 540 mg (0.9 mmol), was dissolved in 40 mL of acetonitrile containing 20 mL of water, and the solution was first cooled in an ice-water mixture and then immediately acidified to pH 1.5

and then adjusted to pH 2.5. The mixture was evaporated and the residue was dissolved in 40 ml of tetrahydrofuran and 80 ml of methanol.

To the solution was then added 540 mg of 5% palladium-on-carbon (pre-reduced in 20 ml of ethanol) over 45 minutes under a hydrogen pressure of 3.5 kg/cm 2 at room temperature), and the solution was hydrogenated at room temperature for 2.5 hours under a hydrogen pressure of 3.5 kg/cm 2 . The catalyst was filtered off and washed on the filter with methanol, THF and with water. The filtrate and washing solution were combined and evaporated to dryness in vacuo. The residue was dissolved in water-ethyl acetate and the pH of the solution was adjusted to 4.5. The aqueous phase was separated, washed with ethyl acetate and then evaporated to a small volume of approx. 2 ml. Upon cooling, 65 mg of the product was precipitated, i.e.: 7-(D-α-phenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid as a crystalline solid.

Example 12

To a suspension of 3.0 g (8.1 mmol) of p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate in 200 ml of tetrahydrofuran (dried with a molecular sieve) was added 2.1 g (8 .3 mmol) N-(t-butyloxycarbonyl)-D-α-phenylglycine and 2.0 g (8.3 mmol) N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The reaction mixture was stirred overnight at room temperature and the solvent was removed under reduced pressure. The residue was dissolved in a mixture of ethyl acetate and water, and the organic phase was separated. It was cooled and successively washed with cold 5% aqueous solution of sodium bicarbonate, a cold solution of 5% hydrochloric acid and then with water. The washed solution was dried over magnesium sulfate, filtered and concentrated by evaporation under reduced pressure to a volume of

about. 50 ml. From this concentrate, 3.7 g (63% yield) of the reaction product was obtained as a crystalline product, namely p-nitrobenzyl-7-[D-2-(t-butyloxycarbamido)-2-phenylacetamido]-3-chloro-3- cephem-4-carboxylate. A new yield of the product of approx. 2 g was obtained by further concentration of the filtrate obtained after filtering off the first yield.

Elemental analysis calculated for C0_,H„_C1N .0oS

z / z / 4 o

Theoretical: C 53.78, H 4.51, N 9.29

Found: C 52.66, H 4.36, N 8.88

UV (acetonitrile) showed a maximum at 268 µm (e = 17,100).

IR: The following significant absorption peaks were observed in the infrared spectrum of the product. Amide NH absorption peak at 3.05 um and carbonyl absorption peaks at 5.59, 5.75 and 6.0 um.

NMR (CDCl 3 ):

Signals at 8.60 (s, 9H, t-BOC), 6.45 (ABq, 2H,

C2-H2), 5.03 (d, 1H, Cg-H), 4.67 (s, 3H, a CH and ester CH2), 4.12 (m, 3H, C7~H and amide NH), and 2.72-1.74 (m, 10H, aromatic H and amide NH) tau.

A solution of 3.0 g (5.0 mmol) of the product in 15 ml of dry tetrahydrofuran (dried with a molecular sieve) and 185 ml of methanol was added to 3 g of pre-reduced 5% palladium-on-carbon. The catalyst was pre-reduced in ethanol for 30 minutes at room temperature under a hydrogen pressure of 3.5 kg/cm . After the pre-reduced catalyst had been added, the product was hydrogenated at room temperature for 1 hour under a hydrogen pressure of 3.5 kg/cm 2 . The catalyst was filtered off and washed on the filter with tetrahydrofuran and methanol. The filtrate and washing solution were combined and evaporated under reduced pressure. The residue was dissolved in ethyl acetate and water was then added. The pH of the mixture was adjusted to 7 by adding 1N sodium hydroxide. The aqueous phase was separated, washed with ethyl acetate and then overlaid with ethyl acetate and back titrated to pH 2.5 with 1N hydrochloric acid. The organic phase was separated from the aqueous phase and washed with water and dried over magnesium sulfate.

