CH628055A5 - Process for the preparation of clavamic acid derivatives - Google Patents

Description

628055

2nd

PATENT CLAIMS 1. Process for the preparation of a compound of the formula:

wherein R3 represents an esterified carboxyl group and X represents an exchangeable atom or group, with an azide.

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^ CH2R

\

II

H

The invention relates to a process for the preparation of new io clavamic acid derivatives which can be used as antibiotics.

DT-OS 26 04697 describes the isolation from fermentations of Streptomyces clavuligerus of the carboxylic acid of the formula I (clavulanic acid):

wherein R represents an amino group -NH2 and R3 represents a carboxyl or esterified carboxyl group, as well as their salts and zwitterionic forms, characterized in that a compound of formula II, wherein R represents an azido group, is subjected to a reduction.

2. The method according to claim 1, characterized in that the reduction is carried out by catalytic hydrogenation.

3. The method according to claim 1, characterized in that the reduction is carried out using a dissolving metal reducing agent.

4. The method according to any one of claims 1 to 3, characterized in that R3 in the starting compound of formula II is a cleavable esterified carboxyl group.

5. The method according to claim 4, characterized in that R3 in the starting compound of formula II is a p-nitrobenzyloxycarbonyl group.

6. The method according to claim 5, characterized in that the p-nitrobenzyloxycarbonyl group is cleaved during the reduction to give a compound of formula II in which R represents an amino group and R3 represents a carboxyl group.

7. The method according to any one of claims 1 to 3, characterized in that R3 in the starting compound of formula II represents an esterified carboxyl group and the reduction of the azido group is carried out essentially without cleavage of the group R3.

8. The method according to any one of claims 1 to 5, characterized in that R3 in the compound of formula II which is initially formed means a carboxyl group and this compound is subjected to esterification and, if appropriate, subsequently reacted with an acid to form the acid addition salt.

9. The method according to any one of claims 1 to 6, characterized in that a salt of the compound of formula II, wherein R is an amino group and R3 is a carboxyl group, is formed by treatment thereof with an acid or a base.

10. Application of the method according to claim 1 to compounds of the formula II, in which R is an azido group and R3 is an esterified carboxyl group, which are prepared by preparing a compound of the formula:

CH2OH

25 and their salts described in pure form.

The compounds in the present application are named with reference to the term "Clavam". This designation became the stem heterocycle of the formula:

40 in analogy to the name “Cepham”, which was used when naming the cephalosporin compounds in J. Amer. Chem. Soc., 1962, 84, 400 was used. Thus the compound of formula I is referred to as (3R, 5R, Z) -2- (2-hydroxyethylidene) -clavam-3-carboxylic acid.

The present invention relates to a process for the preparation of analogs of the compound of the above formula I and its salts and esters which carry an amino group -NH2 instead of its hydroxyl group.

As will be explained in more detail below, these are anti-

50 biotics or as ß-lactamase inhibitors can be used or they can be used as intermediates in the preparation of other active compounds. According to the process of the invention, compounds of the formula

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MR

wherein R is an amino group -NH2 and R3 is a carboxyl group or an esterified carboxyl group, together with their salts and zwitterionic forms

made available.

The esters can generally be present as compounds of the formula II in which R3 is a COOR4 group in which R4 is an organic group which is conveniently derived from an alcohol (aliphatic or ariphatic), a phenol or a stannanol. Such alcohol, phenol or stannanol as used for the esterification of the carboxyl group preferably contain no more than 24 carbon atoms.

The group R4 can thus mean: a straight-chain or branched, unsubstituted or substituted alkyl or alkenyl group, preferably having 1 to 8 carbon atoms, for example a methyl, ethyl, propyl or isopropyl, butyl, sec-butyl, tert- Butyl or allyl group, desired substituents for example alkoxy, for example Methoxy, halogen, e.g. Fluorine, chlorine, bromine or iodine,

Cyano, acyloxy, e.g. Alkanoyloxy such as acetoxy or pivoyloxy or alkoxycarbonyloxy, e.g. Ethoxycarbonyloxy, acyl, e.g. p-bromobenzoyl and alkoxycarbonyl, e.g. Can be ethoxy-carbonyl;

an aralkyl group with up to 20 carbon atoms, in particular an arylmethyl group, for example a benzyl or substituted benzyl group, suitable substituents being each halogen, e.g. Chlorine, nitro, e.g. o- or p-nitro, cyano, alkoxy, e.g. p-methoxy or alkyl, e.g. can be p-methyl groups, a diphenylmethyl or triphenylmethyl group or a fur-2-ylmethyl, thien-2-ylmethyl or pyrid-4-ylmethyl group, the heterocyclic groups of which may also be substituted, for example by a Ci -4- alkyl group, preferably methyl;

an aryl group with up to 12 carbon atoms, e.g. a phenyl or substituted phenyl group, suitable substituents being any halogen, e.g. chlorine, nitro, e.g. o- or p-nitro, cyano, alkoxy, e.g. p-methoxy or alkyl, e.g. can be p-methyl groups;

a cycloalkyl group with no more than 12 carbon atoms, e.g. Adamantyl;

a heterocyclic group having no more than 12 carbon atoms, the heteroatom being, for example, oxygen, such as in the tetrahydropyranyl or phthalidyl group; or a stannyl group with up to 24 carbon atoms, for example a stannyl group with 3 substituents, which may be the same or different, selected from alkyl, alkenyl, aryl, aralkyl, cycloalkyl, alkoxy, aryloxy or aralkoxy groups. Such groups include methyl, ethyl, propyl, n-butyl, phenyl and benzyl groups. If R3 is a carboxyl group and R is an amino group, the compound of the formula II can exist as a zwitterion or form acid addition salts or salts with bases. Esters of the compounds in which R represents an amino group can form acid addition salts and starting materials of the formula II in which R represents an azido group and R3 represents a carboxyl group can form salts with bases. The salts with bases can be salts with inorganic bases, such as alkali metal salts, e.g. Sodium, potassium and lithium salts, alkaline earth metal salts, e.g. Calcium and magnesium salts and ammonium salts or salts with organic bases, for example amine salts. Acid addition salts include salts with inorganic acids, e.g. hydrochloric acid, hydrobromic acid, perchloric acid, sulfuric acid or phosphoric acid salts and salts with organic acids, e.g. Acetic acid, propionic acid, citric acid, maleic acid or toluene-p-sulfonic acid salts.

