DK153154B - PROCEDURE FOR PREPARING 7-ACYLAMIDO-CEPHALOSPORINE COMPOUNDS - Google Patents

Description

The present invention relates to a particular process for preparing 7-acylamido-cephalosporin compounds of the general formula set forth in the preamble of claim.

The invention represents an improvement in an acylation process for the preparation of 7-acylamido-cephalosporins which is the subject of U.S. Patent Application No. 149,364.

It is known to prepare 7-acylamido-cephalosporins with antibiotic activity by first preparing the analog 7-amino-cephalosporin and then acylating it to form the desired product. This method suffers from the disadvantage that it is necessary first to isolate and purify the intermediate 7-aminocephalosporin.

Therefore, other methods for the preparation of 7-aminocephalosporic acid were sought.

Recently, it has been found that cephalosporins with a methoxy substituent instead of the hydrogen atom at the C-7 position are formed by various microorganisms. These cephalosporins contain an aminoadipoyl side chain which is preferably removed to form novel 7α-methoxy-cephalosporins with increased antibiotic activity.

The present invention is based on the recognition that it is possible to exchange or rearrange cephalosporin compounds in accordance with the following reaction schemes.

where B * and R 'represent different acyl groups, with R' being the group

wherein the meaning defined in the claim is R 1 is hydrogen or methoxy and R 1 is hydrogen or an easily cleavable blocking group.

In the process according to the invention, which is characterized by the characterizing part of the claim, the cephalosporin compound I is reacted with an acylating agent in the presence of a molecular sieve catalyst of the kind specified to form the 7-diacylimido-cephalosporin compound (II). which is then cleaved to form the desired 7-acylamido-cephalosporin compounds (III).

The reaction to prepare the diacylated product is carried out by intimate treatment of the cephalosporin compound with an acylating agent in a suitable solvent in the presence of molecular sieve * The temperature at which the reaction takes place is not very critical and temperatures from -20 ° C to 100 ° 0 are usually satisfactorily, although it is preferred to carry out the reaction at temperatures from 50 ° C to 90 ° 0. For carrying out this reaction, solvents containing no active hydrogen are suitable, such as chloroform, acetonitrile, methylene chloride, dioxane, benzene, halogenobenzene, carbon tetrachloride and diethyl ether.

The acylating agent may be an acyl halide, anhydride or a mixed anhydride, but it is usually preferred to use an acyl halide, e.g. an acyl chloride, as an acylating agent.

The molecular sieves suitable for this purpose are aluminum silicate zeolites. The naturally occurring zeolites can be defined as a group of crystalline solids, hydrated aluminum silicates of mono- or divalent bases, capable of losing some or all of their water content without altering crystal structure, as other compounds can be absorbed instead. for the removed water and capable of undergoing base replacement. A synthetic zeolite, on the other hand, can be prepared from a combination of basic oxides (AlCl3, SiCl2, Na2O and KgO) in an aqueous medium to form the hydrated or semi-hydrated crystalline structure. After heat treatment, the zeolites can be considered substantially anhydrous. Synthetic zeolites are characterized and classified mainly by X-ray diffraction methods. Although a systematic chemical nomenclature for synthetic complex aluminum silicates is lacking, there is a tradition of assigning any new synthetic zeolite to any letter or group of letters and numbers. The meaning of these arbitrary symbols is understandable to those skilled in the art.

It has been found that synthetic zeolites of the A and X classes are particularly suitable for use in the above hex-ruptured acylation process. The pore size of the zeolites must be in a range from 3 to 15 Å, such pore size conditional on the desired catalytic effect. The zeolites may be substantially anhydrous or contain some hydration water. The amount of water in the zeolite may be between 0 and 30%.

In compounds I, II and III, the 3-position is substituted by H

however, the transacylation reaction works just as well when using compounds wherein the 3-position represents the -CH 2 A1 group, where A * is hydrogen or an alkanoyloxy group having 2-6 carbon atoms.

The acyl substituents represented by B 'in formulas I, II and III are preferably carboxylic acid radicals. B1 is aminodipoyl when the compounds are produced by certain microorganisms such as S. clavuligerus, S. lipmanii or S. lactamdurans.

However, B 'can also be one of the commonly used acyl groups in cephalosporin compounds. B * may be represented by the general formula R11R10CHCO where R10 represents hydrogen, halogen, amino, guanidino, phosphono hydroxy, tetrazolyl, carboxy, sulfo, or sulfamino, and R11 represents phenyl, substituted phenyl, a monocyclic heterocylic 5- or 6- linked ring with one or more oxygen, sulfur or nitrogen atoms in the ring, substituted heterocycles, phenylthio-heterocyclic or substituted heterocyclic thio groups or cyano. The substituents may be halogen, carboxymethyl, guanidino, guanidinomethyl, carboxaminomethyl, aminomethyl, guanidino, guanidinomethyl, carboxaminomethyl, aminomethyl, nitro, methoxy or methyl. Examples of preferred acyl groups are phenacetyl, 3-bromo-phenylacetyl, p-aminomethylphenylacetyl, 4-carboxymethylphenylacetyl, 4-carboxamidorethylethylphenylaeethyl, 2-furylacetyl, 5-nitro-furylacetyl, 3-furylacetyl, 2-furylacetyl, 2-furylacetyl 5-methoxythienylacetyl, α-guanidino-2-thienylacetyl, 3-thienylethylethyl, 4-methylthienylacetyl, 3-isothiazolylacetyl, 4-methoxyisothiazolylacetyl, 4-isothiazolylacetyl, 3-methylisothiazolylacetyl, 5-isothiazolylacetyl, 5-isothiazolylacetyl methyl 1,2,5-oxadiazolylacetyl, 1,2,5-thiadiazolyl-4-acetyl, 3-methyl-1,2,5-thiadiazolyl-4-acetyl, 3-chloro-1,2, 5-thiazolyl-4-acetyl, 3-methoxy-1,2,5-thiadiazolyl-4-acetyl, phenylthioacetyl, 4-pyridylthioacetyl, cyanoacetyl, "betrazolylacetyl, α-fluorophenylacetyl, D-phenylglycyl, 3-hydroxy-D -plienylglycyl, 2-thienylglycyl, 3-thienylglycyl, phenyl-malonyl, 3-chlorophenylmalonyl, 2-thienylmalonyl, 3-thienylmalonyl, α-phosphonophenylacetyl, α-sulfaminophenylacetyl, α-hydroxyphenylacetyl, α-hydroxyphenylacetyl azolylphenylacetyl and α-sulfophenylacetyl.