The dry organic phase was evaporated under reduced pressure to dryness, whereby the reaction product 7-[D-2-(t-butyloxycarbamido)-2-phenylacetamido]-3-chloro-3-cephem-4-carboxylic acid was obtained as a dry solid residue . The product was crystallized from 70 ml of ether containing 20 ml of petroleum ether and 1.75 g (75%) of crystalline product was obtained.

Elemental analysis calculated for CoriH__ClN^.O,S:

Zv ZZ ob

Theoretical: C 51.34, H 4.74, N 8.98, Cl 7.58

Found: C 51.02, H 4.96, N 8.75, Cl 7.30

UV (acetonitrile):

maximum at 2 68 ym (e = 74 00) .

NMR (CDC1 ):

c

Signals at 8.55 (s, 9H, 5-BOC), 6.48 (ABq, 2H, C2-H2), 5.0 (d, 1H, Cg-H), 4.63 (d, 1H, a -CH), 4.25 (q, 1H, Cy-H), 3.90 (d, 1H, amide NH), and 2.59 (s, 5H, aromatic H) tau.

A solution of 420 mg (2.2 mmol) of p-toluenesulfonic acid in 5 ml of acetonitrile was added to 468 mg (1 mmol) of the crystalline hydrogenolysis product. The solution was left at room temperature for approx. 16 hours and then diluted with 0.5 ml of water. The pH of the solution was adjusted to 4.8, and a precipitate was obtained

320 mg (87%) of the crystalline deblocked product, namely 7-(D-phenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid. The product was collected by filtration and dried.

Elemental analysis calculated for C,rH, .C1N.0.S.0.5 H„0

2 ^ 15 14 3 4 2 Theoretical: C 47.80, H 4.01, N 11.15, Cl 9.40 Found: C 48.04, H 3.82, N 11.18, Cl 9.70

Example 13

7-(D-3-hydroxyphenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid.

N-(t-butyloxycarbonyl)-D-3-hydroxyphenylglycine, 2.9 g (11 mmol), was reacted with 3.7 g (10 mmol) of p-nitrobenzyl-7-amino-3-chloro-3-cephem- 4-carboxylate and 2.6 g (10.5 mmol) of the coupling reagent EEDQ. The reaction was carried out and the product isolated by using the method described in connection with the acylation method from Example 12. The product was obtained as an amorphous solid after treatment with ether, and 2.8 g (46%) were obtained.

Elemental analysis calculated for C^H^ClN^OgS

Theoretical: C 52.39, H 4.40, N 9.05

Found: C 52.16, H 4.59, N 8.79

Ultraviolet absorption spectrum (acetonitrile):

maximum at 270 ym (e = 17,200).

Nuclear magnetic resonance spectrum (CDCl^):

Signals at 8.59 (s, 9H, t-BOC), 6.50 (ABq, 2H, C2~H2), 5.06 (d, 1H, Cg-H), 4.66 (s, 1H, a -CH), 4.09 (m, 2H, C7-H), 3.34-1.70 (m, 9Y, aromatic H and amide NH) tau.

The product, p-nitrobenzyl-7-[D-2-(t-butyloxycarbamido)-2-(3-hydroxy)phenylacetamido]-3-chloro-3-cephem-4-carboxylate, 3.5 g (5.6 mmol ) was hydrogenated in ethanol in the presence of prereduced 5% palladium-on-carbon and followed by the deesterification procedure described in Example 12. The product, 7-[D-2-(t-butyloxycarbamido)-2-(3- hydroxy)phenylacetamido]-3-chloro-3-cephem-4-carboxylic acid was obtained in crystalline form by treating the amorphous crude product with a solution of hexane in diethyl ether. Yield: 1.5 g (55%).