The compounds of formula II generally show a β-lactamase inhibiting activity and can be used for

3,628,055

Protection of β-lactam antibiotics that are amenable to β-lactamase hydrolysis, for example antibiotics such as ampicillin or cephalexin.

In general, the compounds are stable against exposure to β-lactamases produced by gram-positive organisms, for example those produced by Staphylococcus aureus, and β-lactamases produced by gram-negative organisms.

The free acids and their salts and metabolically labile esters generally show antibiotic activity. Examples of such esters include the phthalidyl and acyloxymethyl esters, e.g. the acetoxymethyl and pivaloyl oxymethyl esters, and a-alkoxycarbonyloxyalkyl esters, such as 1-ethoxycarbonyloxyethyl ester.

15 If the carboxylic ester group is split rapidly, for example by hydrolysis or reduction, without considerable degradation of the remaining part of the molecule, the esters can be used as carboxyl-protected derivatives of the parent acids. Certain esters can be used either in the purification or characterization of the acids or as carboxyl-protected intermediates for the production of further derivatives.

Esters which are particularly suitable as carboxyl-protected intermediates and which can be used primarily in this connection include the aryl methyl esters, as explained above, in particular the benzyl, p-nitro-benzyl, benzhydryl and trityl esters.

Of particular value in this regard are the rapidly cleavable esters, for example the arylmethyl esters of the azido-30 do compound, since their catalytic reduction quickly gives the carboxylic acid of the amino compound. The p-nitro-benzyl ester of the azido compound is particularly preferred as the starting product of the process.

A preferred compound of Formula II useful in medicine is the compound in which R is NH2 and R3 is COOH (i.e. the amino acid), which is generally in the zwitterionic form.

It was shown that this compound was active against a range of gram-negative and gram-positive microorganisms, for example against strains of Staphylococcus aureaus, Escherichia coli, Salmonella typhimurium, Shigella sonnei, Enterobacter cloacae, Klebsielle aerogenes, Proteus mirabilis and Proteus vulgaris Proteus morganii has antibacterial activity.

45 This compound has the ability to inhibit β-lactamaseen enzymes. These include enzymes produced by gram positive organisms, for example those produced by Staphylococcus aureus strains. The compound is particularly worth mentioning in connection with the inhibition of β-lactamases, which are formed by an unusually wide range of strains of Gram-negative bacteria, for example strains of Proteus mirabilis, Escherichia coli, Proteus morganii, Klebsiella aerogenes, Salmonella typhimurium, Haemo-55 philus influenzae, Bacteroides fragilis, Proteus vulgaris, Proteus rettgeri.

As is evident from studies on the mouse, this compound is absorbed orally.

The new compounds are of interest for use in connection with β-lactam antibiotics which are sensitive to β-lactamases from gram-positive and / or gram-negative organisms.

In general, it is preferred to use the compounds in conjunction with a broad-spectrum β-lactam antibiotic, which may be of the conventional oral or parenteral type. The use of the amino acid and its salts is preferred and the amino acid in its zwitterionic form is particularly preferred.

628055

4th

Examples of broad spectrum orally absorbable β-lactam antibiotics include cephalexin, cephaloglycin, ampicillin and amoxycillin and their orally absorbable esters, for example the acyloxymethyl and phthalidyl esters and the orally absorbable esters of carbenicillin and ticarcillin, for example the indanyl and phenyl esters . The broad spectrum β-lactam antibiotics that are not orally absorbed include carbenicillin, ticarcillin, cephalotin, cephalidin, cefazolin, cephacetril and cephapirin.

Examples of β-lactam antibiotics with a narrow spectrum of action are penicillin G, penicillin V, mecillinam and pivmecillinam.

A combination of the amino acid, for example with ampicillin, shows a synergistic activity against ß-lactamase-forming organisms, including strains of, for example, Staphylococcus aureus, Escherichia coli, Klebsiella aerogenes, Proteus mirabilis, Salmonella typhimurium, Shigella sonnei, Bacteroidesusorganis and Bacterooteus fragilis vulgaris.

Starting products of the formula II, in which R is N3 and R3 COOH and their salts, have a similar spectrum of activity with regard to that which has the amino acid obtainable according to the invention.

The active compounds of formula II can be used either alone or in combination with another β-lactam antibiotic in the treatment of numerous human and animal diseases caused by pathogenic bacteria, such as infections in the respiratory system or in the urinary system.

Furthermore, pharmaceutical compositions (including veterinary compositions) can be created which contain at least one of the acids, physiologically acceptable salts or metabolically active esters. In view of the protective effect described above, the compositions can advantageously contain one or more further β-lactam antibiotics, preferably orally absorbable. The compositions may also contain a pharmaceutical (including veterinary) carrier or excipient.