The reaction I - * II illustrated above is an equilibrium reaction. An excess of acylating agent is used to increase the yield of the desired final product, III. The diacyl intermediate, II, must be prepared for removal of group B 'to produce the final product III. This cleavage can be accomplished in various ways. First, spontaneous cleavage can occur (in the presence of a molecular excess of the acylating agent) simply by prolonged reaction time. Second, when present in the molecular sieve, the water can act as a decomposition agent and the finished acylamide product is recovered in high yield.

Both of these methods can be characterized as "passive" in the sense that it is not necessary to add a separate cleavage agent to the reaction mixture.

A third method of cleavage consists in the addition of benzyl alcohol, an alkanol or an alkylthiol having 1-6 carbon atoms.

Hydrochloric acid can also be added as a decomposing agent as a fourth reagent.

The process of the invention is illustrated in more detail by the following reaction scheme:

wherein R 1 is hydrogen or methoxy, is carbamoyloxy or acetoxy, and R 2 is a blocking or protecting group. The group COXR ^ denotes a blocking carboxy group or thiocarboxy group (I is 0 or S; R 1 is a blocking group and R 1 is an acyl group.

In said process, the amino group of the cephalosporin starting compound (IX) is first blocked (R2) by reaction with an appropriate reagent to protect the 5'-amino substituent. Thus, the amino group is blocked by amino protecting groups such as acyl, aroyl, alkoxycarbonyl, alkylsulfonyl or arylsulfonyl in a manner known per se. Examples of specific groups suitable for blocking the amino group are the following: trichloroethoxycarbonyl, tertiary butoxycarbonyl, benzoylmethoxycarbonyl, trimethylsilyl, p-methoxybenzyloxy, 2-nitrophenylsulphenyl, 2,4-dinitrophenylsulfenyl, chloroacyl , p-nitrobenzenesulfonyl, p-toluenesulfonyl, methanesulfonyl, benzoyl, p-chlorobenzoyl, p-nitrobenzoyl and toluolyl, but in general it is preferable to use p-toluenesulfonyl or benzoyl derivatives conveniently prepared by reacting the cephalosporin with the cephalosporin. toluene sulfonyl chloride or benzoyl chloride while lowering the pH of the landings, i.e. between 9 and 10.

It is usually preferred to carry out the above-described reactions with an eephalosporin compound, the carboxy groups in the aminoadipoyl side chain and at the 4-position are likewise blocked or protected (XI) since maximum yields of the desired compound are obtained with such derivatives. Furthermore, it is not necessary to block the carboxy group in the aminoadipoyl side chain as it is removed during the cleavage reaction, but the blocking or protecting group is preferably such that it can be easily removed in the 4-position to form the free acid without opening the β-lac-tam group, since the cephalosporin compounds are usually used in the form of salts such as alkali metal salts or amine salts. Protective groups suitable for this purpose are well known. Examples of suitable derivatives are esters of alcohols, phenols, mercaptans and thiophenols, wherein the -COXR group represents esters. In this general formula, X means oxygen or sulfur, and R means radical from an alcohol or a thiol such as methyl, ethyl, tertiary butyl, substituted alkyl such as phthalimidorethyl, succinimidomethyl or phenacyl, substituted phenacyl, e.g. p-bromo * phenacyl, β-substituted ethyl such as 2,2,2-trichloroethyl, 2-methylthioethyl, 2- (p-methylphenyl) ethyl, 2- (p-methylphenyl) sulfonylethyl, 2-methylaminoethyl, 2- chloro (or bromo) ethyl or benzyl, substituted benzyl such as p-nitrobenzyl, p-methoxybenzyl, 3,5-dinitrobenzyl, 2,4,6-trimethylbenzyl or 3,5-diehloro-4-hydroxybenzyl, benzhydryl or substituted benzhydryl , such as p-methoxybenzhydryl, acyloxyalkyl such as acetoxymethyl or pivaloyloxymethyl, alkoxy such as methoxymethyl, or a monocyclic aryl group such as phenyl or substituted phenyl such as p-nitrophenyl or 3,5-dinitrophenyl. It has been found that the most convenient group for blocking the carboxy group is the methoxymethyl group where X is oxygen. These protecting or blocking groups for the carboxy substituents are readily prepared in a manner known per se.

The protective eephalosporin compound is then reacted with an acylating agent in the presence of the molecular sieve described above to form the imide or diacylated product (XII). The acylating agent may be an acid halide (chloride or thromide) or a functional equivalent thereof, such as an acid anhydride, a mercaptide or a mixed acid anhydride with other carboxylic acids, an activated ester of the carboxylic acid such as the p-nitrophenyl ester.

