NO792609L - 3-HALOGENCEPHALOSPORIN COMPOUNDS FOR USE AS INTERMEDIATE PRODUCTS IN THE MANUFACTURE OF THERAPEUTICALLY EFFECTIVE COMPOUNDS - Google Patents

Description

3-halocephalosporin compounds for use as intermediates in the preparation of therapeutically active (compounds gc^^s^^^Iw&a^ v~ vJ&-^

important information

For archival reasons, the Norwegian Patent Office only has access to documents for this generally available patent application that contain handwritten notes, comments or crossing outs, or that may be stamped "Expired" or the like. We have therefore had to use these documents for scanning to create an electronic edition.

Handwritten remarks or comments have been part of the proceedings, and must not be used to interpret the content of the document.

Cross-outs and stampings with "Expired" etc. indicates that newer documents have been received during the proceedings to replace the earlier document. Such crossing out or stamping must not be understood as meaning that the relevant part of the document does not apply.

Please ignore handwritten remarks, comments or crossing outs, as well as any stamps with "Expired" etc. which have the same meaning.

The present invention relates to 3-halocephalosporin compounds for use as intermediates in the production of therapeutically active compounds, more specifically for the production of 7-acylamido- and 7-amino-3-halo-3-cephem-4-carboxylic acids, esters and pharmaceutically acceptable salts of such compounds, whereby a 7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid ester or a 7-amino-3-hydroxy-3-cephem-4-carboxylic acid ester is reacted with a chlorinating or brominating agent. E.g. 7-amino-3-chloro-3-cephem-4-carboxylic acid and 7-phenoxyacetamido-3-chloro-3-cephem-4-carboxylic acid can be produced in this way. Said 7-acylamido-3-halo-cephalosporin acids and their pharmaceutically acceptable salts and esters are valuable antibiotic compounds with desirable and beneficial therapeutic properties.

More particularly, the invention relates to 3"hal°gen"cePhal°~ sporins with the following general formula

where R is hydrogen or an acyl group derived from a carboxylic acid, R-^ is hydrogen, a carboxylic acid protecting ester group or a pharmaceutically acceptable ester or salt, and X represents chlorine or bromine.

Compounds according to the present invention have unique structural properties in that a halogen atom is directly bonded to the carbon atom in the 3-position of the d&hydrothiazire ring. According to the cepham nomenclature system, the above-mentioned compounds can be designated as 7-awino- or 7~acylamido-3_halogen-3-cephem-4-carboxylic acid, salts and esters.

Before this compound, 3-bromomethyl-3-cephem-4-carboxylic acid esters and 3-bromomethyl-2-cephem-4-carboxylic acid esters were known and described. These known 3-bromomethyl compounds have been described as valuable intermediates for the production of cephalosporin antibiotics. In contrast, the present 3-halo-3-cephem-4-carboxylic acids are particularly valuable antibiotics.

According to the present invention, 7'9-mino- and 7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid esters are halogenated under moderate conditions, whereby a 7-amino- or 7-acylamido-3-halo- 3-cephem-4-carboxylic acid ester. By using easily removable ester groups which are well known in connection with cephalosporins, the produced 3-halogen esters can be easily converted into free acids by known methods for removing such ester groups. The produced 7" "amino-3-halo-3-cephem-4-carboxylic acids can be acylated to produce 7~acylamido-3-halo-3-cephem-4-carboxylic acid antibiotics. Such 7~acylamido-3-halo-3-cephem-4- carboxylic acids, whether they are prepared by a direct halogenation of a 7~acylamido-3-hydroxy?3~cephem~4~carboxylic acid ester followed by an ester removal or by an acylation of a 7-amino-3-halo-3-cephem- 4--carboxylic acid or ester are valuable antibiotic compounds that can be used to inhibit the growth of microorganisms that are pathogenic both to animals and plants.

It is thus an aim of the present invention to provide a new group of cephalosporin antibiotics. More particularly, it is an aim of the present invention to provide 7-acylamido-3-halo-3-cephem-4-carboxylic acid antibiotic compounds. Furthermore, it is an aim of the present invention to provide said 3-nal°gen~substituted cephalosporin cores, 7-amino-3-halo-3-cephem-4-carboxylic acids as well as esters and salts thereof. Furthermore, it is an aim of the present invention to provide a method for the production of the 7-amino- and 7-acylamido-3-halo-3-cephem-4-carboxylic acids described here.

3-halo-cephalosporin compounds provided by the present invention can be represented by the following general formula:

where R is hydrogen or an acyl group derived from a carboxylic acid and represented by the formula where R' is C-^-Cg alkyl, C^-C^haloalkyl, C]-C^ cyanoalkyl, phenyl, methylphenyl, hydroxyphenyl, halophenyl, nitrophenyl, aminophenyl, methoxyphenyl, 5~amino"5-carboxybutyl or a 5-substituted-amino-5-carboxybutyl ester group of the formula:

where A is dif en y Imet yl., p-nitrobenzyl, benzyl, 2,2,2-trichloroethyl, t-butyl or p-methoxybenzyl and A' is Cg-C^alkanoyl, Cg-C^halogenalkanoyl, benzoyl, halobenzoyl , 2,4-dinitrophenyl or phthaloyl,

or R' is a group of formula

where a and a' are independently hydrogen, C-^-C^ lower alkyl, G-j^-C^ lower alkoxy, halogen, hydroxy, nitro, amino or carboxy,

■ Z is 0 or S and

m is 0 or 1

or R' is a group of the formula

where P is 2-thienyl, 3-thienyl>phenyl or a substituted phenyl group of the formula

where a and a' are as defined above, Q is hydroxyl, formyloxy, acetoxy, carboxy or sulfo,

or R<*>is a group with the formula

where P has the same meaning as stated previously, and Y is hydrogen, methyl or acetyl,

or R' is a group of the formula

where R" is 2-thienyl-3-thienyl, 2-furyl, 2-oxazyl, 2-thiazyl or 1-tetrazyl,

or R' is a group of the formula'

where a, a', Z and m have the same meaning as stated previously,

R-^ is hydrogen, benzyl, 4-methoxybenzyl, 4-nitrobenzyl, diphenylmethyl, 2,2,2-trichloroethyl, t-butyl or a pharmaceutically acceptable ester of the formula

and X is chlorine or bromine, and when R-^ is hydrogen, pharmaceutically acceptable salts of such compounds.

In the aforementioned definitions of compounds according to the present invention, the term "C-^-Cg alkyl" refers to straight or branched alkyl hydrocarbon groups such as methyl, ethylene n-propyl, isopropyl, n-butyl, sec-butyl, n-amyl, isoamyl, or n-hexyl, "C-^-G^ cyanoalkyl" denotes groups such as cyanomethyl, 2-cyanoethyl, 3-cyanoProPyl or 2-cyanopropyl, "^"^ alkanoyl" denotes groups such as acetyl, propionyl, or butyryl, "Cg"^ haloalkanoyl" refers to chloroacetyl, bromacethyl, 2-chloropropionyl or 3" bromobutyryl, "C-^-C^ lower alkyl" refers to straight or branched lower alkyl hydrocarbons such as methyl, ethyl7n-propyl , isopropyl, n-butyl, or t-butyl, "C-^-C^lower alkoxy" denotes groups such as methoxy, ethoxy, isopropoxy, or n-butoxy. As used herein, "halogen" means either chlorine or bromine. The term "halobenzoyl " refers to chlorine or bromine-substituted benzoyl groups such as 4-chlorobenzoyl, 4-bromobenzoyl or 2,4-dichlorobenzoyl.

Illustrative examples of groups in the above definition represented by the following formula and where m is 0 is

phenylacetyl, 4_methylphenylacetyl, enylacetyl, 4~isoProPyl~phenylacetyl, 2-methylphenylacetyl, 4~chlorophenylacetyl, 4~nitrof enylacetyl, 4~bromophenylacetyl, 2,4-dichlorophenylacetyl, 3~bromophenyl-acetyl, 4-3°dophenylacetyl . phenylacetyl, 4-methoxyphenylacetyl, 3>4~dimethoxyphenylacetyl, 4~t-'butoxyphenylacetyl, 2-carboxyphenylacetyl, 3~chloro-4-methylphenylacetyl or 3-nitrophenylacetyl When m in the above formula is 1

and Z represents -0-, illustrative groups are the following: phenoxyacetyl, 4~hydroxyphenoxyacetyl, 3"hydroxyphenoxyacetyl, 4-chlorophenoxyacetyl, 3"bromophenoxyacetyl, 3~ethylphenoxyacetyl, 4-methylphenoxyacetyl, 3~hydroxy-3-methylphenoxyacetyl, 4~afnin0~ f enoxyacetyl, 3~nitr°f<>>en°xyacetyl, 2-carboxyphenoxyacetyl, 2- chlorophenoxyacetyl, 4~t-butylphenoxyacetyl, 4~methoxyphenoxy-acetyl, 3>4~dimethoxyphenoxyacetyl, 2-aminophenoxyacetyl, 4~iso~propoxyphenoxyacetyl or 4~nitrophenoxyacetyl. When m in the preceding formula is 1 and Z is represented by -S-, the following groups are illustrative: phenylmercaptoacetyl, 4~chlorophenylmercaptoacetyl, 3-hydroxyphenylmercaptoacetyl, 3»4~dimethylphenylmercaptoacetyl, 4~aminophenylmercaptoacetyl, 3>4~dichlorophenylmercaptoacetyl, 3~bromo~phenylmercaptoacetyl, 4~fluorophenylmercaptoacetyl, 2,6-difluorophenyl-

mercaptoacetyl, 4-nitrophenylmercaptoacetyl, or 3~bluorphenyl-mercaptoacetyl .

When Rf in formula I is a 5-substituted amino-5-carboxybutyl group, then R' - C = 0 is representative of esterified amino-protected adipolyl groups, where the ester group is diphenylmethyl, p-nitrobenzyl, benzyl, p- methoxybenzyl, 2,2,2-trichloroethyl or t-butyl and representative substituted amino groups are acetamido, propionamido, chloroacetamido, benzamido, 2,4~dichlorobenzamido, 4~brombenzamido, phthalimido or 2,4~dinitroanilino.