The compositions can be, for example, in the form of powders, tablets, capsules, pills, solutions and syrups which are suitable for oral administration and can be, for example, starch, lactose, talc, magnesium stearate, gelatin, distilled water and suspending, dispersing, Contain emulsifying, flavoring or coloring agents.

The compounds can also be formulated in the form of rectal compositions, such as suppositories or retention clusters.

The compounds can be formulated for parenteral administration. The compounds can be formulated as dry ampoules for reprocessing before use, optionally together with another antibiotic compound.

In general, the weight ratio of the compound to the β-lactam antibiotic to be protected is 10: 1 to 1:10, preferably 5: 1 to 1: 5, and in particular 2: 1 to 1: 2.

The active compounds are generally administered in a total daily dose of 50 mg to 20 g, preferably 100 mg to 10 g, which can be divided into doses which are administered 1 to 4 times a day. If the composition contains a further β-lactam antibiotic, the total amount of the β-lactam antibiotic is advantageously 100 mg to 20 g, which can be administered in divided doses 1 to 4 times a day. The dosage units generally contain 12.5 mg to 5 g, preferably 50 mg to 1 g, of the active compound,

if used alone and 25 mg to 5 g, preferably 100 mg to 1 g, of total β-lactam antibiotic if another antibiotic is present.

According to the invention, the compounds of the formula II in which R is an amino group are prepared from the compound of the formula II in which R is an azido group by reduction, for example by catalytic hydrogenation. The hydrogenation catalyst is usually a noble metal catalyst such as palladium, platinum or rhodium or another transition metal catalyst such as nickel. The catalyst can be applied to a support, for example activated carbon or diatomaceous earth. The metal catalyst is preferably palladium, for example in the form of 10% palladium on activated carbon. If desired, the hydrogenation is carried out in a solvent for the starting material.

Suitable solvents for the hydrogenation include ethers such as diethyl ether or tetrahydrofuran, esters such as ethyl acetate, ketones such as acetone or methyl ethyl ketone, chlorinated hydrocarbons such as methylene chloride, amides such as dimethylformamide or dimethylacetamide, or alcohols such as ethanol or mixtures thereof. Such solvents can advantageously be mixed with water or an aqueous buffer. With some combinations, a two-phase system can arise. If the substrate is sufficiently water-soluble, water or an aqueous buffer can be used alone.

Alternatively, the azido group can be reduced using dissolving metal reducing agents, e.g. Zinc and aqueous hydrochloric acid, with a controlled pH in the range of 2 to 6, preferably 4 to 4.5. Suitable solvents include, for example, water, miscible solvents such as ketones, e.g. Acetone, ether such as tetrahydrofuran, and alcohols such as ethanol.

Certain esters used as the starting material can be cleaved during the reduction, for example the arylmethyl esters, such as the p-nitrobenzyl esters, to give a free carboxyl group, although - as indicated below - a selective reduction of such esters can be carried out without a noticeable reduction in the ester group.

The acid (II) obtained can be isolated from the solution, for example by partitioning, between the solvent (if it is immiscible with water) and water, and after removal of the catalyst the aqueous phase can be lyophilized to obtain the desired acid .

The acidic product can also be isolated from the solution by precipitation or crystallization, either in the form of its zwitterionic salt or as the salt as defined above. Depending on the conditions, the isolated compound can be in the form of a solvate or hydrate.

The azido compounds used as starting products can be prepared by reacting a compound of the formula in which R3 is an esterified carboxyl group as defined above.

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niert means and X is a rapidly exchangeable atom or a rapidly exchangeable group, with an azide, for example an alkali metal azide such as sodium azide or an organic azide such as tetramethylguanidinium azide or a tetraalkylammonium azide such as tetrabutylammonium azide. The reaction is preferably carried out in a solvent medium for the reactants. The solvent is usually polar, for example a substituted amide or imide solvent such as dimethylformamide or dimethylacetamide; a nitrile solution center! such as acetonitrile, a substituted sulf. oxide such as dimethyl sulfoxide, an ester such as ethyl acetate, a cyclic ether such as tetrahydrofuran, a chlorinated hydrocarbon such as methylene chloride or a ketone such as acetone. If desired, water-miscible solvents, such as acetone, can be slightly aqueous to aid in the dissolution of the azide. A small amount of a carboxylic acid such as acetic acid may advantageously be present.

The substituent X can be, for example, a halogen atom (chlorine, bromine or iodine) or an acyioxy group, such as an aliphatic, aromatic or araliphatic carbonyioxy or sulfonyloxy group with, for example, 1 to 20 carbon atoms. The aliphatic or aromatic group of such a sulfonyloxy group may, for example, be an alkyl (e.g. Ci-s) group which is substituted by a halogen atom, e.g. Fluorine or chlorine may be substituted, or an aryl (e.g. Co i5) group which may contain substituents such as alkyl, e.g. Methyl, alkoxy, e.g. Methoxy or halogen, e.g. Bromine. The aliphatic or aromatic group of such a carbonyloxy group may, for example, be an alkyl (e.g. Cs-s) group, preferably substituted by one or more halogen atoms, e.g. Chlorine or fluorine, or an aryl (e.g. Cö-i5) group, optionally substituted by, for example, one or more halogen atoms or nitro groups. Such acyloxy groups can thus include dichloroacetoxy, mesyloxy, fluoromethanesulfonylioxy, tosyloxy or phenylsulfonyloxy.

The compounds of formula III, wherein X is halogen, can be obtained by reacting an ester of the compound of formula I with a Haiogenie-agent. such as a thionyl halide, or with a sulfonylating agent, e.g. a mesylating or tosylating agent in the presence of halide ions. The compounds of formula III, wherein X represents an acyioxy group, can be prepared by reacting an ester of the compound of formula I with a suitable acylating agent, for example an acyl halide or anhydride.