The acylating agents used are those derived from the carboxylic acids containing the group:

wherein R 1 has the meaning defined in the claim.

The acylating agent is used in a molecular excess relative to the cephalosporin compound, preferably 2-6 times as much acylating agent as cephalosporin.

Various molecular sieves known per se can be used as molecular sieves. Preferably, a synthetic zeolite with regular crystal structure and uniform pore size is used. The most commonly used sieves of type 3A, 4A, 5A and 13X are all applicable to the method of the invention. These targets have the following characteristics:

The sieves appear essentially in anhydrous form; they may be used in this form or further dehydrated, to 0 in + 2 io water, by heating to high temperatures (about 500 ° C or above) prior to use; or they can be used with a content of up to 30 io hydration water (weight percent). The hydrated screens are conveniently prepared by standing in a humid atmosphere or slurry in Tooth with subsequent setting of the desired moisture content for vacuum drying or drying at room temperature or elevated temperature.

This drying will usually take 2-5 hours, but these time limits are not critical. The moisture content can be measured according to Karl Fischer, a generally recognized technique, or otherwise known.

The conversion of the protected cephalosporin compound (XI) to the imide of the diacylated product (XII) is conveniently accomplished by intimately mixing the cephalosporin compound with the acylating agent in a suitable solvent in the presence of the selected molecular sieve. The temperature of this reaction is not critical and usually temperatures from -20 ° C to 100 ° C are satisfactory. if the reaction is temperature dependent and proceeds more rapidly at high temperatures, it is preferable to carry out the reaction at temperatures between 50 ° C and 90 ° C. As the reaction medium in this reaction, a solvent containing no active hydrogen such as chloroform, acetonitrile, methylene chloride, dioxane, benzene is used. , halogenobenzene, carbon tetrachloride, 1,2-dichloroethane or diethyl ether. It is important to keep the slurry moving by stirring or shaking during the reaction.

The required amount of sieves for the reaction may vary to suit the operating conditions, it is usually preferred to use approximately equal amounts of starting material and sieves, but it is possible to use a weight ratio of the starting compound to the sieves of 1 - 0.5-2.

As mentioned above, the original acyl group can be split in many different ways. A simple "aging" of the reaction mixture is sometimes sufficient, e.g. when the sieves contain 10 - 30% water, for a period of between 30 minutes and 30 hours. An alkanol, alkylthiol or benzyl alcohol may be added after a shorter aging period. The alkanol or alkylthiol can have 1-6 carbon atoms, and examples of this are methanol, ethanol, isopropanol and t-butanol. Hydrochloric acid may also be added to induce a cleavage. During the acylation reaction, some spontaneous cleavage of the aminoadipoyl group occurs due to the equilibrium nature of the reaction, depending on the conditions under which the acylation takes place. An extended heating of the reaction mixture results in a cleavage of the aminoadipoyl group and formation of the desired 7-acylated cephalic compound, if the sieves contain about 10 µs of water.

The ownership of the "protecting or blocking group by the carboxy function is carried out in a manner known per se. Thus, for example, the methoxymethyl group can be removed using hydrochloric acid at 0-10 ° C; the trichloroethoxycarbonyl group is removed by reaction, with zinc and acetic acid; The butoxycarbonyl and benzhydryl groups are removed by reaction with trifluoroacetic acid.

The method according to the invention will be illustrated in the following by means of some examples.

EXAMPLE 1 3-Carbamoyloxymethyl-7-methoxy-7P-thienylacetamido-3-cephem-4-_________________________-___-__________

Step A: 7β- (D-5-tosylamino-5-carboxyvaleramido) -3-carbamoyloxyl-7-methoxy-3-oephem-4-carboxylic acid

The mono-sodium salt of 7β- (D-5-amino-5-carboxyvaleramido) -3 "Carb-amoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid (45.0 ml, 49.5 mg / ml aqueous solution) mix with acetone (450 ml) and water (450 ml). The pH of the mixture is adjusted to 9.5 - 9.6 with 50 µl of sodium hydroxide solution and 19 g of tosyl chloride in 100 ml of acetone are added in portions. The pH is maintained at 9.5 - 9.6 by frequent addition of sodium hydroxide solution. After 15-20 minutes, the pH becomes stable and the sulfonylation reaction is continued for a total of 1 hour. The temperature of the solution is maintained at 20-23 ° 0 throughout the reaction period.

Next, the solution is cooled in an ice bath and the pH lowered to 7 by the addition of 1: 1 hydrochloric acid (ice cold). The solution is extracted using ethyl acetate. The ethyl acetate layer is washed back with 100 ml of 5 sodium chloride solution. The organic layer is discarded and the aqueous layers together with 500 ml of ethyl acetate are reset to 2.5 and the layers are separated. The aqueous layer is further extracted with 3 x 500 ml of ethyl acetate. Wash the ethyl acetate layer with 100 ml of saturated sodium chloride solution. The extracts are dried over sodium sulfate and the solution is evaporated to a small volume. (Temperature 50 ° C).

The concentrated solution is then dissolved in 200 ml of isopropanol, heated to 40-45 ° 0, and 5.8 ml of acetic acid and 21.6 ml of dioyclohexylalmine are added.

This slurry is cooled slowly upon standing and aged for 18 hours at room temperature. The product is filtered, washed with 100 ml of iso propanol and dried for 18 hours at room temperature under vigorous vacuum.

The product, 7β- (D-5-tosylamino-5 “carboxyvaleramido) -3-earbamoyloxy-methyl-7-methoxy-3-cephem-4-carboxylic acid, dicyclohexylamine salt, is obtained in a yield of 44.5 g; UV: (pH = 7.0 buffer)

Imax. 2620 E # 94.7 Equivalent weight (HClO-titration) 481.5 (theoretical 481.5)

Analysis calculated for 1S2: 0. 58.60 - H 7.74 - N 8.72

Pound * G 58.29 -H 7.29 -N 8.73.