When R' in formula I represents a group of the formula:

then illustrative acyl groups, R' - C = 0, the mandeloyl group with formula and 0-formyl derivatives of these are represented by the following formula the a-carboxyphenylacetyl group with the following formula ■ a-sulfophenylacetyl group with the formula besides the 2-thienyl and 3-thienylacyl groups where the phenyl group in the above formula is replaced by a 2-thienyl or 3-thienyl group. When R' is a group of the formula in the syn or anti form then illustrative acyl groups include the following groups

Examples of preceding acyl groups are 4-methylmandeloyl, 4-hydroxy-, symandeloyl, 3-hydroxymandeloyl, 4-aminomandeloyl, 3-t>rommandeloyl, 4-chloromandeloyl, 3-methyl~4--fluoromandeloyl, 2-fluoromandeloyl, 4 ~fluoromandeloyl, 4_methoxymandeloyl, 3> 4~dimethyl-0-formylmandeloyl, 4-chloro-0-formylmandeloyl, 3~amino~0~formylmandeloyl, 3~bromo-0-formylmandeloyl, 3>4~dimethoxy-0-formylmandeloyl, O -acetyl-mandeloyl, O-acetyl 4-hydroxymandeloyl > a-carboxy-4-niethylphenylacetyl, a-carboxy~35 4~dichlorophenylacetyl, a-carboxy-4-hydroxyphenylacetyl, a-carboxy-2-methoxyphenylacetyl, oc-carboxy- 4-isopropoxyphenylacetyl, α-carboxy-3-hydroxyphenylacetyl, α-carboxy-4-aminophenylacetyl, α-sulfo-4-methylphenylacetyl, α-sulfo-3,4-dichlorophenylacetyl, α-sulfo-4 -chlorophenylacetyl, a-sulfo-4-hydroxyphenylacetyl, oc-sulfo-3-methoxyphenylacetyl, a-oximino-4-hydroxyphenylacetyl,. a-oximino-3-chlorophenylacetyl, a-oximino-4-carboxyphenylacetyl, cc-methoxymino-4-methylphenylacetyl, a-methoxymino-3>5-dichlorophenylacetyl, )a-methoxymino-4-hydroxyphenylacetyl, a-methoxymino-2-aminophenylacetyl , a-acetyloximinophenylacetyl, a-acetyloximino-2-thienylacetyl, oc-acetyloximino-4-hydroxyphenylacetyl, a-oximinop-2-thienylacetyl, a-carboxy-2-thienylacetyl, a-carboxy-3-thienylacetyl, a-methoxyimino-2 -thienylacetyl, α-hydroxy-2-thienylacetyl, α-hydroxy-3-thienylacetyl, α-sulfo-2-thienylacetyl, α-formyloxy-2-thienylacetyl, α-acetoxy-2-thienylacetyl or α-methoxyimino-2-thienylacetyl .

When R' in the preceding formulas represents a group

with the formula R^CHg-, illustrative acyl groups in formula I are the following. 2-thienylacetyl, 3~thienylacetyl, 2-furylacetyl, oxazyl-2-acetyl, thiazyl-2-acetyl and tetrazyl-1-acetyl group with

following formula: 0

When R' in formula I represents the group

then representative examples of the acyl groups R' - C = 0 are the following t N-(phenylacetimidoyl)aminoacetyl, N-(phenoxyacetimidoyl)-aminoacetyl, N-(phenylmercaptoacetimidoyl)aminoacetyl, N -(4-chlorof.nymercapto-acetimidoyl)aminoacetyl, N -(4-methoxyphenylacetimidoyl)aminoacetyl, N-(2,6-dimethoxyphenylacetimidoyl)aminoacetyl, N-(4-hydroxyphenoxyacetimidoyl)aminoacetyl, N-(4-chlorophenoxyacetimidoyl)aminoacetyl, N-(4-nitrophenylacetimidoyl)aminoacetyl, N-(3>4-dimethylphenylacetimidoyl)aminoacetyl, N-(4-fluorophenoxyacetimidoyl)aminoacetyl and similar mono and disubstituted groups. A preferred group 3-halogen cephalosporin antibiotic according to the present invention is represented by the following formula. II where a, a', Z and m have the same meaning as stated above and X is chlorine. Illustrative examples of these preferred compounds are the following: 7~phenylacetamido-3-chloro-3-cephem, 4"carboxylic acid, j-phenox<y>acetamido-3-chloro-3-ce<p>heme-4-carboxylic acid, 7 -(4-hydroxyenylacetamido)-3-chlorotr3-cephem-4- carboxylic acid ,•

7"(4-chlorophenoxyacetamido)-3-chloro-3-cephem-4-carboxylic acid,

7β(4-methoxyphenoxyacetamido)-3-chloro-3-.c.ephem-4-carboxylic acid,

and pharmaceutically acceptable esters and salts of these compounds.

Another preferred group of compounds of formula I are those indicated by formula III where R" represents 2-thienyl, 3-thienyl, 2-furyl and 1-tetrazyl and X represents chlorine.

Illustrative examples of the aforementioned preferred compounds of formula III are the following: 7-(2-thienylacetamido)-3-chloro-3-cephem-4-carboxylic acid, ' 7~(2-furylacetamido)-3-chloro-3-cephem-4 -carboxylic acid, 7-(3-thienylacetamido)-3-chloro-3-cephem-4-carboxylic acid, 7-(1-tetracylacetamido)^3""chloro-3-cephem-4-carboxylic acid, 0£ pharmaceutically acceptable esters and salts of these compounds. Another preferred group of compounds of formula I is the following with formula IV

where P represents phenyl or a substituted phenyl group as defined in formula I, and Q is hydroxy or carboxy. Examples on preferred compounds of formula IV are the following: 7-D-mandelamido-3-chloro-3-cephem-4-carboxylic acid,

7-D-(4-chloromandelamido)-3-chloro-3-cephem-4-carboxylic acid, 7-D-(4-hydroxymandelamido)-3-chloro-3-cephem-4-carboxylic acid,

7~D-(4-Methoxymandelamide-3-chloro-3-cephem-4-carboxylic acid

7-(α-carboxyphenylacetamido)-3-chloro-3-cephem-4-carboxylic acid, and pharmaceutically acceptable esters and salts of these compounds.

3-halo-cephalos.porins described here and indicated by formula I can either be prepared by a direct halogenation of a 7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid ester or by an acylation of a 7-amino ~3~hal°gen""3-cePhem-4-carboxylic acid or an ester thereof (formula I, R=H) . The 7-amino°-3-halo-3-cephem-4-carboxylic acid or ester is prepared either by a direct halogenation of the corresponding 7-amino-3-hydroxy ester or by cleavage of the 7-acylamido chain in a 7- acylamide°-3-halogen-cephem ester.

Compounds of formula I where X is chlorine or bromine are prepared by reacting a 7-acylamido-3-hydroxy-3-cephem-.

ester or a 3-hydroxy-3~cephem core ester. in dimethylformamide

. (DMF) with a reactive chlorine or bromine compound which with DMF forms the chlorine or bromine-dimethyliminium chloride or bromide of the formula:

where X and X~ represent chlorine or bromine and the chloride or bromide respectively. The reactive halogen-iminium halide with the above formula is formed in situ and is a highly reactive chlorination or bromination intermediate. Chlorine and bromine compounds which form the above iminium halide 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). Bronzing agents which can be used in the present invention include carbonyl dibromide, ox.allyl bromide, thionyl bromide (sulfur oxybromide) and phosphorous bromides, such as. phosphorus oxybromide and phosphorus tribromide. Phosphorus pentachloride can be used in the preparation of 3-chloro-3~1cephem compounds according to the present invention, but this reagent reacts simultaneously with the 7-acylamido side chain in the starting material, so that imino chloride is formed, the reactive intermediate in the well-known cephalosporin side chain cleavage reaction . Accordingly, it is preferred to use one of the other chlorinating agents.

The chlorination and bromination of a 3-hydroxycephem ester can conveniently be carried out by using a dry DMF as solvent. DMF is preferably dried over a molecular sieve before it is used. A co-solvent can be used with an excess of DMF, although this is not necessary. One can e.g. as. cosolvent use tetrahydrofuran-dioxane, methylene chloride, dimethylacetamide or dimethylsulfoxide. The brominating or chlorinating agent is preferably used in an amount corresponding to two equivalents of the amount of 3-hydroxy-cephem ester used. The reaction is carried out by adding the halogenating agent to a. solution of 3-hydroxycephem ester in dry DMF which is maintained at a temperature of from 5° to 15°C, after which the reaction mixture is allowed to stand at room temperature for 4 to 8 hours or longer. The reaction is initially exothermic and the reaction vessel should preferably be kept in an ice-water bath so that the temperature is kept below approx. 25°C during the first part of the reaction. The reaction mixture can then be allowed to stand at or above room temperature for the rest of the reaction. How along the reaction is. progress can be determined by thin-layer chromatography.

Alternatively, the chlorination or bromination can be carried out by first preparing a mixture of the halogenating agent in DMF to form halogeniminium halide beforehand, after which a solution of the 3-hydroxy-3-cephem ester in DMF is added to this mixture, a mixture of DMF and a cosolvent or in a solvent such as dimethylacetamide or tetrahydrofuran.

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

The preferred chlorinating and brominating agents are phosphorus trichloride and phosphorus tribromide.

7-Amino-3-halo-3-cephem-4-carboxylic acid is preferably prepared by cleaving the 7-acyl group in a 7-a-cylamido-3-halo-3-cephem-4-carboxylic acid ester followed by a removal of the carboxylic acid protecting ester group.

As mentioned earlier, 3~chloro- or 3""bromo-cephalosporin compounds according to the present invention can be prepared either by halogenating a 7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid ester or by acylating a 3- Halogen~7-amino-3~cephem-4-carboxylic acid ester. When using the first-mentioned method for producing compounds according to the present invention, said (7-acylamido group in the starting material is preferably one that does not react with the halogenating agent under the conditions used. When, for example, the acyl group in the 7"" position in the starting material contains reactive functional groups such as a carboxyl group, an amino group or a sulphonic acid group, such groups should be blocked by the formation of an unreactive derivative before starting the above-mentioned halogenation reaction. Illustrative examples of 7-acylamido-3 -hydroxy-3-cephem-4-carboxylic acid esters which can be halogenated without simultaneous halogenation of the side chain are compounds of formula I, where R is 2~7alkanoyl, C8-C8haloalkanoyl, C8-G8cyanoalkanoyl , phenoxyacetyl, 2-thienylacetyl, 3-thienylacetyl and 2-furylacetyl.

7-amino-3-halo-3-cephem compounds of formula I, where R=H, can best be prepared by cleaving the 7-acylamido side chain from a 7-acylamido-3-halo-3-cephem-4-carboxylic acid ester by a well-known cleavage reaction using phosphorus pentachloride,

When e.g. rJ-(2-£-thienyl)-acetamido)-3-chloro-3-cephem-4-carboxylic acid p-nitrobenzyl ester is reacted with phosphorus pentachloride in methylene chloride in the presence of pyridine, then an eto imino chloride derivative is produced of this connection. This intermediate: is converted rried an alcohol, e.g. methanol or isobutanol, whereby the corresponding imino-ether derivative is obtained. A hydrolysis of the imino ether gives p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate hydrochloride.

The starting compounds used in the preparation of compounds according to the present invention can be prepared by reacting a 7-acylamido-3-exomethylenecepham-4-carboxylic acid ester or a 7-amino-3"exomethylenecepham-4-carboxylic acid ester with ozone in an inert solvent with temperature between -80 and 0°C, thereby forming the ozonide derivative at the 3-exomethylery double bond. This ozonide product which is not isolated can be decomposed by reacting it in situ with a mild reducing agent such as sodium bisulfite or preferably sulfur dioxide, whereby one "obtains the corresponding 3-hydroxy-3-cephem-4-carboxylic acid ester.

The ozonolysis of 7-amino-3-exomethylenecepham-4-carboxylic acid ester or of 7-acylamido-3-exomethylenecepham-4-carboxylic acid ester of formula V is carried out by passing ozone through a solution of 3-exomethylenecepham-ester in an inert solvent with temperatures between -80 and 0°C. The exomethylene double bond reacts with ozone so that an intermediate ozonide is formed in situ which decomposes as described below to form a 3-hydroxy-3-cephem ester of formula VI:

In the above formulas, R is hydrogen or an acyl group derived from a carboxylic acid and where the acyl group is non-oxidizable under the ozonolysis conditions described. R-^ is an ester-forming group and preferably one which readily lends itself to hydrogenolysis or under acidic or basic hydrolysis conditions.