Is in the connection of the forms! III X halogen, it is preferably chlorine or bromine.

If desired, the compound of formula III can be reacted with the azide without isolation from the medium.

In the above reactions where the starting product is an ester and the appropriate acid or salt is desired, the compound can be subjected to removal of the ester group. For this purpose, rapidly cleavable esters, for example aryl methyl esters, which can be opened or cleaved by reduction, for example by hydrogenolysis, are preferred. The cleavage of an arylmethyl ester, e.g. a p-nitrobenzyl ester, can be carried out by hydrogenolysis, e.g. using a metal catalyst e.g. a noble metal such as platinum, palladium or rhodium. The catalyst can be applied to a support, for example activated carbon or diatomaceous earth. It can also have a p-nitrobenzyl group

Reduction of the nitro group (e.g. using a dissolving metal reducing agent such as zinc in acetic acid or zinc in aqueous tetrahydrofuran or acetone) by controlling the pH range within a value of 3 to 6, preferably 4.0 to 5.5 Addition of aqueous hydrochloric acid; aluminum amalgan in moist ether, e.g. tetrahydrofuran; or iron and ammonium chloride in an aqueous ether, e.g. tetrahydrofuran) and subsequent hydrolysis, either under reduction conditions or by subsequent treatment with acid. Alternatively, a stannyl ester can be cleaved by very mild solvolysis, for example by reaction with water, alcohols, phenols or carboxylic acids, such as acetic acid.

If a reductive cleavage is used to remove the ester group of an ester of the formula II in which R is N3, the azido group may tend to be reduced at the same time. However, careful use of selected methods enables the azido acid of formula II to be prepared. For example, the p-nitrobenzyl ester of azido acid can be cleaved using a dissolving metal reducing agent such as zinc and hydrochloric acid under pH control as described above to obtain the azido acid or a salt thereof.

The esters of clavulanic acid used as starting materials in the above processes can be prepared from clavulanic acid or a reactive derivative thereof by reaction with an alcohol, phenol or stannanol or a reactive derivative thereof to form the desired ester. If desired, the reaction is carried out under mild conditions to prevent the dicyclic core from breaking. It is therefore preferred to use neutral or mild acidic or basic conditions at temperatures between -70 and + 35 ° C.

The alkyl, alkoxyalkyl and aralkyl esters can be prepared by reacting the acid of the formula I with the suitable di-azoalkane or diazoaralkane, for example diazomethane or diphenyldiazomethane. The reaction is generally carried out in an ether, ester or halogenated hydrocarbon solvent, e.g. Diethyl ether, ethyl acetate or dichloromethane performed. In general, lower temperatures, such as -15 to + 15 ° C, are preferred.

The alcohol-derived esters can be obtained by reacting a reactive derivative of the alcohol, for example a halide, such as a chloride, bromide or iodide, or a hydrocarbon sulfonyl derivative, such as a mesyl or tosyl ester, with a salt of the acid of the formula I, for example an alkali - Or alkaline earth metal salt, such as a lithium, sodium, potassium, calcium or barium salt or an amine salt, such as a triethylammonium salt. This reaction is preferably carried out in a substituted sulfoxide or amide solvent, for example dimethyl sulfoxide or dimethylformamide or hexamethylphosphoramide.

The stannyl esters can suitably be carried out by reacting the carboxylic acid of formula I or a salt thereof with reactive tetravalent tin parts. Trialkyltin oxides are preferred for the synthesis of tin compounds in view of their accessibility and low toxicity.

If desired, the acids of the formula II can be esterified using the methods described above for the preparation of the esters of clavulanic acid. In particular, if desired, the azido acid of Formula II can be re-esterified to one of those initially

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present ester group to introduce various ester groups into the azido ester product made from the compound of formula III. It is thus possible to introduce an ester group which is inert to the conditions used in the subsequent reduction of the azido group, which facilitates the formation of the esters of the amino acid of the formula II (R = NH2; R3 = COOH). Such esters can be of a type that cannot be removed by reductive cleavage, e.g. Alkyl esters, such as methyl esters, or may be amenable to reductive cleavage, but may be less reactive than the azido group.

In the formation of the salts, an acid initially formed in solution in a suitable solvent can be reacted with a suitable base, preferably under conditions which favor the precipitation of the salt. In the formation of alkali metal salts, e.g. Sodium salts, is an alkanoate, e.g. a 2-ethylhexanoate, a preferred base.

The acid addition salts of the compounds of formula II, wherein R represents an amino group, can be rapidly prepared by reacting the free amino compound of formula II with an acid.

The invention is illustrated by the following preparations and examples.

The following preparations illustrate the means by which the starting materials for the preparation of the compounds of formula II can be obtained.

Manufacturing 1

4-nitrobenzyl- (3R, 5R, Z) -2- (2-hydroxyethylidene) clavam-3-carboxylate

A mixture of 10 g (3R, 5R, Z) -2- (hydroxyethylidene) clavam-3-carboxylate, 9.5 g of 4-nitrobenzyl bromide and 65 ml of hexamethylphosphoramide was stirred at room temperature for 18 hours. The suspension was then partitioned between 800 ml of ethyl acetate and 800 ml of 50% saturated aqueous sodium chloride solution. The organic layer was separated, washed successively with water, 0.5 M aqueous sodium hydrogen carbonate solution and water, dried and concentrated to obtain a slurry. The colorless crystals were collected and 8.39 g of title ester were obtained, F = 117.2 ° C (Mettler), /, £ ° H 264 nm (e 11000), Vmax (CHBn) 1781 (β-lactam), 1738 (ester ), 1680 cm- '(0-C = C), x values (CDCb) include 4.30 (d, J 2 Hz, C-5H), 4.61 (s, benzylproons), 5.09 (t, J 7 Hz, = C-) and 5.78 (d, J 7 Hz, -CH2OH).