Step B * Dimethoxymethyl ester of 7β- (D-5-tosylamino-5-earboxy- - * valeramido) -3-carbamoyloxymethyl-7-methoxy-3-oephem-4-carboxylic acid --------

The toosyl salt of step A, 20 g, is introduced into a 3-neck flask. Methylene chloride (200 ml) is added and the slurry is cooled to 0 ° in an ice bath under nitrogen. Chloromethyl methyl ether (4.1 ml) in 30 ml of methylene chloride is added to the reaction mixture over 90 minutes under good stirring and ice-cooling. After adding for 1 hour, a solution of collidine (1.58 ml) is introduced into 5 ml of methylene chloride.

After the addition, the mixture is stirred for an additional 2 hours, filtered and the filter cake washed with dry methylene chloride. After extraction with aqueous phosphoric acid, sodium chloride, sodium numbicarbonate and sodium chloride solutions, the filter cake is washed back with methylene chloride. The organic layer is dried, filtered, evaporated to a small volume and crystallized. The product is the dimethoxymethyl ester of 7β- (D-5-tosylamino-5-carboxy-valeramido) -3-carbamoyloxy-methyl-7-methoxy-3-cephem-4-carboxylic acid, 9.6 g (yield 85.5 1 °). Both ultraviolet and thin layer chromatography show only a single component of the product.

grin Π; 3-carbamoyloxymethyl-7-methoxy-7.beta.-thienylacetamido-3-cet> hem-4 - carbox.yls.vre_

To a slurry of 6.9 g of Step B tosylmethoxymethyl ester and 7.5 g of Type 4A powdered molecular sieve (600 mesh, hydrated to 17 $ + 2 $ water) in 85 ml of 1,2-dichloroethane was added 5 ml of distilled 2-thienylacetyl chloride. . The slurry is heated with stirring at 65 ° for 16 hours under a nitrogen atmosphere.

The reaction is controlled using thin layer chromatography. After the specified time, the main component of the reaction mixture consists of the desired intermediate. Methanol (0.8 ml) is then added and the slurry is aged for a further 2 hours. At this point, the 4-methoxymethyl ester of the desired product is the main compound in the slurry.

The ester is hydrolyzed by cooling the solution to ca. 25 ° C, filter and wash with cold methanol, the filtrate and washings are combined and cooled to 0 ° C. A solution at 0 ° 0 of 20.8 ml of concentrated hydrochloric acid and 23.6 ml of methanol is added and the solution is heated to 15 ° C. and stirred at this temperature for 2 hours and 40 minutes. At this point, thin layer chromatographic analysis is performed. The pH of the mixture is adjusted by first introducing, if necessary, gaseous ethylene oxide to adjust the pH of 2 - 2.5, after which the pH is raised to 5-6 by the addition of solid sodium hydroxide. The mixture is filtered and the dichloroethane layer is separated. The cold aqueous layer contains the sodium salt of the product. This layer is purified by column chromatography using an IRA-68 nitrate resin, while the eluent is 0.02 M phosphate buffer, pH = 7.0. The final yield of the product is 1.74 g, exhibiting a single spot by thin layer chromatography. The angle of rotation is 192 ° C. The identity of the product is confirmed by NMR and by chemical analysis. The product is the desired 3- carbamoyloxymethyl-7-methoxy-7β-thienylacetamido-3-cephem-4-carboxylic acid, mp: 165-167 ° C.

The starting material, the sodium salt of 7β- (D-5-amino-5-earboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-eephem-4-carboxylic acid, used in the above example, is prepared as follows:

Preparation of monosodium salt of 7β- (D-5-amino-5-carboxy-valeramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4- __________________________________________________________

Modified fermentation process:

Ir in 1-: Oblique cultures

A lyophilized tube culture of Streptomyces lactamdurans (NRB.Ii 3802) was aseptically opened and the organism transferred to a medium of the following composition:

Medium Kl: 1 io Blackstrap molasses 1 io National Brewer's yeast 2.5 in Difco agar pH = 7.0 Remaining water

The cultures are grown for 7 days at 28 ° C. When stored in the cold, the cultures become stable for more than 13 weeks.

Step 2: Grafting step: lotion system First nodal material: The first graft material is seeded directly from a culture of steps 1 to 40 ml of primary dried yeast NE, pH = 7.0 (derived from the Xeast Product Corporation) in a 250 ml aerated conical flask . The flasks were then shaken at 220 rpm. per minute in a rotary shaker with a stroke of 50 mm at 28 C for 2-3 days.

Second grafting: A 2.5 in grafting material from the first grafting stage was added to a flask containing a 2 in Eleischmann S-150 yeast autolysate, pH = 7.0. The growth at this stage is typically light and the cultivation carried out as indicated in step 1 was not extended beyond 48 hours.