Although 3-exomethylene cephalosporins can also undergo oxidation with ozone to form the sulfoxide, the exo double bond under the described ozonation conditions will preferentially react with ozone to form the ozohide. The formation of the sulfoxide occurs as a result of overoxidation. While the exo double bond reacts rapidly with ozone, the reaction at the sulfur atom in the dihydrothiazine ring to form sulf oxide takes place at a much slower rate. The following over-oxidation products can, however, be formed by ozonolysis reaction.

Ozone gas is produced using an ozone generator of the type that is usually used in synthetic and analytical chemical work for the production of ozone, and where the ozone is produced by an electrical discharge in oxygen. Such an ozone generator is manufactured by Welsbaek Corporation. 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 be varied as desired, e.g. by varying the flow rate of the oxygen through the ozone generator, in addition to varying the intensity of the electrical discharge. The percentage content of ozone in the oxygen stream can be determined iodometrically by titrating with sodium thiosulphate the quantity of iodine released from a standard solution of potassium iodide by ozone from the generator. The percentage content of ozone in the oxygen stream is not critical, but a determination of the amount of ozone that flows into the reaction mixture will, in the present method, make it possible to determine the time when the desired reaction is complete, whereby a minimal formation of over- oxidation products.

Alternatively, the ozonolysis reaction can be followed chromatographically. E.g. a small part of the reaction mixture can be taken out, the ozonide is decomposed and the amount of unreacted starting material and possibly the 3" hydroxy-3-cephem product in the sample can be examined using thin-layer chromatography, where one compares with a known amount of starting material and 3 ~the hydroxy-3-cephem compound.

Inert solvents that can be used in ozonolysis are those solvents in which the 3-exomethylene-cepham esters are at least partially soluble and which are unreactive with ozone under the conditions described. Generally, satisfactory results will be achieved with organic solvents such as methanol, ethanol,

•ethyl acetate, methyl acetate and methylene chloride.

The concentration of the starting material in the inert solvent is not critical and it is preferred to use a sufficient amount to form a complete solution.

The preferred temperature during the ozonolysis reaction

is between -80 and -50°C.

When ozonide formation is complete as determined by one of the above methods, excess ozone can be driven out of the reaction mixture by bubbling nitrogen or oxygen through the mixture. After any excess of ozone has been removed, the ozonide can be decomposed by adding to the reaction mixture a mild reducing agent selected from the group consisting of sodium bisulphite, sulfur dioxide and trimethyl phosphate, whereby the 3-hydroxy-3-cephem-4-carboxylic acid ether is obtained. The Decomposition is carried out by adding an excess of the reducing agent and stirring the reaction mixture at a temperature from -80 to d°C, until the reaction mixture is negative at the potassium iodide-starch test.

A preferred agent for decomposition of the intermediate ozonide is gaseous sulfur dioxide. This reagent is preferred as it can be completely expelled from the reaction mixture during the subsequent work-up and thus does not complicate the recovery of the reaction product.

The 7_acylamido-3-hydroxy-3-cephem-4-carboxylic acid esters produced can be recovered from the reaction mixture by first evaporating the mixture to dryness and then extracting the product from the residue. Alternatively, cari N-acylated 3~hydroxy-3-cephem esters are recovered from the organic liquid phase in the decomposition mixture by separating the liquid phase from insoluble solids, and after washing and drying, the organic layer can be evaporated, thereby obtaining 3~ hydroxy ester.

Said 3-hydroxy core ester, i.e. a 7-amino-3-hydroxy-3-cephem-4-carboxylic acid ester, can best be isolated in the form of a salt, e.g. to form a hydrochloride or hydrobromide.

When an ester of 7-amino-3-exomethylenecepham-4-carboxylic acid (formula V, R=H) is ozonized, it is preferred to use a salt of this nucleus, e.g. the hydrochloride or the p-toluenesulfonate salt.

J- .

In a specific example of. the preparation of a 3-hydroxy-3-cephem ester, p-methoxybenzyl " J-phenoxyacetamido-3-exomethylenecepham-4-carboxylate was dissolved in ethyl acetate and reacted with ozone at a temperature of about -78°C. The excess of ozone was driven out by bubbling oxygen into the cold solution. The ozonide was decomposed by adding an excess of sodium bisulfite at 0° G with stirring. The organic layer was separated from the insoluble solids and washed, dried and evaporated to give p-Methoxybenzyl J-phenoxyacetamido-3-hydroxy-3-.cephem-4-carboxylate.

In another example, p-nitrobenzyl 7-amino-3-methylenecepham-4-carboxylate hydrochloride was dissolved in methanol and ozone bubbled through the solution at temperatures of about -78°S. Excess ozone was expelled from the mixture with nitrogen, and the ozonide was decomposed by bubbling through sulfur dioxide. The reaction mixture was evaporated to dryness and the residue, i.e. p-nitrobenzyl 7-amino-3-hydroxy-3-cephem-4-carboxylate, was obtained in the form of the hydrochloride salt.

The starting compounds for the preparation of the 3-exomethylene cepham esters can be prepared by reacting a 7-acylamido-cephalosporanic acid with a sulfur-containing nucleophilic compound using known methods, whereby a nucleophilic displacement of the acetoxy group in the cephalosporanic acid is obtained. and a 7~acylamido-3^thiosubstituted-methyl-3-cephem-4-carboxylic acid is obtained. This 3~tnisubstituted cephem product was then reduced with hydrogen in the presence of Raney nickel or with zinc/formic acid in the presence of dimethylformamide, whereby 3-exomethylenecephamic acid is produced. Thus, for example, J-phenylacetamido-cephalosporanic acid can be reacted with potassium ethylxanthate, whereby 7-phenylacetamido-3-ethoxythionocarbonylthiomethyl-3-cephem-4-carboxylic acid is obtained as by reduction with zinc/formic acid i. presence of DMF gives J-phenylacetamido-3-exomethylenecepham-4-carboxylic acid of the formula nu In a similar manner, the 3-exomethylenecepham core of the formula

prepared by reacting a 7-acylamido-3-exomethylenecepham-4-carboxylic acid ester with phosphorus pentachloride (PCl^) in methylene chloride in the presence of pyridine, whereby the intermediate imino chloride was obtained. This was reacted with methanol in the cold, so that the imino ether was obtained. The imino ether can easily be subjected to hydrolysis. ■ whereby the 7-amino-3-exomethylenecepham-4-carboxylic acid ester hydrochloride is obtained. The ester group can then be removed, whereby 3-exomethylenecepham nuclei are obtained.

As stated previously, compounds according to the present invention with formula I can be prepared by acylating a 7-amino-3-halo-3-cephem-4-carboxylic acid or an ester of such a compound.

The acylation of such nuclei can be carried out by known methods which are used for the acylation of 7-aminocephalosporinic acid or 7-aminodeacetoxycephalosporinic acid. Cephalosporin 3~halogen core acids or esters (formula I, R=H) can be acylated under anhydrous acylation methods as well as in the presence of water. Accordingly, the cephalosporin t3~halogen core in the form of a free acid or an ester thereof can 4cylated with a carboxylic acid halide in an aqueous solvent system, eg aqueous acetone,

in the presence of a hydrogen halide acceptor such as propylene oxide, pyridine or sodium bicarbonate. The acylation can also be carried out by reacting an ester of the halogen nucleus with a carboxylic acid in the presence of a condensing agent such as e.g. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) or dicyclohexylcarbodiimide. Furthermore, the 3-halogen core ester can be acylated by means of a mixed anhydride. In another known acylation method, the halogen core compound can be acylated with an active ester of a carboxylic acid, e.g. the pentachlorophenyl ester of a carboxylic acid.

E.g. can p-nitrobenzyl-7-amino-3-chloro-3-ceph.em-.4-. carboxylate is reacted with phenylacetyl chloride in cold aqueous acetone containing sodium bicarbonate, whereby p-nitrobenzyl - 7-phenylacetamido-3-chloro-3-cephem-3-carboxylate is obtained.

Diphenylmethyl 7-amino-3-bromo-3-cephem-4-carboxylate

is reacted with phenoxyacetyl chloride in the presence of pyridine, whereby diphenylmethyl " J-phenoxyacetamido-3-bromo-3-cephem-4-carboxylate is obtained.

p-Methoxybenzyl 7-amino-3-chloro-3-cephem-4-carboxylate can be reacted with mandeline O-carboxylic anhydride in ethyl acetate, whereby p-methoxybenzyl 7"(D-a-mandelamido)-3-chloro-3- cephem-4-carboxylate.

Examples of these derivatives of the acyl groups R'-C=0 which can be used for acylation of the halogen core esters or the free acids are the following: thiophene-2-acetyl chloride, phenoxyacetyl chloride, phenylacetyl chloride, oxazole-2-acetyl- bromide, thiazole-2-acetyl chloride, tetrazole-1^-acetic acid, mandelic acid O-carboxylic acid anhydride, 4-hydroxymandelic acid O-carboxylic anhydride, '4-chlorophenoxyacetyl bromide, benzoyl chloride, 2.6 -dimethoxybenzoyl chloride, thiophene-3-acetyl chloride, furyl-2-acetyl chloride, the pentachlorophenyl ester of phenylmercaptoacetic acid or 4-chlorophenylmercaptoacetyl chloride. Illustrative examples of antibiotic cephalosporins of formula I, where R' is represented by R"CH2 -, are the following compounds: 7~(2-(2-thienyl)acetamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(2-(3-thien<y>l)acetamido)^-chloro^-ce<p>hem^-carboxylic acid,

7~(2-(2-furyl)acetamido)-3-chloro-3-cephem-4-carboxylic acid,

7~(2-(2-furylJacetamido)-3-bromo-3-cephem-4-carboxylic acid,

7-(2-(2-oxazyl)acetamido)-3-chloro-3-.cephem-4-carboxylic acid7

7~(2-(2-thiazyl)acetamide dp)-3-chloro-3-cephem-4~carboxylic acid,

7"(2-(2-thiazyl)acetamido)-3-brpm-3-cephem-4-carboxylic acid,

7-(2-(1-tetrazyl)acetamido)-3-chloro-3-cephem-4-carboxylic acid,

and benzyl, diphenylmethyl, m-methoxybenzyl, p-nitrobenzyl, 2,2,2-trichloroethyl and tert-butyl esters and pharmaceutically acceptable esters and non-toxic base addition salts of the above compounds. Compounds with formula I, fevpr Q is carboxylic acid group can e.g. is prepared by acylating a 3~halo-3-cephem core ester with tert-butyl-phenylmalonic acid chloride or with tert-butyl 2-thienyl-malonic acid chloride in the presence of a hydrogen halide acceptor such as e.g. sodium bicarbonate. Thus, e.g. tert-butyl 7-amino-3""chloro-3-cephem-4-carboxylate is reacted with 2 equivalents of tert-butyl-phenylmalonyl chloride in acetone at approx. 5°C°S in the presence of an excess of sodium bicarbonate, whereby tert-butyl 7~(α-tert-butyloxycarboxylphenylacetamido)-3-chloro-3-cephem-4~carboxylate is obtained. Removal of both tert-butyl ester groups with JOfo formic acid gives the diacid, 7~(α-carboxyphenylacetamido)-3-chloro-3-cephem-4-carboxylic acid.