Manufacturing 2

4-nitrobenzyl- (3R, 5R, Z) -2- (2-chloroethylidene) clavam-3-carboxylate

A solution of 1.0 g of 4-nitrobenzyl- (3R, 5R, Z) -2- (2-hydroxyethylidene) -clavam-3-carboxylate in 20 ml of ethyl acetate, which contained 0.32 ml of pyridine, was cooled to -60 ° C , stirred and treated with a solution of 0.26 ml thionyl chloride in 2.0 ml ether. The mixture was warmed to -10 ° C. and stirred for a further 10 minutes at -10 ° C. to 0 ° C. and then diluted with 250 ml of ether. The mixture was washed successively with 0.5 N aqueous hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate solution (until the wash water was colorless) and water. The ethereal layer was dried and concentrated to a slurry of colorless needles, which were collected by filtration, washed with ether and dried to obtain 320 mg of chloroester. [a] d + 30 ° (c 0.49, DMSO) 264 nm (8 10 550), Vmax (CHBn) 1800 (β-lactam), 1753 (ester), 1692 cm1 (0-C = C), x (CDCb) values include 4.25 (d, J 2 Hz, C-5H), 4.7 (s, benzylproons), 5.08 (t, J 8 Hz, = CH-), 5.82 (d, J 8 Hz, CH2CI).

Manufacturing 3

Methyl- (3R, 5R, Z) -2- (2-hydroxyethylidene) -clavam-3-carboxylate

A suspension of 4.00 g of lithium (3R, 5R, Z) -2- (2-hydroxyethylidene) -clavam-3-carboxylate was acidified in a mixture of 80 ml of salt water and 200 ml of ethyl acetate with 20 ml of 2N hydrochloric acid shook. The separated aqueous phase was extracted further with 200 ml of ethyl acetate and the combined organic solutions were dried, filtered and concentrated under reduced pressure to about 200 ml. The resulting ethyl acetate solution of free acid was stirred, cooled to less than 5 ° C. and treated with excess ethereal diazomethane. The solvents were removed under reduced pressure to leave an oil which was chromatographed on a silica gel column. Elution with ethyl acetate-petroleum ether (b.p. 40-60 ° C) (3: 1) gave 2.32 g of crude title ester in the form of yellow crystals (yield 56%).

A portion of this product (350 mg) was dissolved in a mixture of 30 ml ether and 20 ml petroleum ether (bp 40-60 ° C), the solution was treated with activated carbon and filtered through diatomaceous earth. The filtrate was concentrated to about 20 ml and set aside for crystallization at 0 ° C. The colorless needles obtained were collected, washed with petroleum ether (bp. 40-60 ° C.) and dried in vacuo to give 312 mg of title ester. F. = 63.5 ° C (Mettler). [ö] d + 76 ° (c 1.03, DMSO), A, max. (0.1 N NaOH) 258 nm (s 18300) Vmax. (CHBn) 3590 (OH), 1798 (β-lactam), 1748 (CO2R), 1694 cm- '(0-C = C) x values (CDCb) include 4.29 (d, J 2.5 Hz, C-5 H), 5.77 (d, J 7 Hz, CH2OH), 6.21 (s, CHs).

Manufacturing 4

Benzyl- (3R, 5R, Z) -2- (2-hydroxyethylidene) -clavam-3-carboxylate

A mixture of 10.25 g of lithium (3R, 5R, Z) -2- (2-hydroxyethylidene) clavam-3-carboxylate and 8.55 g of benzyl bromide in 50 ml of hexamethylphosphoramide was stirred for 22 hours at room temperature. The mixture was then diluted with 11 ethyl acetate and washed successively with 1150% saturated saline, twice with 500 ml of water, with 500 ml of 5M NaHCOs and three times with 250 ml of brine. The organic layer was dried over Na2SO4 and concentrated in vacuo to give an oil which was chromatographed on a silica gel column (150 g) and eluted with chloroform and then with ethyl acetate. The appropriate fractions were combined and concentrated in vacuo to give 8.8 g of title ester in the form of an oil. vmax. (CHBn) 3590 (-OH), 1786 (β-lactam), 1732 (ester), 1684 (-O-C-CH), 734 cm- '(phenyl). x (CDCb) 2.68 (s, phenyl), 4.38 (d, J 3 Hz, C-5 H), 4.85 (s, CH-aPh), 4.95 (s, C-3 H) ), 5.16 (t, J 7 Hz, = CH), 5.85 (d, J 7 Hz, -CH2OH), 6.55 (dd, J 17 and 3 Hz, C-6 a H), 6 , 98 (d, J 17 Hz, C-6 β H), 8.29 (s, -CH2OH).