Step 3: Production medium

The production medium contains per. liter of distilled water 30 g distillation residue, 7.5 g primary dry yeast N.F. and 0.25% v / v of an emulsified (Mobilpar-S) petroleum-based anti-foaming agent. The medium is adjusted to a pH of 7.0 with a small amount of concentrated sodium hydroxide solution, transferred into conical flasks and autoclaved for 15-20 minutes at 121 ° C. After cooling, the medium is added 2.5 µ g of seed material formed in the above step. 2. Cultivation may vary between 50 and 100 hours, but preferably 72 hours is used. The volume of the medium in each flask may vary between 30 and 50 ml and the preferred amount is 40 ml. The amount of seed material can vary between 1 and 5 #, but in practice 2.5 frins are usually used 4 · Testing

After fermentation was complete, the cells were removed by centrifugation and the liquid diluted with phosphate buffer, pH = 7.0. The concentration of 7β- (D-5-amino-5-carboxyvaleramido) -3-carbamoyloxy-methyl-7-methoxy-3-cephem-4-oreboxylic acid in the fermentation liquid was determined by the standard biological slice test method. The sample organism was Vibrio percolans (ATCC 8461). Eilter paper slices were immersed in the diluted liquid and placed on the surface of agar-containing Petri dishes grown with the sample organism Vibrio percolans (ATCC 8461). were also placed on these Petri dishes, which were pre-dipped in standard solutions containing known concentrations of 7β- (D-5-amino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid. The slices were grown for 18 hours at 28 ° C, and the zone inhibition diameters were measured. The concentration of the product and the fermentation liquid is calculated by interpolation from standard curves over the ratio of the zone diameter to known concentrations of standard solutions of the product. By this method, it was calculated that Streptomyces lac-tamdurans NRR1-3802 produces 78.6 pg / ml of 7β- (D-5-amino-5-carb-oxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem 4-carboxylic acid by the modified fermentation process.

Irin 5? Isolation

The filtered liquid is adjusted to pH = 7.0 with dilute hydrochloric acid and 2900 ml of it is passed through a column containing a highly basic anion exchange resin (100 g) with a styrene-divinyl-benzene matrix (Dowex 1 x 2 ehloride cycle resin ) at 10 ml / minute. The spent solvent is collected in 500 ml fractions.

The resin column is washed with water and eluted with 3 µm ammonium chloride in 90 µm methanol. The eluate is collected in 100 ml fractions. The activity of the fraction is measured, the most active fractions are combined, the pH is adjusted to 7.2 - 8.0 with dilute sodium hydroxide and adsorbed on a strong basic anion exchange resin (100 g) with a styrene-divinylbenzene matrix (Dov / ex 1x2 chloride cycle resin) at 14 ml / min. The column is washed with water and eluted with 5 $ aqueous sodium chloride. The concentrate is diluted to 500 ml, adjusted from pH = 8.8 to pH = 2.0 with dilute hydrochloric acid, and adsorbed on 25 ml of strongly acidic cation exchange resin of the sulfonate type with a styrene-end vinyl benzene matrix (Dowex 50 x 2 hydrogen cycle resin) at 2.5 ml / minute. The column is washed with 25 ml of water, then eluted with 2 $ pyridine until the pH of the drain from the column increases to 7.0 (54 ml). The eluate thus formed is adjusted to pH = 8.0 with dilute sodium hydroxide and evaporated in vacuo to remove the pyridine and to form the monosodium salt of 7β- (ϋ-5-amino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3 -cephem-4-carboxylic acid.

Elemental analysis for C-j g ^^ N N N₂SO Na Na:

Calculated: 0 41> 0 - H 4.5 - N 12.0 - S 6.8

Found: C 39> 31- H 4 »76 - N 11.16 - S 6.46.

EXAMPLE 2 3-Carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3 "-cephem-4-carboxylic acid ________________________________________________________

Step A: Limethoxymethyl ester of 7β - [(D-5-tosylamino-5-oreboxyvaleryl) phenylacetamido] -3-carbamoyloxymethyl-7-methoxy-3-cenhem-4-carboxylic acid ......

A solution of the dimethoxymethyl ester of 7β- (L-5-tosylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid (9.3 g, 10 mmol), type 12A powdered molecular sieve (hydrated to 20 fo + 2 <fo water) (10.8 g) and phenylacetyl chloride (5.3 ml, 40 τητηηΐ) in 50 ml of acetonitrile are heated to 40 ° C for 20 hours. After this period, the mixture is cooled to room temperature and filtered. The filtrate is evaporated to dryness and triturated with hexane. Insoluble residue containing the dimethoxymethyl ester of 7β - [(D-5-tosylamino-5-carboxyvaleryl) in> henylacetylamino] -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid is used without purification in the next step.

Step B: 3-carbamoylo: Kymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylic acid

The crude product from step A is dissolved in 50 ml of 1,2-dichloroethane.

1.0 ml of methanol is added and the solution is stirred for 1 hour. The methoxymethyl ester is hydrolyzed by adding at 0 ° C a solution of 20 ml of concentrated hydrochloric acid in 25 ml of methanol and stirring at 15 ° C for 3 hours. The product is isolated and purified by the same general procedure as in Example 1. There is thus obtained 3-carbamoyl-oxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylic acid, mp: 159-161 ° C, and UV and SMR spectra corresponding to the structure indicated.

EXAMPLE 3 3-Carbamoyloxymethyl-7-methoxy-7β- (2-furylacetamido) -3-cephem-4-carboxylic acid

The dimethoxymethyl ester of 7β- (D-5-tosylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid is reacted with 2-furyl acetyl chloride in the presence of 12 g of hydrated type 4A molecular sieve (hydrated to 1 ° + 2 $ water), followed by the procedure just described. The side chain and the ester blocking group are also removed by the procedure described. The product formed is 3-carbamoyloxymethyl-7-methoxy-7β- (2-furylacetamido) -3-cephem-4-carboxylic acid, mp: 156-161 ° C, UV (pH = 7.0 buffer). 265 pm. & 7200 and with IR and NMR similar to structure.