Examples of compounds of formula I, where Q is a carboxy group are 7-(α-carboxyphenylacetamido)-3-chloro-3~cephem-4-carboxylic acid, 7~ (2-carboxy-2-(2-thienyl)-acetamido) -3-chloro-3-cephem- 4~carboxylic acid, (α-carboxy-4-hydroxyphenylacetamido)-3-chloro-3-cephem-4-carboxylic acid, 7~(α-carboxyphenylacetamido)-3-br,om-3 -cephem-4-carboxylic acid, 7~(oc-carboxy-4-chlorophenylacetamido) - 3-chloro-3-cephem-4-carboxylic acid, J-[α-carboxy-3,4-dimethbxyphenyl)-acetamido)-3- chloro-3-cephem-4-carboxylic acid, 7-(2-carboxy-2-(3-thienyl)acetamido)-3-chloro-3-cephem-4-carboxylic acid, 7-(α-carboxy-3-hydroxyphenylacetamido) -3-chloro-3-cephem-4-carboxylic acid and benzyl, diphenylmethyl, p-methoxybenzyl, p-nitrobenzyl, 2,2,2-trichloroethyl and tert-butyl esters of these compounds, as well as pharmaceutically acceptable non-toxic base addition salts of said connections.

α-sulfo-acylamido-3-halo-cephem compounds of formula I, wherein. Q is a sulfo group (-SO^H) can be prepared by using the acylation methods used for the preparation of α-sulfobenzylpenicillins as described in J.Med.Chem.

15 (11), 11-5 (1972), ibid.p. 1108. E.g. can 7-aminop-3-chloro-3-cephem-^-carboxylic acid be reacted with α-sulfophenylacetyl chloride in

a mixture of acetone and water containing an excess of sodium bicarbonate, whereby 7~(oc-sulf of enylacetamido)-3-chloro-cephem-4-carboxylic acid is obtained. Examples of α-sulfoacylamido-3-halogen-cephem according to the present invention are the following: 7-(o-sulfo-3-chlorophenylacetamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(α-sulfo-4-hydroxyphenylacetamido)-3-chloro-3-cephem-4-carboxylic acid,

7~(α-sulfo-4-methoxyphenylacetamido)~3-bromo-3-cephem-4-carboxylic acid,

7-(2-sulfo-2-eph-thienyl)acetamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(2-sulfo-2-(3-thienyl)acetamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(oc-sulf 6-3>4-dimethylphenylacetamido)-3-chloro-3-cephem-4-carboxylic acid and benzyl, diphenylmethyl, p-methoxybenzyl, p-nitrobenzyl, 2,2,2-trichloroethyl. and tert-butyl esters of the above-mentioned compounds, as well as pharmaceutically acceptable non-toxic base addition salts of said compounds..

Compounds of formula I where R is an acyl group with the formula:

can be prepared by acylating a 3~halo-3-cephem core ester with an α-methoxyimino or an α-acetyloximino glyoxamoyl chloride, where Y is methyl or acetyl, the α-oximino compounds Y=H, can. is produced by base hydrolysis of the α-acetyloximino compounds. Thus, e.g. p-nitrobenzyl 7-(α-methoxyminophenylglyoxamido)-3-chloro-3-cephem-4-carboxylate is prepared by acylation of p-nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylate with α-methoxyimino-phenylglyoxamoyl -chloride in acetone-water containing an excess of sodium bicarbonate.

p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate can then be acylated with α-acetyloximinophenylglyoxamoyl chloride in acetone in the presence of pyridine, thereby obtaining p-nitrobenzyl 7-(α-acetyloximinophenylglyoxamido)-3 -chloro-3-cephem-4-carboxylate.

The a-oximino compounds Y=H can be prepared by mild base hydrolysis of the a-acetyloximino compounds. Thus, e.g. p-nitrobenzyl 7_(α-acetyloximinophenylglyoxamido)-3-klbr-. 3-cephem-4-carboxylate in aqueous acetone reacts within 12 hours

at room temperature with one mole of sodium hydroxide, whereby p-nitrobenzyl 7-(α-oximinophenylglyoxamido)-3-chloro-3-cephem-4-carboxylate is obtained.

The aforementioned methods are illustrated by the following reaction core, which has arbitrarily indicated the syn form.

where. P, R-j^ and X have the same meaning as indicated previously.

Illustrative examples of 7-α_°ximino, α-acetyloximino- and α-methoxyminoarylglyoxamido-3-halo-oephalosporins which can be prepared with the help of the present invention are the following: 7_(α-°ximinepphenylglyoxamido)-3-chloro-3-cephem~ 4-carboxylic acid, 7-(α-methoxyimino-4-chlorophenylglyoxamido)~3-chloro-3-cephem-4-carboxylic acid,

7~ (α-ximino-4-hydroxyphenylglyoxamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(α-oximino-2-(2-thiehyl)glyoxamido)-3-chloro-3-cephem-4-carboxylic acid,

7~(α-Methoxyminophenylglyoxamido)-3-bromo-3-cephem-4-carboxylic acid,

7-(a-oximino-2-(3-thienyl)glyoxamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(α-acetyloximinophenylglyoxamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(a-acetyloximino-2-(2-thienyl)glyoxamido)-3-chloro-3-cephem-4-carboxylic acid and benzyl, diphenylmethyl, p-methoxybenzyl, p-nitrobenzyl, 2,2,2-trichloroethyl and tert -butyl esters of these compounds as well as pharmaceutically acceptable esters and non-toxic base addition salts of these compounds.

Antibiotic compounds of formula I, where R' is a substituted imidoyl-aminoacetamido group of the formula:

Can be prepared with a 7_(3_Denzyi>3~phenoxymethyl, or 3-phenyl-mercaptomethyl<->1,2,4-oxdiazole-5-bn-4-acetamido)-3'-halo-3-cephem-4-carboxylic acid by hydrogenation in the presence of a Raney nickel catalyst at neutral pH. The following reaction core is illustrative of this reaction:

where a, a', Z, m and X are as defined previously.

Substituted oxadiazole-3-halocephalosporins can be prepared by acylating a 3-nal core acid or ester, e.g. 7-amino_3-chloro-3-cephem-4-carboxylic acid with a 3-SUD substituted

1,2,4-oxadiazol-5-one-4.-acetyl chloride under usual acylation conditions. Alternatively, these intermediates can be prepared by reacting a 7-halogenacetamido-3-halo-3-cephem-4-carboxylic acid or ester (formula I, R=chloro- or bromoacetyl) with a 3-substituted 1, 2,4~oxadiazol-5-one- in the presence of a hydrogen halide acceptor such as e.g. pyridine.

Illustrative examples of 7-substituted-imidoyl-aminoacetamido-3-haiogen-cephalosporin antibiotics according to the present invention are the following: 7-(N-(phenoxyacetimidoyl)aminoacetamido)-3-chloro-3-cephem-4-carboxylic acid,

7~(N-(phenylacetimidoyl)aminoacetamido)-3-chloro-3~cephem-4-carboxylic acid,

4^" 7-(N-(phenylmercaptoacetimidoyl)aminoacetamido)-3-chloro-3-cephem-carboxylic acid-,

7-(N-(4-chlorophenylmercaptoacetimidoyl)aminoacetamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(N-(phenoxyacetimidoyl)aminoacetamido)-3-bromo~3-cephem-4-carboxylic acid,

7-(N-(4-Hydroxyphenoxyacetimidoyl)aminoacetamido)-3-chloro-3-cephem-4-carboxylic acid,

7~(N-(4-chlorophenoxyacetimidoyl)aminoacetamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(N-(4_nitrophenylacetimidoyl)aminoacetamido)-3-chloro-3-cephem-4-carboxylic acid and benzyl, diphenylmethyl, p-methoxybenzyl, p-nitrobenzyl, 2,2,2-trichloroethyl and tert-butyl esters of these compounds, as well as pharmaceutically acceptable esters and non-toxic

base addition salts of these compounds..

Particularly useful compounds according to the present invention are 3-halogen core esters and. acids of formula I, where R is hydrogen. Such 3-halogen iron esters and acids can be represented by the following formula: where X and R-j^ are as defined above. When R- is hydrogen, the zwitterionic form of the compound can exist as shown below.

Other particularly useful compounds with. formula I are those where R is 5_amino-5-carboxyvaleryl or an esterified amino-protected 5~amino°-carboxyvaleryl group of the formula: where A and A' are as defined above, Such compounds can be represented by the following formula: n

where X and R-^ are as defined previously.

The above-mentioned compounds are valuable intermediates for the preparation of 3-halo91-nucleic acids and esters in that they can be subjected to the well-known 7-acylamido side chain cleavage reaction with phosphorus pentachloride and pyridine using the cleavage method by which 7-ananocephalosporanic acid esters are prepared with cephalosporin C.

They are prepared with cephalosporin G in the following way. ""■ First, the side-chain amino group and both carboxyl groups in cephalosporin C are protected with groups such as A and A', after which the protected molecule is reacted with a sulphur-containing nucleophilic compound, e.g. potassium methyl xanthate or thiourea , whereby the 3-thio-substituted methyl derivative is formed by a nucleophilic displacement of the acetoxy function in the 3-acetoxymethyl group. Subsequently, the 3"thio-substituted methyl-3-cephem ester is reduced, either with a Raney nickel in the presence of hydrogen or zinc/formic acid 1 in the presence of DMF, whereby a reductive displacement of the 3~thio-substituent is obtained and The 3-exomethylene cepham ester.

The 3-exomethylene cepham ester is then reacted with ozone

and the ozonide decomposed to produce the 3-hydroxy-3-cephem ester by the method which. is described above. The 3-hydroxy ester is then halogenated using the present method, whereby a compound with the formula VIII is obtained.

Cleavage of the protective 7-acyl side chain with PCl^/pyridine/isobutanol or methanol yields the 3-halogen core ester of formula VII. Cleavage of the 7"*acyl group can be performed with nitrosyl chloride when the aminoadipoyl group is unprotected.

Illustrative examples of compounds with the above-mentioned formula VIII are the following: diphenylmethyl-7~(5-diphenylmethyloxycarbonyl-5-(2,4~di-chlorobenzamido)valeramido)-3-chloro-3-cephem^4~carboxylate,

diphenylmethyl-7~(5~diphenylmethyl-5-chloroacetamido-valeramido)-3-chloro-3-cephem-4-carboxylate,

p-nitrobenzyl-7-(5-p-nitrobenzylcarbonyl-5-propionamido-valeramido)-3-chloro-3-cephem-4-carboxylate,

diphenylmethyl-7-(5-diphenylmethyloxycarbonyl.-5-tho--chloro-berizamido)valeramido)-3-chloro-3-cephem-4-carboxylate,

p-nitrobenzyl-7-(5_P-nitrobenzyloxycarbonyl-5-acetamido-valeramido)-3-chloro-3-cephem-4-carboxylate and

diphenylmethyl-7-(5-diphenylmethyloxycarbonyl-5-(2,4-dichlorobenzamido)valeramido)-3-bromo-3-cephem-4-carboxylate.

Examples of 7-amino-3-halo-3-cephem acids and esters that can be prepared are the following: 7-amino-3-chloro-3-cephem-4-carboxylic acid, 7-amino-3-bromo-3-cephem-4-carboxylic acid,' .diphenylmethyl-7-amino-3-chloro-3-cephem-4-carboxylate, p-nitrobenzyl-7-amino-3-chloro-3-cephem-4- carboxylate, p-nitrobenzyl-7-amino-3-bromo-3-cephem-4-carboxylate, 2,2,2-trichloroethyl-7-amino-3-chloro-3-cephem-4-carboxylate, benzyl-7-amino- 3-bromo-3-cephem-4-carboxylate and

the like.