Manufacturing 5

4-nitrobenzyl- (3R, 5R, Z) -2- (2-azidoethylidene) clavam-3-carboxylate

A mixture of 0.5 g of 4-nitrobenzyl- (3R, 5R, Z) -2- (2-chloroethylidene) -clavam-3-carboxylate, 0.092 g of sodium azide, 15 ml of acetone, 1.5 ml of water and approx 1.0 ml of acetic acid for 15 minutes at room temperature and then partitioned between ether and water. The organic phase was washed with water, dried over sodium sulfate and concentrated to about 10 ml. The residue was applied to a dry silica gel column, which was then eluted with ether. The fractions were collected and pooled on the basis of a thin layer chromatographic analysis, after which the solution was concentrated. At Ste6

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When left at 0 ° C, pale yellow crystals separate from the solution. The crystals were collected, washed with ether and dried in vacuo to give 0.078 g of title ester, mp = 63-64 ° C, [cc] d + 38 ° (c, 0.9; EtOAc), Vmax. (CHBn) 2120 (N3), 1796 (ß-lactam), 1750 (ester), 1690 (enol ether), 1524 and 1345 cm- '(NCh), t (CDCb) 1.70 and 2.50 (doublets, J 8 Hz, aromatic protons), 4.22 (d, J 2 Hz, C-5 H), 4.64 (s, CH2Ar), 4.80 (s, C-3 H), 5.14 (t, J 7 Hz, = CH-), 6.10 (d, J 7 Hz, CH2N3), 6.40 and 6.88 (dd, J 2 and 17 Hz; and d, j 17 Hz, C-6 protons) .

Manufacturing 6

4-nitrobenzyl- (3R, 5R, Z) -2- (2-azidoethylidene) clavam-3-carboxylate

A stirred solution of 0.5 g of 4-nitrobenzyl- (3R, 5R, Z) -2- (2-hydroxyethylidene) -clavam-3-carboxylate in 5 ml of N, N-dimethylformamide was treated at 0 ° C. with 0. 44 ml of collidine and then 0.26 ml of methanesulfonyl chloride for 5 minutes. The mixture was stirred and kept at a temperature of -5 to 0 ° C for 30 minutes, after which it was treated with 0.195 g of sodium azide. After a further 2 hours at 0CC, the mixture was partitioned between ethyl acetate and water. The organic phase was washed successively with water, saturated aqueous cupric nitrate, water, 50% saturated aqueous sodium hydrogen carbonate and brine. The solution was dried over sodium sulfate and evaporated to dryness. The residue was redissolved in chloroform and evaporated to give 0.671 g of gummy material.

The rubbery material was chromatographed on a dry silica gel column and eluted with ether. Appropriate fractions were collected and pooled and evaporated to give 0.11 g of title ester in the form of an oil. The spectral properties of the product were similar to those described in Example 1.

example 1

(3 R, 5 R, Z) -2- (2-aminoethylidene) -clavam-3-carboxylic acid

0.2 g of 4-nitrobenzyl- (3R, 5R, Z) -2- (2-azido-ethylidene) -clavam-3-carboxylate was hydrogenated under atmospheric pressure at room temperature in a mixture of 10 ml of ethyl acetate and 10 ml of ethanol over 10 % palladium on activated carbon (0.2 g) for 2 hours. The mixture was partitioned between 20 ml of ethyl acetate and 50 ml of water and the catalyst was removed by filtration. The aqueous phase was washed with ethyl acetate and lyophilized to obtain 0.092 g of the title ester in the form of a leather-colored powder, vmax (Nujol) 2600 (NH3 +), 1780 (β-lactam), 1690 (enol ether) and 1600 cnr1 (carboxylate), x- Values (D2O) include 4.22 (d, J 2 Hz, C-5 H), 4.99 (s, C-3 H), 5.14 (t, J 7 Hz, = CH-), 6, 30 (d, J 7 Hz, CH2N + H3), 6.39 and 6.86 (dd, J 17 and 2 Hz; and d, J 17 Hz, C-6 protons).

Example 2

(3R, 5R, Z) -2- (2-aminoethylidene) -clavam-3-carboxylic acid

10.0 g of 4-nitrobenzyl- (3R, 5R, Z) -2- (2-azido-ethylidene) -clavam-3-carboxylate were hydrogenated under atmospheric pressure at room temperature in a mixture of 250 ml of ethyl acetate and 250 ml of ethanol over 10 % palladium on activated carbon (15g) for 15 minutes. The mixture obtained was filtered through diatomaceous earth and the filter bed was washed well with ethyl acetate. The bed was then washed with several portions of methanol and the combined methanolic filtrates were concentrated to approximately 30 ml. The crystalline product obtained was collected, washed with methanol and dried to obtain 2.013 g of title ester. Xmax. (0.1 N NaOH aq) 258.5 nm (s 15 500).

200 mg of this material was dissolved in 3 ml of water and the solution obtained was diluted with 18 ml of acetone until it became cloudy. While standing at 0 ° C, acicular crystals separated from the solution, which were collected, washed and dried to obtain 171 mg of the title ester.

[a] D + 61.5 ° (c 1.07; H2O), A, max. (0, IN NaOH aq) 258 nm (e 16410) Vmax. (Nujol) 2800-2500 (NH3 +), 1806 (ß-lactam), 1698 (enol ether) and 1588 cm- '(carboxylate), x (D2O) 4.20 (d, J 3 Hz, C-5 H), 4.98 (s, C-3 H), 5.14 (t, J 8 Hz, = CH-), 6.30 (d, J 8 Hz, CH2NH2), 6.39 and 6.86 (dd, J 17 and 3 Hz; d, J 17 Hz, C-6 protons).