Ultraviolet absorption spectrum (acetonitrile) :

maximum at 272 ym (e = 8.280).

Electrometric titration (66% aqueous DMF):

pKa at 4.5.

The above product, 1.3 g (2.7 mmol), was reacted with

1.1 g (5.9 mmol) of p-toluenesulfonic acid in 28 ml of acetonitrile to remove the t-butyloxycarbonyl protecting group. The procedure was carried out as described in example 12.

The product, 7-(D-3-hydroxyphenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid, was crystallized from the reaction mixture, filtered off and then vacuum dried. Yield 700 mg (64%).

Elemental analysis calculated for C^H-^CIN-jOj-S . 1^0

Theoretical: C 44.83, H 4.01, N 10.46

Found: C 45.12, H 4.06, N 10.31

Ultraviolet absorption spectrum (pH 6 buffer):

maximum at 268 ym (e = 9750).

Nuclear magnetic resonance spectrum (D20/DC1):

Signals at 6.31 (ABq, 2H, C2~H2), 4.81 (d, 1H, Cg-H), 4.52 (s, 1H, ct-CH) , 4.26 (d, 1H, C ?-H) and 3.1-2.5 (m, 4H, aromatic H) tau.

Example 14

7-(D-4-chlorophenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid.

Using the procedures described in Example 12, 850 mg (2.3 mmol) of p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate was acylated with 700 mg (2.5 mmol) N-t-butyloxycarbonyl-D-4-chlorophenylglycine and 567 mg (2.3 mmol) EEDQ, whereby 1,2 p-nitrobenzyl-7-[D-2-(t-butyloxycarb-amido)-2-(4- chlorophenylacetamido)]-3-chloro-3-cephem-4-carboxylate.

The product, 1.2 g (1.9 mmol) was hydrogenated at room temperature in the presence of pre-reduced 5% palladium-on-carbon to remove the p-nitrobenzyl ester group to give 450 mg of 7-[D-2-(t-butyl -oxycarbamido)-2-(4-chlorophenylacetamido)]-3-chloro-3-cephem-4-carboxylic acid as a crystalline product. The product was obtained in crystalline form by treating the amorphous residue with diethyl ether.

Elemental analysis calculated for C^H^C^N^OgS

Theoretical: C 47.82, H 4.21, N 8.36, Cl 14.11

Found: C 47.75, H 4.43, N 8.11, Cl 14.15 Electrometric titration (66% aqueous DMF): pKa at 4.4, apparent molecular weight = 508, calculated molecular weight = 502.

The de-esterification product, 450 mg (0.9 mmol) was reacted with p-toluenesulfonic acid in acetonitrile using the procedure described in previous examples to remove the t-butyloxycarbonyl protecting group, yielding 160 mg (44%) crystalline 7-(D-4-chlorophenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid in the zwitterionic form.

Elemental analysis calculated for C15N13C12N304S.1H20

Theoretical: C 42.86, H 3.59, N 9.99, Cl 16.87

Found: C 43.07, H 3.63, N 9.69, Cl 16.75 Ultraviolet absorption spectrum (pH 6 buffer): maxima at 265 ym (e = 8100) and 2.25 ym (e = 13900). Electrometric titration (66% aqueous DMF): pKa = 4.15 and 6.8, apparent molecular weight is 407, calculated molecular weight 403.

Example 1 5

7-(D-4-hydroxyphenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid.

N-(t-butyloxycarbonyl)-D-4-hydroxyphenylglycine, 2.9 g (11 mmol), was reacted with 3.7 g (10 mmol) of p-nitrobenzyl-7-amino-3-chloro-3-cephem- 4-carboxylate and 2.6 g (10.5 mmol) of the coupling reagent EEDQ in dry tetrahydrofuran. The reaction was carried out and the product isolated as described in Example 12. The product of 3.7 g (60% yield) was obtained in crystalline form from cold diethyl ether.