By the term "pharmaceutically acceptable" esters are understood here C^-C^ straight or branched alkanoyloxymethyl esters such as acetoxymethyl, propionyloxymethyl and pivaloyloxymethyl esters. These esters of 7-acylamido-3-halo-3-cephem-4-carboxylic acids have antibiotic activity at a similar level to the free acids of the cephalosporin compounds and can be used instead of antibiotics in the free acid form. In contrast to this, the other ester groups within the definition of R-^, namely benzyl, benzhydryol, p-methoxybenzyl, p-nitrobenzyl, 2,2,■ 2-trichloroethyl and the t-butyl esters do not have much antibiotic activity.

The specified 7-acylamido-3-halo-3-cephem-4-carboxylic acids (formula I, R=R'-C=0, Rn=H) are useful antibiotic compounds for combating infections that are flushed gram-positive and gram-negative organisms. The compounds can be administered by injection, (subcutaneously or intramuscularly) in the free acid form or in the form of a pharmaceutically acceptable ester or non-toxic acid addition salt. Salts formed with the free acids and inorganic bases such as sodium bicarbonate, potassium carbonate, sodium hydroxide and calcium hydroxide give sodium, potassium and calcium salts respectively which can be worked up for supply, e.g. in the form of isotonic solutions or as liquid suspensions.

Pharmaceutically acceptable esters, e.g. acetoxymethyl esters, can be prepared by reacting a 7~acyl-amiclo-3-halo-3-cephem-4-carboxylic acid alkali metal salt, e.g. in the form of the potassium salt, with an alkanoyloxymethyl halide such as acetoxymethyl-

chloride in aqueous acetone.

The in vitro antimicrobial activity for 3-halo-cephalosporins is indicated by data in Table I. Table I contains antimicrobial activity for 7-(2-(2-thienyl)-acetamido)-3-chloro-3-cephem-4. -carboxylic acid as. was obtained in a standard plate method. The numerical values are the diameter in millimeters of inhibition zones that were observed with the specified organisms.

The following Table II indicates the minimum inhibitory concentration (MIC) of 7-(2-(2-thienyl)acetamido)-3-chloro-3-cephem-4-carboxylic acid against penicillin-resistant Staphylococcus both in the presence and absence of serum.. MIC- the values were obtained by the so-called gradient plate technique which was carried out essentially as described by Brysdndg Szybålski, Science, 116, 45 (1952).

In Table III below, MIC values for the test compound from Tables I and II against representative gram+-negative organisms are indicated. The indicated data were obtained by the gradient plate technique.

In Table IV below, the in vitro antimicrobial activity of 7-(2-(2-thienylacetamido)-3-bromo-3-cephem-4-carboxylic acid is indicated for several Illustrative microorganisms. These data were obtained by a standard plate method, where the numerical values indicate the diameter in millimeters of the inhibition zones obtained with the indicated microorganisms.

The following Table V indicates the minimum inhibitory concentration (MIC) of the antibiotic 7-(2-(2-thienyl)acetamido)-'3-brbm-5-cephem-4-carboxylic acid against penicillin-resistant strains of Staphylococcus in the absence of serum.

The inhibitory concentrations were obtained by the gradient plate method.

Minimum inhibitory concentrations for 7~(2-(2-thienyl)acetamido)-3-bromo~3-cephem-4-carboxylic acid against representative gram-negative bacteria are indicated in Table VI. These data were obtained by the Gradient plate method.

The previously stated 7-acylamido-3-halo-3-cephem-4-carboxylic acid esters (formula I, R=R'-C=0, R-,= ester) can be used as intermediates for the preparation of the compounds in the form of the free acids. Ester groups that fall within the definition of R-1 are all well-known groups commonly used to protect the C1-carboxylic acid group in the cephalosporin molecule while carrying out reactions involving other groups in the molecule. These ester-forming groups can be easily removed, whereby the free acid is obtained by reduction or hydrolysis. Thus, e.g. The p-nitrobenzyl ester group is removed via catalytic hydrogenolysis over palladium and carbon, the diphenylmethyl group (benzhydryl) can be removed with trifluoroacetic acid in anisole at approx. 10°C, the p-methoxybenzyl group can be removed with trifluoroacetic acid at approx. 10°C.

(J.Org.Chem. 36, 1259•(1971)), the 2,2,2-trichloroethyl group can•■ be removed with zinc and acid (J.Am.Chem.Soc. 88, 852 (1966)), The benzyl ester group can be removed via catalytic hydrogenolysis over palladium catalyst (J.Org.Chem. 27 > 1381 (1962)) and the tertiary butyl group can be removed as described in J.Org.Chem, 31 > 444 (1966 ).

7-Amino-3-halo-3-cephem-4-carboxylic acids and esters (formula I, R=H) are valuable intermediates that can be used for the production of 3-halogen-an'bibiotics. As described

previously, these 3" halogen core acids and esters can be acylated by

to use standard N-acylation methodology, whereby 7-acylamido-3-halo-3-cephem-4-carboxylic acids or esters are obtained.

The following examples illustrate the invention.

A. Preparation of starting material.

Example 1

p-nitrobenzyl 7-amino-3-methyleneepham-4-carboxylate hydrochloride

A solution of 965 mg (2 mmol) of p-nitrobenzyl-7-phenoxyacetamido-3-methylenecepham-4-carboxylate in 10 ml of methylene chloride was added. 175 mg of dry pyridine and 460 mg of phosphorus pentachloride were added, and the mixture was stirred at room temperature for 6 hours. F,n ml iso- . butanol was added to the mixture which was then stored at 0°C overnight. The reaction product p-nitrobenzyl 7-amino-3-methylenecepham-4-carboxylate hydrochloride which had formed as a crystalline precipitate was filtered off and the yield was 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 help

Carbonyl absorption at 5j65 ((3-lactam) and 5»75 (ester) micron.

N.M.R. signals at 6.34 (2d, 2H, 62~H2), 4.98 (d, 1H, C6-H), 4.7-4.4 U> 6H, C^-H, ester CH2, G^-CH2 and C^- H) and

2.4-1.6 (m,4H, aromatic H) tau.

Example 2

p-nitrobenzyl-7-amino-3-niethylenecepham-4-carboxylate p-toluenesulfonate salt.

To a solution of 965 mg of p-nitrobenzyl-7-phenoxy-acetamido-3-inethylenecepham-4-carboxylate in 10 ml of methylene chloride was added 175 mg of dry pyridine and 46"0 mg of phosphorus pentachloride and the mixture was stirred for 5 hours at room temperature. It was then cooled to 0° C. and 50 ml of cold methanol was added. After stirring for one hour at room temperature, the mixture was evaporated in vacuo to remove the solvents, and the residual reaction product was dissolved in a fine mixture of ethyl acetate and water. The pH was adjusted to 7°g. The ethyl acetate layer was separated and washed with water and dried. One equivalent of p-toluenesulfonic acid was added, the dried solution, and on cooling gave 600 mg of p-nitrobenzyl-7 -amino-3-methylenecepham-4.-carboxylate-p-toluenesulfonate as a crystalline precipitate The product was purified by recrystallization from a mixture of 12 ml of methanol and 24 ml of ether and 15 ml of petroleum ether.

Elemental analysis for C^H^N^OgS^

Theoretical: C 50.66 H 4.45 N 8.06 Found: C 50.41 ' H 4.51 N 7.86

I.R. (Nujol Mull)

Carbonyl absorption at 5>65.(p-lactam) and 5\71 (ester) micron

N.M.R. (DMSO dg)'

Signals at 7.70 (s, 3H-p-methyl), 6.39 (s, 2H,

C2-H2), 4.9<8>(d, 1H,C6-H), 4.-7-4.3(m, 6H,C4-H,

ester CHg, C^-CHg, and C^-H) and 2.93-1.68 (m, BH, aromatic H) tau.

UV (pH 6 buffer)

Maxima at 219' miy, (g, = 19,600) and

268 mu. 9,400).

Example 3.

p-Methoxybenzyl-7-amino-3-methylenecepham-4-carboxylate hydrochloride

A solution of 4>3S p-methoxybenzyl-7-phenoxy-acetamido-3-methylenecepham-4-carboxylate in %0 ml of methylene chloride was added to 880 mg of dry pyridine and 2.3 g of phosphorus pentachloride and the mixture was stirred under reflux for 3 hours. It was then cooled in an ice-water bath and 5 ml of isobutanol was added. The mixture was stirred in the cold for several hours and a total of 2.2 g of the reaction product, p-methoxybenzyl-7-amino-3-methylene-cepham-4-carboxylate hydrochloride, precipitated. The product was filtered off and

washed with cold methylene chloride and then dried in vacuo.

Elemental analysis for C-^gH-j^NgSCl Theoretical: C 51.82, H 5.16, N 7.55 Found: C 51.65, H. 5.04, N 7.72

Example 4

p-Methoxybenzyl-7-amino-3-methylenecepham-4-carboxylate p-toluenesulfonate

To a solution of 937 mg of p-methoxybenzyl-7-phenoxyacetamido-3-methylenecepham-4-carboxylate in 10 ml of methylene chloride was added 0.18 ml of dry pyridine and 460 mg of phosphorus pentachloride. The mixture was stirred at room temperature for 2 hours and then cooled to 5°C. The cold mixture was added with 50 ml of cold methanol and allowed to warm to room temperature. The mixture was evaporated in vacuo and the residue was dissolved in a mixture of ethyl acetate and water. The pH of the solution was adjusted to 7. The ethyl acetate layer was separated, washed with water and dried. To the dried ethyl acetate layer was added one equivalent of p-toluenesulfonic acid. Upon cooling, a crystalline solid precipitated approx. 600 mg p-Methoxybenzyl-3-methylenecepham-4-carboxylate-p-toluenesulfonate....

Elemental analysis for Cgj^gNgOgS^

Theoretical: C 54.53. H 5.17. N 5.53 Found: C 54.33, H 5.05, N 5.47

I.R. (Nujol Mull): Carbonyl absorption bands at 5.^5 (p-lactam) and 5>79 (ester) micron.

N.M.R. (DMSO dg):

Signals at 7.^9 (s, 3^. para-methyl)

6.41 (s, 2H, C2-H2)

6.23 (s, 3^, para-methoxy)

5.0 (d, 1H, Cg-H)

4.82 (s, 2H, ester CHg) 4.7H.55 (m, 4H, c4-h, c3-ch2 and C?-H) 3.2-2.0 (m, 8H, aromatic H)tau'.

Example 5

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

A solution of 3.85 g of p-nitrobenzyl-7-amino-3-methylenecepham-4-carboxylate hydrochloride prepared as described in Example 1, in .600 ml of methanol was cooled in an acetone-dry ice bath. Ozone was bubbled through the reaction mixture for approx. 20 minutes,

and the mixture at this point took on a faint brown colour. Nitrogen was passed through the reaction mixture to drive off an excess

of ozone. The intermediate ozonide was then decomposed by passing sulfur dioxide gas through the reaction mixture until it gave a negative potassium iodide starch test.

The reaction mixture was evaporated in vacuo, and the residue was dissolved in 200 ml of 0.1 N hydrogen chloride in methylene chloride. The solution was evaporated to dryness and the remaining reaction product was dissolved in acetone. On cooling, 3.15 g of p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride were precipitated as a crystalline solid.

I.R. (Nujol Mull):

Carbonyl absorption by

5.55 (p-lactam carbonyl) and

5.°2 (ester-carbonyl hydrogen-bonded to. 3

hydroxy)micron.

Electrometric titration (66% DMF) pKa 4.0 and 6.3.

Example 6

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

A solution of 4 g of p-nitrobenzyl-7-amino-3-niethylene-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 purged of residual ozone by passing nitrogen through the solution, and 10 g of sodium bisulfite was added.