Preparation 7 sodium (3R, 5R, Z) -2- (2-azidoethylidene) -clavam-3-carboxylate

1.9 g of zinc dust were added in small portions over a period of 2 hours to a stirred solution of 1.5 g of 4-nitrobenzyl- (3R, 5R, Z) -2- (2-azidoethylidene) -clavam-3-carboxylate in 80 ml of tetrahydrofuran and 50 ml of water, which was kept at pH 4.3 by cooling at 0 ° C. and by dropwise addition of n-hydrochloric acid. The resulting mixture was stirred for a further 1½ hours, filtered and partitioned between 150 ml of ethyl acetate and 100 ml of water. Hydrogen sulfide was passed through the two-phase mixture and the pH of the aqueous phase was kept at 2.5 to 4.5 by adding n-sodium hydroxide solution. After the pH had reached a constant value of 4.5, the mixture was filtered through diatomaceous earth and the separated organic layer was extracted three times with 0.5N aqueous sodium hydrogen carbonate solution. These extracts were combined with the separated aqueous layer, washed with ethyl acetate, acidified to pH 2 with 2N hydrochloric acid and extracted with ethyl acetate. The organic extract was dried over magnesium sulfate, filtered and treated with a solution of 0.6 g of sodium 2-ethylhexanoate in ethyl acetate. The solution obtained was concentrated to about 2 ml and slowly diluted with ether. The precipitate was collected, washed with ether and dried to give 0.45 g of the title salt.

[a] D + 510 (c 0.95; H2O), Xmax. (0.1 N NaOHaq) 258 nm (e 14100), Vmax. (Nujol) 2100 (azide), 1782 (β-lactam), 1688 (enol ether) and 1614cm- '(carboxylate); x (D20) 4.19 (d, J3 Hz, C-5H), 4.92 (s , C-3 H), 5.01 (t, J 8 Hz, = CH-), 6.03 (d, J 9 Hz, -CH2N3), 6.36 and 6.84 (dd, J 17 and 3 Hz, d, J 17 Hz, C-6 protons).

Preparation 8 methyl- (3 R, 5 R, Z) -2- (2-azidoethylidene) -clavam-3-carboxylate

An aqueous solution of 0.2 g of sodium (3R, 5R, Z) -2- (2-azidoethylidene) -clavam-3-carboxylate was acidified with 2N hydrochloric acid and extracted with ethyl acetate. The organic solution was dried over magnesium sulfate and treated at 0 ° C. with an excess of ethereal diazomethane. The solution was flushed with a stream of nitrogen for 10 minutes and then evaporated to give 0.16 g of title ester as an oil. [a]? "+ 66 ° (c 0.85; CHCb), W. (0.1 N NaOH aq) 258 nm (e 15 700), Vmax. (CHBn) 2100 (azide), 1798 (β-lactam ), 1748 (ester) and 1690 cm- '(enol ether); t (CDCb) 4.27 (d, J 3 Hz, C-5 H), 4.88 (s, C-3H), 5.15 ( t, J 8 Hz, = CH-), 6.13 (d, J 8 Hz, -CH2N3), 6.20 (s, CH3), 6.49 and 6.92 (dd, J 17 and 3 Hz, d, J 17 Hz, C-6 protons).

Preparation 9 methyl- (3R, 5R, Z) -2- (2-azidoethylidene) -clavam-3-carboxylate

A stirred solution of 2.0 g of methyl

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(3R, 5R, Z) -2- (2-hydroxyethylidene) -clavam-3-carboxylate under nitrogen to -50 ° C and treated with 2.17 ml of pyridine and then 0.92 ml of thionyl bromide in 10 ml of ethyl acetate. The resulting mixture was stirred at -45 ° C for 10 minutes and then poured into 80 ml of water. The separated organic phase was added to a stirred suspension of 0.71 g of sodium azide in a mixture of 8 ml of water and 55 ml of acetone at -5 ° C. 0.2 ml of acetic acid was added dropwise and the mixture was allowed to warm to room temperature. After 15 minutes, the solution obtained was between! 50 ml of water and 100 ml of ether distributed. The separated organic layer was washed with water and with saturated saline. The solution was dried over magnesium sulfate and evaporated to give 0.79 g of the title ester as an oil. Amax. (0.1 N NaOH aqj 258 nm (e 15260). The NMR and IR spectra were similar or analogous to those described in Example 6.

Manufacturing 10

Benzyl-GR, 5R, Z) -2- (2-azidoethylidene) -clavam-3-

carboxyla:

A stirred solution of 1.0 g of benzyl- (3R, 5R, Z) -2-f2-hydroxyethylidene) -clavam-3-carboxylatin 20 ml of ether was cooled to -55 ° C. under nitrogen and treated with 0.8 ml of pyridine and then with 0.34 ml of thionyl chloride in 5 ml of ether. The mixture obtained was stirred for 10 minutes at approx. 45 ° C. and then poured into water. The organic phase was washed with water and then treated with a solution of 0.26 g of sodium azide in 3 ml of water and 20 m! Acetone. The mixture obtained was concentrated by evaporation until it became homogeneous, treated with about 0.2 ml of acetic acid and left to stand at room temperature for 10 minutes. The solution was partitioned between ether and water and the organic layer washed with water and brine. The dried solution was concentrated to about 10 ml and chromatographed on silica gel acid with ether as the eluent. The appropriate fractions were pooled and evaporated to give an oil. The oil was redissolved in chloroform to give 0.395 g of title ester in the form of an oil. /. «>« (CHBn) 2100 (azide), 1792 (ß-lactam), 1744 (ester) and 1688 cm- '(enol ether), x (CDCb) 2.62 (s, aromatic protons), 4.29 (d, J 3 Hz, C-5 Hl, 4.79 (s, CH: Ph), 4.86 (s, C-3 Hj, 5.20 (t, J 8 Hz, = CH-), 6J 7 (d, J 8 Hz, -CIL'N?), 6.50 and 6.96 (dd, J 17 and 3 Hz, d, J 17 Kz, C-6 protons).