Similarly, the product is prepared 3-carbamoyloxymethyl-7-methoxy-7β-thiophenoxyacetamido-3-cephem-4-carboxylic acid, using phenylthioacetyl chloride in place of 2-furylacetyl chloride. The product has a melting point of 119-123 ° C, UV (pH = 7.0 buffer), Xmax. 247 um. £ 10400 and an MRI spectrum similar to the structure.

EXAMPLE 4 4-ace ΐοχγηθΐ ^ 2ΐ-7β- £ 2-ΐ1ιΐθηγΐ8θθΪ8, ω.ϊάο2-3-οθ £ ^ θΜ .-- 4-θΒΓ ^ ο ^ ΐ82Γθ grin Αϊ 7β- (ΐ> -5-ΐΐϊο1ι1θΓ ? 1αΐ] ΐίηο-5-οαΓΐ3θΧ3Γνα1βΓΒΐΕΐάο) - 5-Acetylmethyl-3-ee-phem-4-earboxylic acid to a solution of 7β- (D-5-amino-5-carboxyvaleramido) -3-acetoxy methyl 3-cephem-4-carboxylic acid (2.5 g, 0.53 mol) in acetone (13 ml) and aqueous 10 $ dichloromethane phosphate (40 ml) are added trichloroethoxycarbonyl chloride (3.35 g, 0.159 mol). During the addition, the pH value of the solution is maintained at 8.5 - 9.0 by the gradual addition of a 17 $ 6 aqueous solution of sodium hydroxide. After 30 minutes, the mixture is washed with ethyl acetate and the aqueous layer acidified to pH = 2.5 with concentrated hydrochloric acid. One precipitated product is extracted into ethyl acetate, the solution is dried over sodium sulfate, filtered and the solvent is evaporated to give 2.7 g of 7β- (5-5-trichloroethoxycarbonylamino-5-carboxyvaleramido) -3-acetylmethyl-3-cephem-4-carboxylic acid.

Step Bs Dihenzhydryl ester of 7- (D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido) -3-acetoxymethyl-3-cephem-4-carboxylic acid

To a solution of 7β- (D-5-trichloroethoxyearbonylamino-5-carboxy-valeramido) -3-ethylmethyl-3-cephem-4-carboxylic acid in ethyl acetate (30 ml) is added diphenyldiazomethane (2.0 g) in ether (25 g). mL). The mixture is stirred for 18 hours and the solvent is evaporated to give 4.0 g of crude product. The crude product is purified by chromatography on silica gel using chloroform as the eluent to give 2.3 g of the substantially pure dihenzhydryl ester of 7- (D-5-trichloroethoxycarhonylamino-5-carhoxyvaleramido) -3-acetate. oxymethyl-3-cephem-4-oarboxylsyre.

NMR: - (Solvent - CDCl3) δ = 2.0 (methyl, s), 4.9 (10-H2, quartet), 3.2 (2-H2, quartet), 4.95 (6-H, d), 5.92 (7-H), 7.0 (benzhydryl protons, 2 s).

Step C: Dihenzhydryl ester of 7 - [(D-5-trichloroethoxycarbonylamino-5-carb oxyvaleryl) -2-t-enylacetylamina] -3-acetoxymethyl 1-3-cenhem-4-carboxylic acid

A mixture of the dibenzhydryl ester of 7β- (Ε-5- ^ ίι11ιιι-vinylamino-5-carboxyvaleramido) -3-acetoxymethyl-3-cephem-4-carboxylic acid (2.0 g, 0.02 11.0 g of type 5A molecular sieve (hydrated to 23 io + 2 µl - thienylacetyl chloride (1.31 g, 0.0815 mol) and methylene chloride (6 ml) are heated to 40 - 45 ° C on an oil bath under The reaction mixture is poured into hexane (100 ml) and filtered. Evaporation of the solvent gives the dibenzhydryl ester of 7 - [(M-trichloroethoxycarbonylamino-5-carbo: nyvaleryl) -2-thienylacetylamino] -3-acetoxymethyl-3- cephem-4-carboxylic acid.

Step D: Benzhydryl ester of 3-acetoxymethyl-7- (2-thienylacetamido) - 5-cephem-4-carboxylic acid

The dibenzhydryl ester of 7 - [(DS-trichloroethoxycarbonylamino-5-carboxyvalerylM-thienylacetylamino] -acetoxymethyl 3 -cephem 3 - carboxylic acid is dissolved in ethyl acetate (10 ml) and added to a mixture of 90 io aqueous acetic acid (10 ml), The reaction mixture is filtered to remove zinc The reaction mixture is washed successively with two portions of water, a cold sodium bicarbonate solution and then with saturated sodium chloride solution (15.0 ml). The ethyl acetate solution is dried over sodium sulfate, filtered and the solution evaporated to give 1.9 g of crude product which is chromatographed over silica gel using a mixture of chloroform and ethyl acetate (50: 1) as eluent to give 0.380 g of product which, after recrystallization from ethyl acetate, has a melting point of 141.5 - 143 ° 0.

UV: (CH 2 OH) \ max. 263. £ 7580.

Elemental analysis for C29H26N2 ° 6S2:

Calculated: C, 61.91 - H4.6, - N4.98

Bound: C, 62.14 - H, 4.84, N, 4.91.

Step E: 3- (Acetoxymethyl) -7- (2-thienylacetamido) -3-cephem-4-carboxylic acid

A cold solution of benzhydryl ester of 3-acetoxymethyl-7- (2-thienylacetamido) -3-cephem-4-carboxylic acid (100 mg) in anisole (1.0 ml) and trifluoroacetic acid (0.5 ml) is stirred at 0 ° C. for 35 minutes. Carbon tetrachloride (50 ml) is added and the reaction mixture is evaporated to dryness. The residue is triturated with hexane.