The product, p-nitrobenzyl-7-[D-2-(t-butyloxycarbamido)-2-(4-hydroxy)phenylacetamido]-3-chloro-3-cephem-4-carboxylate gave the following percent elemental analysis and nuclear magnetic

resonance spectrum.

Elemental analysis calculated for <C>27<H>27N4OgSCl

Theoretical: C 52.39, H 4.40, N 9.05

Found: C 52.12, H 4.26, N 8.91

Nuclear magnetic resonance spectrum (DMSO db,)

showed signals at: 8.62 (s, 9H, t-BOC-CH3), 6.16 (ABq, 2H, C2-H2), 4.81 (d, 1H, Cg-H), 4.75 (d , 1H, α-CH), 4.53 (s, 2H, ester-CH2), 4.18 (q, 1H, C?-H), 7.04 and 2.0 (2q, 8H, aromatic H) , 0.76 (d, 1H, C7~NH) and 0.58 (s, 1H, p-OH) tau.

The product of 2.2 g (3.5 mmol) was hydrogenated in ethanol in the presence of pre-reduced 5% palladium-on-carbon catalyst to remove the p-nitrobenzyl group. The deesterified product, 7-[D-2-(t-butyloxycarbamido)-2-(4-hydroxy)phenylacetamido]-3-chloro-3-cephem-4-carboxylic acid, was obtained in crystalline form from a mixture of diethyl ether and hexane. 1 g of the product was obtained and this corresponds to a 59% yield:

Elemental analysis calculated for C2qH22C1N307S

Theoretical: C 49.64, H 4.58, N 8.08

Found: C 48.92, H 4.40, N 8.24

Nuclear magnetic resonance spectrum (DMSO dg)

shows signals at 8.61 (s, 9H, t-B0C-CH3), 6.26 (ABq, 2H, C2H2), 4.89 (d, 1H, Cg-H), 4.78 (d, 1H, a-CH),

4.28 (q, 1H, C?-H), 3.06 (q, 4H, aromatic-H) and 1.20 (d, 1H, C?-NH) tau.

Said t-BOC protecting group was removed from the deesterification product by reacting this in acetonitrile with p-toluenesulfonic acid. From 1 g of product, 330 mg (40%) of the final product was obtained, i.e. 7-(D-4-hydroxyphenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid with the following elemental analysis, nuclear magnetic resonance spectrum and titration values.

Elemental analysis calculated for C^j-H-^ClN^Oj-S . 1^0

Theoretical: C 44.83, H 4.01, N 10.46

Found: C 44.92, H 3.45, N 10.63 Electrometric titration in 66% aqueous DMF gave

pKa values of 4.2, 7.7 and 12.4. Apparently

molecular weight was 384, the calculated molecular weight was 383.8. Nuclear magnetic spectrum (D2O/DCI)

showed signals at: 6.32 (ABq, 2H, C2~H2), 4.84 (d, 1H, Cg-H), 4.27 (d, 1H, C?-H), and 2.79 (q , 4H, aromatic H) tau.

Claims (2)

1. Analogous process for the preparation of therapeutically effective 3-chloro-7-a-aminoacylcephalosporin compounds of the formula: where R is phenyl, 3- or 4-hydroxyphenyl or 4-chlorophenyl, as well as pharmaceutically acceptable, non-toxic salts thereof, characterized in that a 3-chloro-3-cephem ester with the formula: where R^ is benzyl, p-methoxybenzyl, p-nitrobenzyl, diphenylmethyl, 2,2,2-trichloroethyl, trimethylsilyl or t-butyl, is reacted with an acylating agent of the formula: where R has the above meaning, and R2 is t-butyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, trichloroethoxycarbonyl or a trityl group, or enamines formed with methyl acetoacetate and acetylacetone, or a reactive derivative thereof, after which the carboxylic acid protecting and amino protecting groups are removed , and, if desired, converting a compound thus obtained into a pharmaceutically acceptable, non-toxic salt thereof. 2. Analogy method according to claim 1, characterized in that a compound of formula I is prepared where R is phenyl.