Example 3

«3îl, 5R, Z) -2 - (2-aminoethylidene) -clavam-3-carboxylic acid

10.0 g of zinc powder was added in small portions over a period of! Hour to a stirred solution of 2.5 g of 4-nitrobenzyl-f3R, 5R, Z) -2- (2-azidoethylidene) -clavam-3-carboxylai in a mixture of 130 ml of tetrahydrofuran and 80 ml of water by cooling 0 ° C and was kept at pH 4.5 by the dropwise addition of n-hydrochloric acid. The reaction mixture was stirred for a further 2! : Stand and then spread between 60 ml of water and 69 ml of ethyl acetate. Hydrogen sulfide was passed through the mixture and the pH of the aqueous phase was kept at about 3 by adding n-sodium hydroxide solution. After the pH had not changed, the mixture was filtered through diatomaceous earth and the separated aqueous phase was washed with ethyl acetate and lyophilized . The residue was stirred with methanol and some insoluble material was removed by filtration. The solution was concentrated to about 25 ml and the precipitated sodium chloride was removed by filtration. The filtrate was diluted with ether and the resulting precipitate was collected, washed and under

Obtained 0.94 g of dried title acid. The NMR and IR spectra were similar to those described in Example 4.

Example 4

Benzyl- (3R, 5R, Z) -2- (2-aminoethylidene) -clavam-3-carboxylate

A stirred solution of 0.1 g of benzyl- (3R, 5R, Z) -2- (2-azidoethylidene) -clavam-3-carboxylate in 2 ml of tetrahydrofuran and 1 ml of water was treated with 0.2 g of zinc dust in small proportions for a period of 5 minutes, the pH being kept between 2 and 4 by the dropwise addition of 2N hydrochloric acid. The resulting mixture was decanted from the zinc residue and partitioned between brine and ethyl acetate. The aqueous phase of pH 5 was extracted with ethyl acetate. The organic extract was dried over sodium sulfate and evaporated to give a gummy material. This was redissolved in chloroform and evaporated to give 0.092 g of the title ester in the form of a foam. Vmax. (CHBn) 3320 (NH2), 1798 (β-lactam), 1740 (ester) and 1696 cm- '(enol ether); x (CDCb) 2.62 (s, aromatic protons), 4.31 (d, J 2 Hz, C-5 H), 4.80 (s, CH2PI1), 4.86 (s, C-3 H), 5 , 08 (d, J 7 Hz, —CH-), 6.41 (d, J 7 Hz, -CH2NH2), 6.58 and 6.80 (dd, J 17 and 2 Hz, d, J 17 Hz, C-6 protons).

Example 5

Methyl (3R, 5R, Z) -2- (2-aminoethylidene) clavam-3-carboxylate

1.2 g of zinc powder were added in portions over a period of 20 minutes to a stirred solution of 0.5 g of methyl (3R, 5R, Z) -2- (2-azidoethylidene) -clavam-3-carboxylate 25 ml of tetrahydrofuran and 15 ml of water, which was kept at pH 4 to 4.5 by cooling at 0 ° C. and by dropwise addition of n-hydrochloric acid. The mixture was stirred for an additional 20 minutes and then filtered and partitioned between saturated pH 7.2 brine and ethyl acetate. The separated aqueous phase was extracted with further ethyl acetate and the combined organic extracts were dried over magnesium sulfate and evaporated to give the title ester in the form of a rubber-like material. Vmax. (CHBr3) 1790 (β-lactam) and 1750 cm- '(ester); x (DMSO-de) 4.17 (d, J 2 Hz, C-5H), 4.68 (s, C-3H), 5.16 (t, J 7 Hz, = CH-), 6.21 (s, CHb), 6.54 (m, CH2NH2), 6.3-6.9 (m, C-6 protons).

Example 6

(3R, 5R, Z) -2- (2-ammoniumethylidene) -clavam-3-carboxylic acid-4-toluenesulfonate

A solution of 100 mg (3R, 5R, Z) -2- (ammonium ethylidene) -clavam-3-carboxylic acid and 90 mg of 4-toluenesulfonic acid monohydrate in 10 ml of water was lyophilized to obtain the title salt. Vmax. (Nujol) 2700 (NH3 +), 1794 (ß-lactam), 1730 (carboxyl), 1700 (enol ether) and 1200 cm- '(sulfonate) x (D2O) 2.30 and 2.65 (doublets, J 8 Hz, aromatic protons), 4.20 (d, J 3 Hz, C-5H), 4.75 (s, C-3 H), 5.08 (t, J 7 Hz, = CH-), 6.31 ( d, J 7 Hz, CA-NH + j), 6.39 and 6.82 (dd, J 17 and 3 Hz, d, J 17 Hz, C-6 protons) and 7.61 (CH3).

Example 7

(3R, 5R, Z) -2- (2-aminoethylidene) -clavam-3-carboxylic acid A solution of benzyl- (3R, 5R, Z) -2- (2-aminoethylidene) -clavam-3- carboxy! at in 10 ml ethyl acetate from 0.49 g benzyl- (3R, 5R, Z) -2- (2-azidoethylidene) -clavam-3-carboxylate analogously to the manner described in Example 4 see

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forth. The solution was diluted with 10 ml of ethanol and the mixture was hydrogenated over 10% palladium on activated carbon (0.35 g) for 20 minutes at room temperature and atmospheric pressure. 10 ml of water were added and the Kata628055 was removed

lysator by filtering. The aqueous phase was washed with ethyl acetate and lyophilized to give 0.04 g of title acid in the form of a foam. The NMR and IR spectra were similar to those described in Example 3.

B