The hexane is removed by decantation and the residue is dissolved in 10 ml of ethyl acetate, evaporated to 1 ml and diethyl ethers are added to form a precipitate. This precipitate is recrystallized from a mixture of diethyl ether and ethyl acetate to give 0.025 g of 5- (ac ethoxymethyl) -7- (2-thienylacetamido) -3-cephem-4-carboxylic acid, mp: 164 ° C. authentic sample was 163 ° C.

EXAMPLE 5

Sodium 7- (2-thienylacetamido) -7-methoxy-3-carbamoyloxymethyl-3-cephem-4-carboxylate ___________________________________________

Irin A: 7β- (D-5-benzoylamino-5-carboxyvaleramido) -3-carbamoyloxy-methyl-7-methoxy-3-cenhem-4-carboxylic acid, disodium salt

To 500 ml of an aqueous solution containing 48.5 mmol of monosodium 7β- (D-5-amino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7 “methoxy-3-cephem-4-carboxylate is added as much as 50 $. sodium hydroxide that the pH becomes 9 * 5. To this solution is added 15 ml (128 mmol) of benzoyl chloride with vigorous stirring. The pH is maintained at 9.5 for 30 minutes with the addition of sodium hydroxide if needed.

The pH of the solution is then adjusted to 4.0 with concentrated hydrochloric acid and washed twice with ethyl acetate.

The aqueous fraction is cooled to 0 ° C and 200 ml of isopropanol and 300 ml of ethyl acetate are added with stirring. The pH of the solution is adjusted to 2.0 with hydrochloric acid. The organic fraction is discarded and the aqueous fraction is re-extracted three times with ethyl acetate.

The combined extracts are washed with sodium chloride solution, dried over sodium sulfate and evaporated in vacuo to give 43.0 g of a dark oil.

This oil is dissolved in 200 ml of ethanol and a solution of 30 g of 2-ethylhexanoic acid sodium salt is added. The slurry is cooled to 0 ° C, filtered, washed with ethanol and dried in vacuo to give 28.8 g of [102 $] disodium 7P- (D-5-benzoylamino-5 "Carboxyvaleramido) -3-carbamoyloxymethyl -7-methoxy-3-cephem-4-carboxylate, which was 67 56 pure by chromatography compared to a pure standard.

Step B: Dimethoxymethyl ester of 7β- (1-benzoylamine-5-carboxy-valeramido) -3-carbamoyloxymethyl-7-methoxy-3-eephem-4-carboxylic acid

To a slurry of 20 g disodium 7P- (B-5-benzoylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl-17-methoxy-3 16 ml of 6 M chlorine thylmethy le ther in 9 minutes. After an addition time of one hour, 6 ml of S-collidine is added. The slurry is stirred for an additional two hours at 0 ° C. The mixture is then diluted with 500 ml of methylene chloride and washed twice with dilute phosphoric acid, once with dilute sodium bicarbonate solution and once with 5 ° Jo sodium chloride. The aqueous fractions are washed back with 50 ml of methylene chloride. The organic phase is dried over sodium sulfate and evaporated in vacuo to ca. 10Θ ml.

The solution is passed through 200 ml of silica gel G, washed with 200 ml of methylene chloride, then eluted with 800 ml of ethyl acetate. The ethyl acetate eluates are evaporated in vacuo to give 18.5 g of an SU1 oil.

The crude substance is recrystallized from 50 ml of ethyl acetate to give 10.0 g of [67 µl dimethoxymethyl ester of 7β- (D-5-benzoylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carb - oxylic acid.

Step 0: Sodium 7- (2-thienylacetamido) -7-methoxy-3-carbamoyloxymethyl-3-cephem-4-cafboxylate

A slurry of 192 mg (0.3 mmol) of dimethoxymethyl ester. of 7β- (p-5-thienzoylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid, 225 mg powdered lime type 4A molecular sieve with 10 fo + 2 fo water, 0.3 ml 2-thienylacetylchlo -Rid and 4 ml of dichloroethane are boiled with vigorous stirring:! 4 hours. The mixture is cooled to 65 ° C and 10 ml of 0.05 M t -butanoyl dichloroethane is added over two hours, then heated for an additional hour at 65 ° C. The reaction mixture is cooled to 0 ° C, filtered and washed with 5 µL methanol, the filtrate is cooled to 0 ° 0 with stirring, then 1.4 ml of a 1 ml

hydrochloric acid: methanol and the resulting mixture is stirred at ca. 15 ° C

1 3 hours. The mixture is poured into 10 ml of water containing 1.6 g of sodium bicarbonate. Bone organic phase is discarded. Aqueous fractions contained a 65-fold yield of the product, sodium 7- (2-thienylacetamido) -7-methoxy-3-carbamoyloxymethyl-3-cephem-4-carboxylate.

Irin_B: By analogy, p-chlorobenzoyl chloride, p-nitrobenzoyl chloride or toluoyl chloride is used in step A. The final yield of the desired product, sodium 7- (2-thienylacetamido) -7-methoxy-3-carbamoyl oxymethyl-3-cephem -4-carboxylate, in each case are 70 fo, 68 fo and 72 fo respectively.