The reaction mixture was stirred for one hour at ice bath temperature, at which time the mixture gave a negative potassium iodide starch test.

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

I.R. (Nujol Mull):

Carbonyl absorption bands at 5, 60 (p-lactam) and 6.04 (ester carbonyl hydrogen bonded to 3 hydroxy) micron.

N.M.R. (DMSO dg):

Signals at 7.92 (s, 3H, l/2 mol acetone),

6,22-(2d, 2H, C2-H2)

5.07 (d, 1H, CgH),

4q8-4, 5 (m, 3H, ester GH2 andC^H),

2,4-1j6 (m, 4H, aromatic H)tau.

Example 7

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

By applying the ozonation methodology described in Example 5 and 6, 3.5 g of p-nitrobenzyl. 7-amino-3-methylenecepham-4-carboxylate hydrochloride ozonized in methanol, and the intermediate ozonide was decomposed at a temperature of 0°C with 3.5 ml of trimethyl phosphite. The reaction mixture was evaporated and the residue was dissolved in 100 ml of 0.1 N HCl in methylene chloride. Said solution was then evaporated and the residue was crystallized from acetone, whereby 2.8 g of p-nitrobenzyl 7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride was obtained.

Example 8

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

Four millimoles of p-nitrobenzyl 7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride, prepared as described in Example 5, were dissolved in water and ethyl acetate was added to the solution. The pH of the suspension was adjusted from pH 2.2 to pH 5 with IN sodium hydroxide. The ethyl acetate layer was separated and washed with water and dried over magnesium sulfate. The dried ethyl acetate layer was evaporated to dryness to give 1.2 g of p-nitrobenzyl 7-amino-3-hydroxy-3-cephem-4.-carboxylate as a crystalline residue.

Elemental analysis, for: C-^H-^N^OgS:

Theoretical: C 47.86, H 3.73, N 11.96

Found: C 47.87, H 4.00, N 12.11

I.R. (Nujol Mull): Carbonyl absorption at 5>65 (broad, (3-lactam and ester) and 6.0 (amide) micron.

N.M.R. (DMSO dg):

Signals at 6.63 (2d, 2H, CgH),

5.31 (d, 1H, CgH),

4.89( d,1H, (yi) ,

4.62 (s, 2H, ester GU^),

4.30 (broad s, 2H, J N-H),

2.5~1>8 (m, 4H, aromatic H) and 1.2 (d, 1H, C^OH) tau.

Example.

Methyl 7-phenoxyacetamido-3-hydroxy-3-cephem-4-carboxylate..

A solution of 1.6 g of methyl J-phenoxyacetamido-3-methylenecepham-4-carboxylate in 300 ml of methylene chloride was cooled in an acetone-dry ice bath. Ozone was bubbled through the cold solution for three minutes and by this time the reaction mixture had acquired a faint blue color. Excess ozone was driven off with a stream of oxygen and 10 g of sodium bisulphite was added. The reaction mixture was stirred and allowed to warm to 0°C. The liquid phase was separated by Hekanlyering and washed successively with a 5% solution of hydrochloric acid, water and a saturated solution of sodium chloride. The washed mixture was dried and evaporated to give 1.5 g of impure methyl J-phenoxyacetamido-3-hydroxy-3-cephem-4-carboxylate as an amorphous solid.

The crude product was dissolved in ethyl acetate and was extracted with a 5%-0PPl solution of sodium bicarbonate. Ethyl acetate was added to the extract, which was then acidified to pH 2 with 1N hydrochloric acid.

The organic phase was separated and washed with a saturated solution of sodium chloride and then dried. The dried extract was evaporated

to dryness, thereby obtaining 7^9 mg of the reaction product contaminated with a small amount of the corresponding 3-hydroxy-3-cephem sulfoxide, an overoxidation product. The product was separated from the sulfoxide impurity and obtained in a pure state by preparative thin layer chromatography on silica gel with chloroform.methanol (9:1).

Elemental analysis for: C]_6<H>l<6N>2<0>6S*H2°

Theoretical: C 50.26, H 4.75, N 7.33, , S 8.38 Found: . C 51.03, H 4.62, N 7.06, S 8.37 I.R. (chloroform): absorption peaks at 2.8 (amide NB), 5.6 ((3-lactam carbonyl), 5.^5 (broad, amide and ester carbonyl and 6.6 (amide II)micron.

N.M.R. (CDCl^): signals at

6.65 (s, 2H, C2-H2),

6.13 (s, 3H, methyl ester),

5.40 (s, 2H, side chain GB2),

4.93 (d, IB,<g>6b),

4.32 (q, IB, C„H),

3.15-2.38 (m, oB, aromatic and amide-B) and 1.60 (broad s, IB, 3~0B)tau.

Electrometric titration (66% aqueous DMF):

pKa 5.6.

Example 10

p-Methoxybenzyl J-phenoxyacetamido-3-hydroxy-3-cephem-4-carboxylate.

A solution of 2.5 g of p-methoxybenzyl 7-phenoxy-acetamido-3-methylenecepham-4-carboxylate in 350 ml of ethyl acetate was cooled in an acetone-dry ice bath. Ozone was bubbled through the cold solution for 8 minutes, after which oxygen was passed through the mixture to drive off any excess ozone. The intermediate ozonide was decomposed by adding 25 g of sodium bisulphite to the reaction mixture while stirring at approx. 0°C. The reaction solution was poured off and washed successively with water,

5% hydrochloric acid and a saturated solution of sodium chloride. The washed solution was dried and evaporated to give the reaction product p-methoxybenzyl J-phenoxyacetamido-3-hydroxy-3-cephem-4-carboxylate as an amorphous solid.

N.M.R. (CDCl^): signals at

6.7.3 (s, 2H, c2H2), 6.23 (s, 3H, p-methoxy),

5.53 (s, 2H, side chain CHg)',.

5.03 (d, 1H, C6H), 4.87 (s, 2H, ester CHg),

4.47 (q, 1H, C?H),

3.40-2.50 (m, 9H, aromatic H),

2.33 (d, 1H, amide NH) and

1.53 (wide s, 1H, 30H) rope.

Example 11

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 3^4 mg (0.5 ml, 3>6 mmol) of triethylamine was added .9É>2 mg urea. With stirring at room temperature, a solution of 730 mg (4.4 mmol) of 2-thiophene-acetyl chloride in 20 ml of acetone was added dropwise to the mixture. After 2.5 hours, the reaction mixture was filtered and evaporated. The residue was dissolved in ethyl acetate and the solution 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 to give 1.2 g of the reaction product as a crystalline residue. The product was recrystallized from ethyl acetate, whereby pure p-nitrobenzyl 7-(2-(2-thienyl)acetamido)-3-hydroxy-3-cephem-4-carboxylate was obtained with the following spectral properties. I.R. (Nujol Mull): absorption peaks at 3'0 (amide NH, 5.68 ((3-lactam carbonyl), and 6.1 (amide, and ester hydrogen bonded to 3 0H)micron.

N.M.R. (CDCIyDMSO dg): signals at

6.54 (2d, 2H, C2H2),

6,l6 ( s, 2H, side chain GH2),

4.90 (d, 1H, CgH), 4.60 (d, 2H, ester CH2),

(signals by:)

4.43 (q,1H, GrjH), 3.1-1.6 (m, 7H5aromatic H) and 1.30 (d, 1H, amide NH)tau.

Example 12

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

p-nitrobenzyl 7~&min°-3-methylenecephem-4-carboxylate hydrochloride, 3.5 g, was reacted with ozone in methanol as indicated in Example 5 for the preparation of the ozonide. This was decomposed with sulfur dioxide, whereby the 3-hydroxy product was obtained which was isolated as one crude product. This crude product was dissolved in 175 ml of tetrahydrofuran and 50 ml of water. 2.1 g of sodium bisulphite was suspended in the solution, after which a solution of 4.8 g of 2-thiophene-acetyl chloride in 200 ml was added dropwise.

THF.

The mixture was stirred for two hours at room temperature and then evaporated to an aqueous residue. This was stirred with ethyl acetate, the organic layer separated and washed with 5% hydrochloric acid and with water. The washed layer was dried and evaporated to dryness, whereby the reaction product was obtained as a crystalline residue. This residue was treated three times with diethyl ether to remove impurity 2-thiophene acetic acid, and 2.9 g of purified crystalline product was obtained, i.e. p-nitrobenzyl J-(2-(2-thienyl)acetamido)-3-hydroxy -3~cephem-4-carboxylate.

Electrometric titration (66%' aqueous DMF)

pKa 5.9

N.M.R. (CDC1^/D2O): signals at 6.60 (s, 2H, C2H2), 6.13 (s, 2H, side chain GH2), 4.96 (d, 1H, CgH), 4.62 Cd, 2H, ester CH2), 4.46 (d, 1H, CLH) and 3.1-1.7 (m, 7H, aromatic H)

' rope.

Example 13

p-nitrobenzyl 7-acetamido-3-hydroxy~3-cephem-4-carboxylate •

A solution of 10 mmol of p-nitrobenzyl 7-amino-3-hydroxy<y>-3-ce<p>heme-4-carboxylate hydrochloride in a mixture of 325 ml of acetone and 125 ml of water was cooled in an ice- water bath. While stirring, a stream of ketene gas was bubbled through the solution for 30 minutes. Then the mixture was evaporated to remove acetone and the aqueous residue was stirred with ethyl acetate. The ethyl acetate layer was separated and washed with 5% hydrochloric acid and a saturated solution of sodium chloride. The washed extract was dried and evaporated in vacuo to give a crystalline residue. This was treated with diethyl ether and vacuum-dried, whereby 3.55 g of p-nitrobenzyl 7-acetamido-3-b.hydroxy-3-cepb.em-4-carboxylate was obtained which melted at approx. 146-152°C with decomposition.

Elemental analysis for: C-^gH^N^OyS:

Theoretical: C 48.85, H 3.84, N 10.68 Found: G. 48.97, H 3.96, N10.42 I.R. (CHClo): absorption bands at 2.9 and 3.0 (amide NH and OH), 5.63 (p-lactam carbonyl) and 5.95 (broad, amide and ester carbonyl hydrogen bonded to 3 OH) micron.

N.M.R. (CDGl^): signals at.

7.90 (s, 3H, 7-acetamido CH 2 ),

<6.>55 (s, 2H, C2H2), 4.92 (d, 1H, C5H),

4.63 (m,2H, ester GH2), 4.30 (q, 1H, C^H),

2.8l (d, 1H, amide NH),

2.5-1.8 (m, 4H, aromatic H) and 2.8 (s, 1H, CoOH)

rope.

Electrophoretic titration (66% aqueous DMF)

pKa 5.9-

Example 14-

p-nitrobenzyl J-phenylacetamido-3-hydroxy-3-cephem-4-carboxylate.

By using the ozonation method described

In Example 9, a solution of 350 mg of p-nitrobenzyl 7-phenyl-acetamido-3-methylenecepham-4-carboxylate in 250 ml of methylene chloride was cooled to -78°C and ozonized. The intermediate ozonide was decomposed in situ with sulfur dioxide, and the reaction product was recovered and obtained crystalline by extraction with ethyl acetate.

Elemental analysis for CggH-^N^OyS .