EXAMPLE 6

Sodium 7- (2-thienylacetamido) -7-methoxy-3-carbamoyloxymethyl-3-c ephem-4-carboxy;

A mixture of 2.76 g (4 mmol) of dimethoxymethyl ester of 7β- (Β-5-tosylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7α-methoxy-3-cephem-4-carboxylate, 3 g of dry Linde type 4A molecular sieve with less than 2 fo water, 2 ml of thienylacetyl chloride (16 mmol) in 34 ml of dichloroethane is stirred at reflux for 5 hours. Add tertiary 0.38 ml (4 mmol) of t-butanol and stirring is continued for 2 hours. After this time, an additional 0.095 ml (1 mmol) of t-butanol is introduced and the reaction mixture is stirred at reflux for an additional half hour. The reaction mixture is cooled to 0-5 C in an ice-water bath. The molecular sieves are removed by filtration on suction filter and then washed with 40 ml of ice-cold methanol. The filtrate and washings are combined and cooled to 0 ° C. An ice-cold solution of 8.3 ml of concentrated hydrochloric acid and 9.5 ml of methanol is added and the solution is heated to 15 ° C and stirred at 15 ° C for 2 hours and 40 minutes. After the hydrolysis is complete, the reaction is stopped by adding a suspension of 22 g of sodium bicarbonate in 120 ml of water at 0-5 ° C. The two-phase solution is stirred for 10 minutes. Bet formed precipitate of a heavy salt is filtered off and washed with a small amount of 5 µl sodium chloride solution containing 0.5 µl sodium bicarbonate. The bichloroethane layer is separated and extracted with 2 x 20 ml of a solution of 0.5 µl sodium hydrogen carbonate plus 5 µl sodium chloride. The aqueous fractions are combined and washed with 20 ml of dichloroethane. The bicarbonate solution was found by liquid chromatography to contain 73 $ of sodium 7- (2-thienylacetamido) -7-methoxy-3-carbamoyloxymethyl-3-cephem-4-carb oxylate and 2.196 unchanged starting material.

EXAMPLES 7-20

Sodium 7- (2-thienylacetamido) -7a-methoxy-3-carbamoyloxymethyl-3 -__ cephem-4-carho ^ lat ____________________________________________

The procedure of Example 6 is repeated with 4 mmol (2.8 g) of dimethoxymethyl ester of 7β- (D-5-tosylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7α-methoxy-3-cephem-4-carboxylate> reacting with the amounts of reagent given in the following table and under the reaction conditions stated therein. In each case, the total reaction time is approx. 16 hours. The molecular sieves used are in each case the Linde type 4A, which is heated to 700 ° C prior to the reaction, exhibiting a weight loss of approx. 3 $ on drying. The sieves are intended to be substantially anhydrous or to have less than 2 µl water content by weight. In each case, the reaction temperature is 67 ° 0.

a) Relate between batch addition of imide-splitting reagents *

Id) Addition after 3 hours of cleavage with isopropanol, continuing the reaction for a further 16 hours before the final imide cleavage.

c) An additional 1.5 g of sieve was added after the end of the transacylation period and prior to the addition of alcohol.

d) The transacylation period was 1.5 hours.

e) An additional 1 g of sieve was added after completion of the transacylation.

EXAMPLES 21-25

The same general procedure as used in Examples 6-20 is used with the change that the reaction temperature is as given in the following table. The reaction mixture consists of 4 mmol (2.8 g) of the dimethyl oxymethyl ester of 7β- (D-5-tosylamino-5-carboxyvaleramido) -3-carlaminoloxymethyl] 7-methoxy-5-cep] im-4 carboxylate, 3 g of dried Linde type 4A sieves (less than 2 $ water by weight) and 16 mmol of phenylacetyl chloride in 34 ml of dichloroethane.

The final product is in all cases sodium 7- (2-thienylacetamido) - 7α-methoxy-3-carbamoyloxymethyl-3-cephem-4-carboxylate.

EXAMPLE 26 7- (2-Thienylacetamido) -7-methoxy-3-carbamoyloxymethyl-3-cephem-4-______________________________________________________________

One gram of sodium 7β- (L-5-amino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylate is introduced into a reaction vessel containing 20 ml of diclilorethane. To this is added 1 g of a Linde molecular sieve type 4A in powder form with 15 1 <> water and 8 mmol (1 ml) of thienyl acetyl chloride. The slurry is heated under reflux for 8 hours. After filtration, the molecular sieve is washed with methanol. The methanol wash liquid from the sieves is analyzed and contains a yield of 25 l <> of the desired product, 7-thienylaeetamido-7a-methoxy-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid, as determined by thin layer chromatography and liquid chromatography. The lichloroethane layer of the reaction mixture is extracted with 5 L of sodium hydrogen carbonate and found to contain, by thin layer chromatography and liquid chromatography, an additional $ 5 of the desired product. Thin layer chromatography of the organic layer at this time shows the presence of the mixed anhydrides. After removal of the solvent by evaporation, the mixed anhydride is hydrolyzed in 50 acetone water (50 ml) in the presence of 10 mole percent pyridine. Yed thin layer chromatography and liquid chromatography show the formation of an additional $ 13 of the final product as free acid. Slight total yield is 156 mg of the acid, 23 in ° yield.

Claims (1)

Patent claim: Process for the preparation of 7-acylamido-cephalosporin compounds of the general formula: wherein R 1 is hydrogen or methoxy; A 'is carbamoyloxy or alkanoyloxy of 2-6 carbon atoms, R3 is phenyl, 2-thienyl, 3-thiehyl, 2-furyl or 3-furyl, and M is hydrogen or an alkali metal. with the formula wherein B is blocked aminoadipoyl or an acyl group other than the desired substituent in the final product, and R "is a conventional blocking group, reacted with an acylating agent containing the group R3CH2CO- where R3 is as defined above, in the presence of a natural or synthetic zeolite having a pore size of from 3 to 15 Å, whereupon the B substituent and the blocking group R "are cleaved to form the final product, optionally transferring any free acid, if desired, into a corresponding alkali metal salt.