Theoretical: C 56.28, H 4.80, . N 8.95

Found: C 56.11, H 4.15, N 8.74

N.M.R. (CDCl^): signals at

6.68 (2d, 2H, C2H2), 6.37 (s, 2H, side chain CHg),

5.03. (d, 1H, CgH), 6.66 (d, 2H, ester CHg),

. 4.4CK«(q, 1H, C^H), 2.7 (m, 6H, amide NH and aromatic H), 2.53-1j70 (q, 4H, aromatic H) and a singlet in the low field integrating for 1H in G^hydroxyl group, tau. I.R. (Nujol Mull): absorption peaks at 3.04 (amide), 5.6"0 and 6.0 ((3-lactam, ester and amide carbonyl groups) micron.

Example 15

p-nitrobenzyl J-(D-oc-phenyl-α-formyloxyacetamido)-3-hydroxy-3-cephem-4-carboxylate.

A solution of 1.54 g of p-nitrobenzyl. 7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride in 120 ml of acetone and 40 ml of water was added 936 mg of sodium bisulphite. While stirring, a solution of 960 mg of O-formyl-D-mandelic acid chloride in 20 ml of anhydrous acetone was added dropwise to the solution at room temperature. The reaction mixture was stirred at room temperature for 16 hours and then evaporated to remove acetone. The aqueous residue was stirred with ethyl acetate and the organic layer separated. The extract was washed with water and dried and then evaporated. The crystalline residue was treated with diethyl ether and dried, whereby 1g of p-nitrobenzyl 7-(1~a-phenyl-a-formyloxyacetamido)-3-hydroxy-3-cephem-4-carboxylate was obtained.

Elementary analysis for COQH1 oNo0nS :

Theoretically: . C 53.80, H 3.73, N 8.l8 Found: G. 53.51, H 3.8l, N 8.46

I.R. (CHCl^): carbonyl absorption peaks at 5>55>5>73>

5 j85 and 5»93 microns.

N.M.R. (CDCl^): signals at

6.61 (s, 2H, C2H2), 4.95 (d, 1H, C6H),

4.61 (d, 2H, ester CH2), 4.39 (q, 1H.C^H),'. 3.70 (s, 1H, α-CH(, and 2.80pl,70 (m, 11H, amide NH and aromatic H)tau.

B. Preparation of 3-halo-3-cephem acids and esters.

Example 16

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. methylene chloride

21.4 g (110 mmol) of diphenyldiazomethane were added and the resulting mixture was stirred for two hours at room temperature. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate. The ethyl acetate solution was washed with 5% sodium bicarbonate solution, then with water and then dried over magnesium sulfate. The dried solution was concentrated to a small volume. On standing, 40 6 of diphenylmethyl 7~(2-(2-thienyl)acetamido)-3-methylenecepham-4-carboxylate was separated which melted at 132-133°^ as a crystalline solid.

IR (chloroform): absorption peaks at 2.9 (amide N-H),

5.<6>5, 5.75°g 5j93((3-lactam, esters and amide carbonyl groups respectively) and 6.62 (amide II) micron.

N.M.R. (CDCl^): signals at

6.72 (ABq, 2H, C2-H2), 6.21 (s, 2H, α-CHg), 4.83-4.65 (m, 4H, C4-H, Cg-H and C^CHg) ,

4.39 (q, 1H, C7-H), 3.4-2.65 (m, 15H, C2-NH, ester CH and aromatic H)tau. b) A solution of 8.1 g (16 mmol) of the above-mentioned ester in 80 ml of methylene chloride was added 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 two hours at room temperature and then cooled in an ice-water bath. The cold mixture was treated with 8 ml of isobutanol while stirring. Stirring was continued for two hours while 3 g of diphenylmethyl 7-amino-3-methylene-cepham-4-carboxylate hydrochloride were precipitated as a crystalline precipitate. The product was filtered off and washed with methylene chloride and vacuum-dried.

Elemental analysis (percent) for C^H^N^^SCl: 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.

N.M.R. (DMSO dg): signals at

6.45 (ABq, 2H, C2-H2), 5.00 (d, 1H, Cg-H).,

4.68 (d, 1H, C7-H), 4.60 (d, 2H, 3-CH2),

4.44 (s, 1H, C^-H), 3.10 (s, 1H, ester CH), and 2.6l (s, 10H, aromatic H) tau.

c) 7-amino-3-exomethylene cepham ester hydrochloride salt, 2.1 g (5 mmol) was dissolved in 200 ml of methanol, and. the resolution

was cooled in an acetone-dry ice bath. Ozone was bubbled into the cold solution for 7 minutes to form the intermediate ozonide. This was then decomposed by passing a stream of sulfur dioxide gas through the reaction mixture for two minutes. The mixture was then evaporated and the residue treated with diethyl ether to give 1.6 g of diphenylmethyl 7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride as a crystalline solid.

N.M.R. (CDCl^): signals at

6.4 (ABq,2H, C2-H2), 5.0-4.5 Cm, 2H,Cg-H and C^-H), 3.2-2.4 (m, UH, ester CH and aromatic H) rope.

I.R. (chloroform): carbonyl absorption peaks at

5 j 57°S 5.70 ((3-lactam and ester carbonyl respectively)micron. UV (pH7 buffer) : Tv max 275Jt,<=>755<0.>Electrometric titration (60% aqueous DMF): titratable groups at 4.5°g 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, while 800 mg of thiophene-2-acetyl chloride in 10 ml of acetone was added dropwise. The mixture was stirred for 4.5 hours at room temperature and then evaporated under reduced pressure. The residue was dissolved in a mixture of ethyl acetate and a 5% aqueous 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 diphenylmethyl 7-(2-(2-thienyl)acetamido)-3-hydroxy-3-cephem-4-carboxylate.

N.M.R. (CDCl^): signals at

6.79 (s, 2H, G2-H2), 6.16 (s, 2H, α-GH2),

5.0 (d, 1H,C5-H), 4.32 (q, 1H, ^-H),' 3.05-2.46 (m, 15H, C7-NH, ester CH: and aromatic H)

rope.

I.R. (chloroform): absorption peaks at 2.9 (amideNH), 5.6, 5.73 and 5.95 (P-Iactam, ester and amide carbonyl groups respectively) and 6.65 (amide II) micron. e) A solution of 4.2 g of diphenylmethyl 7"(2-(2-thienyl)acetamido)-3-hydroxy-3-cephem-4-carboxylate in 44 ml of dry dimethylformamide was added with 865 mg of phosphorus trichloride. The mixture was stirred 1.5 hours at room temperature and then poured into a mixture of ethyl acetate and 5% aqueous hydrochloric acid. The ethyl acetate layer was evaporated, washed with 5% hydrochloric acid, with water and then dried. The dried solution was concentrated in vacuum and the product crystallized out.3-Chloroesters were filtered off, washed with cold ethyl acetate and dried to give 2.2 g.

Elemental analysis (percent) for G26H21N2^4^2G1: 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. N.M.R. (CD.Cl^): 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. I.R. (CHCl^): absorption peaks at 2.9 (amide NH), 5.55, 5.72 and 5.9° ((3-lactam, ester and amide carbonyl groups)) and 6.60 (amide II) micron.

UV (dioxane): max 275 m#,<&>=87OO.

Example 17

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

A resolution 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 to 950 mg (0.58 mL, 8 mmol) freshly distilled thionyl chloride. The mixture was stirred at room temperature for 6.5 hours and 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 ca.164 -166°C. Elemental analysis (percent) for Cj<pøH>^<gN>^<Og>S<j>p<C>l: 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 band at 2.9 (amide NH),

5.59 ((3-lactam carbonyl), 5.75 (ester carbonyl) and 5.92 micron (amide carbonyl).

UV absorption spectrum (acetonitrile) showed maxima at ?V max 235 mn, £ = 12,100

7\ max 268mp, I5.8OO

The mass spectrum of the product showed a molecular ion of 493 m/e.

N.M.R. (CDCl^) showed signals at

6.39 (ABq, 2H, C2-H2), 6.17 (s,2H, cc-CHg), 4.99 (d, 1H, C5-H), .4.64 (s, 2H,. ester CHg), 4.19 (q, 1H, C?-H), 3.45 (d, 1H, C7-NH), 3.1-1.67 (m, 7H, aromatic H)tau.

Example 18

7~(2-(2-thienyl)acetamido)-3-chloro-3-cephem-4-carboxylic acid..

A solution of 995 mg (2 mmol) of p-nitrobenzyl 7~(2-(2-thienyl)acetamido)-3-chloro-3-cephem-4-carboxylate, prepared as described in Example 17, in 60 ml of tetrahydrofuran and 100 ml methanol containing. 5 drops of IN hydrochloric acid were added to 1 g of a 5% palladium on carbon catalyst. The catalyst was pre-reduced in a suspension of 40 ml of ethanol at room temperature under a hydrogen pressure of 3.5 kg/cm^.

The suspension 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 THF and then with water. The combined filtrate and catalyst wash solutions were evaporated to dryness, and the reaction product residue was dissolved in a mixture of ethyl acetate and water. The pH of the solution was adjusted to 2.5 and the ethyl acetate layer was separated. The acid reaction product was extracted with water from the ethyl acetate solution at pH 7«

The aqueous phase was separated, overlaid with ethyl acetate and acidified to pH 2.5. The ethyl acetate layer was separated, washed with water, dried over sodium sulfate and dried in vacuo. The amorphous residue was treated with ether to give 165 mg of 7-(2-(thienyl)acetamido)-3-chloro-3-cephem-4-carboxylic acid as a crystalline solid, mp 114-120°C with decomposition and softening at approx. 110°C..

This reduction product had the following physical characteristics. I.R. (Nujol Mull): showed absorption bands at 3.1 (amide NH, 5.64 and 5.75 ((3-lactam and carboxylic acid carbonyl groups respectively) and 6.1 (amide II). micron..

UV (acetonitrile): absorption maxima at

>v max 235, £<=>IO.7OO.

>max 268,. £ = 7,200

N.M.R. (CDCl^) showed signals at

6.38 (ABq, 2H, C2-H2), 6.16 (s, 2H, α-CHg),

4.98 (d, -1H, C6-H), 4.2a (q, 1H, C?-H), and 3.1-2.5 (m, 4H, aromatic H and C^-NHjtau

Percent elemental composition for C-^H-^NgO^SCl : Theoretical: C 43.52, H 3.09, N 7.8l, Cl 9.88 Found:. C 43.55, H 3.79, N 7.27, Cl 9.28.

Example 19

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

A cooled solution of 439 mg (0.93 mm°i) of p-nitrobenzyl 7~(2-(2-thienyl)acetamido)-3~hydroxy-3-cephem-4-carboxylate in 4.4 ml of DMF was slowly added 85 mg (0.05 ml, - 0.63 mmol) phosphorus trichloride. The reaction mixture was allowed to stand for four hours at room temperature, after which the reaction product mixture was worked up as described in Example 17, and a total of 374 mg of p-nitrobenzyl (2-(2-thienyl)acetamido)~3-chloro-3- cephem-4-carboxylate. NMR spectrum of the product was consistent with the expected product and with the corresponding for the compound from Example 17.

Example 20

7" Phenoxyacetamido-3-chloro-3-cephem-4-carboxylic acid.

By applying the chlorination method from Example 19,. p-nitrobenzyl 7-phenoxyacetamido-3-chloro-3-cephem-4-carboxylate was prepared with phosphorus trichloride. The p-nitrobenzyl ester group was removed by acid hydrogenolysis as described in Example 18, and 3-chloro-cephalosporanic acid was produced in an antibiotic compound.

Example 21

p-nitrobenzyl J-(2-(2-thienyl)acetamido)-3-chloro-3-cephem-4-carboxylate (via phosphorus oxychloride).

A solution of 325 mg (0>7 mmol) p-nitrobenzyl 7~(2-(2-thienyl)acetamido)-3-hydroxy-3~cephem-4-carboxylate in 3.3 ml DMF cooled in an ice-water bath was slowly added 212 mg (0.13 ml, 1.4 mmol) of phosphorus oxychloride. The mixture was left for four hours at room temperature and the product, which was 225 mg in total, was recovered by using the method described in Example 17. The nuclear magnetic resonance spectrum of the product was in agreement with the spectrum of the previously characterized compound.

Example 22

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

A 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 cooled in an ice bath was added dropwise 118 mg (0.07 ml, 0.93 mmol) of oxalyl chloride. The reaction mixture was allowed to stand for four hours at room temperature and was 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, yielding the reaction product p-nitrobenzyl 7~(2-Kienyl)acetamido)-3-chloro-3-cephem-4-carboxylate as an amorphous solid. The product was obtained in crystalline form by treating the amorphous residue with ester. Yield 360 mg. The infrared spectrum and the NMR spectrum of the crystal steel-like product were consistent with a spectrum from authentic .

material.

Example. 23

7~(2-(2-thienyl)acetamido)-3-bromo-3-cephem-4-carboxylic acid.

A solution of 19 g (40 mmol) of p-nitrobenzyl 7~(2-(2-thienyl)acetamido)-3-hydroxy-3-cephem-4~carboxylate in 300 ml of dry DMF was added to 15 g (56 mmol) of osph or tribromide. and

the reaction mixture was stirred at room temperature overnight.

It was then poured into a mixture of ethyl acetate and water, the organic phase was separated and washed several times with water and finally dried over magnesium sulfate. The dry organic phase was evaporated in vacuo to dryness. The impure reaction product weighing approx. 9 g were purified by chromatography above

500 g of silicon dioxide gel, using ethyl acetate-hexane (55:45 v:v) as eluent. The eluate was evaporated to dryness under reduced pressure, and the product p-nitrobenzyl 7-(2-(2-thienyl)acetamido)-3-bromo-3-c.ephem-4-carboxylate was obtained crystalline by treating the residue with diethyl ester.

UV (ethanol) >> max 27O m/i ( £ = I3.3OO)

and ?i max 243 mya (£ = 12-700)

Elemental analysis calculated for C^H-^gBrN^OgSr,: Theoretical: C 44.61, H 3.00, N 7.81, Br 14.84 Found: C 44.78, H 3.03, N 7, 65, Bro. 14.91..

Nuclear magnetic resonance spectrum (DMSOdg)

showed signals at 6.21 (s,2H, cc-CH<g>),

5.98 (ABq,' 2H,' C2-H2), 4.72 (d, 1HCg-H),

451 (s, 2H, ester-CH2), 420 (q, 1H, C?-H), 3.04-1.74 (m, 7H, aromatic H) and 0.66 (d,1H, C^- CHjtau.

The above 3" bromo ester was de-esterified as follows. The ester of 545 mg (1.0 mmol) was hydrogenated at room temperature in ethanol in the presence of a pre-reduced 5% palladium-on-carbon catalyst. The catalyst was filtered off, the filtrate was evaporated under reduced pressure to dryness.The remaining product was treated with diethyl ether to give 180 mg (44%) of a crystalline product, that is, 7-(2-(2-thienyl)acetamido)-3-bromo -3-cephem-4-carboxylic acid.

Electrometric titration (66% aqueous DMF)

showed a pKa of 4.4°g an apparent molecular weight of 393.

Calculated molecular weight = 403'

Elemental analysis, calculated for C-^H-QBrNgO^Sg.

l/2 diethyl etherate:

Theoretical: C 40.91, H 3.66, N 6.36, Br 18.15.' Found: C 41.29, H 3.20, N 6.29, Br l8£50.

Nuclear magnetic resonance spectrum (CD.Cl^) showed signals at 8.8 fciiethyl ether-CH^),

6.68-5.86 (m, C2-H2, α-CH2 and diethyl ether-CH2), 4.90 (d, 1H, C5-H), 3.0-2.63 (m, 3H, aromatic H) , and 1.9 (d, 1H, amide NH). rope.

Example 24

By applying the bromination method from Example 23, p-nitrobenzyl 7-phenoxyacetamido-3-bromo-3-cephem-4-carboxylate was prepared with phosphorus tribromide.

Example 25

Using the bromination method indicated in Example 23, 2,2,2-trichloroethyl 7-acetamido-3-bromo-3-cephem-4-carboxylate was prepared with phosphorus tribromide.

Example 27

p-nitrobenzyl 7-amino-3*"chloro-3-cephem-4-carboxylate.

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 to 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-warm bath to approx. 5°C> and 0.6 ml of isobutyl alcohol was added. By continued cooling and stirring, the reaction product crystallized out, p-nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylate hydrochloride. This was filtered off, washed with: cold methylene chloride and dried, and a total of 200 mg of crystalline product was obtained which melted with decomposition at approx. l68°C.

Percent elemental composition for C-^^H^^CIN^O^S.HC1

Theoretical: C 4-1.39, H 3.20, N 10.34, Cl 17.45 Found: C 41.14»H 3.31, N 10.44, Cl 17.29

I.R. (Nujol Mull): showed absorption bands at 5'55 ((3-lactam carbonyl) and'

5.78 (ester carbonyl) micron.

UV (pH 7 buffer): showed absorbance maximum

max 268 mu (£ = I3.8OO.)

N.M.R.(DMSOdg): signals at

5.97 (s, 2H, C2-H2), 4.8-4.5 (m„ 4H, Cg-H, Cy-H and ester CH2), and

2.35-1.6 (q, 4H, aromatic H)tau.

Example 28

7-amino-3-chloro-3-cephem-4-carb,oxylic acid.

To a solution of 750 mg (1.85 mmol) p-nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylate hydrochloride in 2.0 ml tetrahydrofuran and 40 ml methanol was added a suspension of 750 mg pre-reduced % palladium on carbon, catalyst in 20 ml of ethanol,

and the suspension was hydrogenated under a hydrogen pressure of 3>5 kg/cm at room temperature for 45 minutes. The catalyst was filtered off and washed with THF and water. The filtrate and catalyst wash were combined and evaporated to dryness. The residue was dissolved in a mixture of water and ethyl acetate and the pH adjusted to 3* Det

insoluble product was filtered off and treated with acetone. The product was then dried, and 115 mg of 7-amino-3-chloro-3~ was obtained

cephem-4-carboxylic acid.

I.R. (Mull): absorption peaks at

5.6l ((3-lactam carbonyl) and 6.2(carboxylic acid).

N.M.R. (DgO-NaHCO^j: signals at.

6.25 (ABq, 2H, C2-H2)

.4.88 (d, 1H, C6-H) and 4.54 (d, 1H, C7-H) tau.

UV (pH 7 buffer): absorption maximum at

Tvmax 265 mp £ = 755<0>.

Example 29

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 two hours at room temperature and then cooled in an ice-water bath. To the cold mixture was added 0.6 ml of isobutanol and after 30 minutes the reaction mixture was evaporated. The residue was dissolved in ethyl acetate, and the soln

■washed with 5% sodium bicarbonate and then with water and then dried. The dried solution was evaporated to dryness and the residue treated with ether to give 190 mg of 3-chloro-nuclear ester, that is, diphenylmethyl 7-amino-3-chloro-.3-cephem-. 4-carboxylate.

I.R. (Mull): absorb ion peaks at 5.7°g 5.9

lactam and ester carbonyl)micron.

N.M.R. (CDCl^): 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 30

Using the 7-acyl side chain cleavage reaction described in Example 29, diphenylmethyl 7-amino-3-bromo-• 3-cephem-4-carboxylate was prepared with diphenylmethyl 7-phenoxy-acetamido-3 -bromo-3-cephem-4-carboxylate.

Example 31

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

A suspension of 812 mg (2 mmol) of p-nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylic acid hydrochloride in 40 ml of ethyl acetate was added to a solution of 520 mg (5 mmol) of sodium bisulfite in 40 ml of water. The mixture was stirred vigorously, while 395 mS (2.2 mmol) of D-mandelic acid O-carboxylic anhydride was added. The mixture was stirred for 1.5 hours at room temperature, and the aqueous layer was separated from the ethyl acetate layer and washed with ethyl acetate. The ethyl acetate wash solution was combined with the ethyl acetate layer, and these were washed several times with water and then dried and evaporated, whereby the reaction product was obtained as a dry residue. This was treated with ether and 685 mg of p-nitrobenzyl 7-(D-mandelamido)-3-chloro-3-cephem-4-carboxylate was obtained which melted at approx. 158-164°C with decomposition.

Elemental analysis for CggH^gN^O^SCl:

Theoretical: C 52.44, H 3.60, N 8.34, Cl 7.04% ■Found: C 52.25, H 3.45, N 8.58, Cl 6.82% N.M.R. (CDCl^): signals at

6.24 (ABq, 2H, C2-H2), .

. 5.0-4.7 U, 2H, C6-H and oc-H),

4.57 (s, 2H, ester CHg),

6.23 (q, 1H, C?-H) and

2.8-1.2 (m, 10H, aromatic H and C7~NH)taU.

UV (acetonitrile) : A max 265 mp ('£.= 18,600).

The reaction product of 200 mg was reacted with hydrogen in the presence of 5% palladium on carbon to remove the p-nitrobenzyl ester group, and 75 mg of 7-(D-mandelamido)-3-chloro-3~cephem-4- carboxylic acid) N.M.R. (D20 sodium bicarbonate): signals at

6.42 (ABq, 2H, C2-H2), 4.90 (d, 1H, Cg-H), 4.68 (s, 1H, a-CH), 4.37 (d, 1H, ^-H ) ,bg 2.49 (s, 5H, aromatic H) tau.

Claims (1)

1. 3-Halogenocephalosporin compounds for use as intermediates in the production of therapeutically active (compound dei-se-r^,^^^r^a^k terized where R |ex„hydrogen or an acyl group with the formula: ^ where R' is C 1 -C 8 -alkyl, C 1 -C 4 -haloalkyl, or C 1 -C 3 -cyanoalkyl; or R' is a group with the formula: where a and a' are independently hydrogen or halogen; Z is 0 or S; ' and.m is/ O or 1; / or/ R <1> is a group with the formula: wherein Q is hydroxy or formyloxy; - - - -"' / ■~e-1-l.er_.Ri_ er-thienylacetyl; and where R 1 is benzyl, p-methoxybenzyl, p-nitrobenzyl, diphenylmethyl, 2,2,2-trichloroethyl or t-butyl; ^g—X— e-r—kior—eirlrer-brom-.y 2. Intermediate product according to claim 1, characterized in that it consists of diphenylmethyl-7-[2-(2-thienyl)acetamido]-3-chloro-3-cef em- 4-carboxy lat . 3. Intermediate product according to claim 1, characterized in that it consists of p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate. 4. Intermediate product according to claim 1, characterized in that it consists of p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-bromo-3-cef em-4-carboxylate. 5. Intermediate product according to claim 1, characterized in that it consists of p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate. 6. Intermediate product according to claim 1, characterized in that it consists of p-nitrobenzyl-7-[2-(2,5-dichlorobenyl)-thioacetamido]-3-chloro-3-cephem-4-carboxylate. 7. Intermediate product according to claim 1, characterized in that it consists of p-nitrobenzyl-7-phenoxyacetamido-3-chloro-3-cephem-4-carboxylate.