DE2456528A1 - METHOD FOR PRODUCING ANTIBIOTICA - Google Patents

Description

The invention relates to an improvement on that disclosed in the United States patent application Serial No. 149 364 of June 2, 1971 described acylation process for the preparation of 7-acylamidocephalosporins.

A process for the preparation of 7-acylamidocephalosporins, which are used in medicine as antibiotics consists in producing the analogue 7-aminocephalosporin and then acylated to the desired product. However, this method has the disadvantage that the 7-aminocephalosporin is first used must isolate and purify as an intermediate product. One has therefore looked for other methods in which there is no need to produce 7-aminocephalosporanic acid.

More recently it has been found that cephalosporins with a Methoxy substituents instead of hydrogen in the place

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lung C-7 can be produced by various microorganisms. These cephalosporins contain an aminoadipoyl side chain, which is preferably split off, new 7 <x-methoxycephalosporins of increased antibiotic activity.

It has now been found that cephalosporin compounds are as follows can be converted into each other:

H? L ¹ N. /

-N

CH "0CNH _o B '

CH ₂ OCNH ₂

COOR "

COOR "

II

O CH ₂ OCNH ₂

COOR "

III

In the above reaction scheme, B ¹ and R ¹ denote different acyl groups, R ₁ denotes hydrogen or a substituent such as methoxy, and R ″ denotes hydrogen or an easily cleavable protective group.

In the Pliess diagram above, the cephalosporin compound I with an acylating agent in the presence of a molecular sieve as a catalyst for the 7-diacylimidocephalosporin compound (II), which is then cleaved to form the new T-acylamidocephalosporin compound (III).

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The diacylated product is made "best." by intimately mixing the cephalosporin compound with one Acylating agent in a suitable solvent in the presence of the molecular sieve. The temperature at which this reaction occurs is not particularly critical; Temperatures from about -20 to + 100 ° C. are generally satisfactory; however, the reaction is preferably carried out “at temperatures of about 50 to 90.degree. C.”. Solvent, which are suitable for carrying out this reaction are those which do not contain any reactive hydrogen, such as chloroform, acetonitrile, methylene chloride, dioxane, benzene, halobenzenes, carbon tetrachloride, diethyl ether and the like.

As the acylating agent, an acyl halide, an anhydride or a mixed anhydride can be used; generally works however, it is preferred to use an acyl halide, e.g., an acyl chloride, as the acylating agent.

The molecular sieves that can be used for the purposes of the invention are aluminosilicate zeolites. The zeolites occurring in the door can be referred to as a group of crystalline, solid, hydrated aluminosilicates of monovalent and divalent bases which can lose their water of hydration in whole or in part without changing the crystal structure , can adsorb other compounds instead of the separated water and are able to undergo a base exchange. A synthetic zeolite, on the other hand, is synthesized from a combination of basic oxides (A102> ^s ^ ° 2 '^ a ₂ O, K ₂ 0', ^etc. ) in an aqueous system. This results in hydrated or semi-hydrated crystalline structures. ] times the heat treatment, the zeolites can be regarded as essentially anhydrous. Synthetic zeolites are mainly characterized and classified by X-ray diffraction on powders. Although there is no systematic chemical method for designating the synthetic, composite

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plexen aluminosilicates; however, it belongs to the tradition, every new synthetic zeolite with an arbitrarily chosen one To denote letters or a group of letters and numbers. The importance of this arbitrarily chosen Symbols are well known to those skilled in the art.

It has been found that Class A synthetic zeolites and X for use in the acylation procedure described above are particularly advantageous. The pore size of these zeolites can be in the range from about 3 to 15 α. The zeolites can be essentially anhydrous or contain some water of hydration. The amount of water that the zeolites can contain, can be 0 to 30 percent by weight.

The substituent at position No. 3 in the above compounds I, II and III is the group.

H
-CHpOCNHp. The transacylation reaction can be carried out just as well if one starts out from compounds in which position no. 3 can be represented by -CHgA, where A is hydrogen or other known substituents. When A is a hydroxyl group, the general formula also includes the lactone which forms with the 4-position carboxyl group, and when A is an amino group, the formula also includes the lactam which forms with the 4-position carboxyl group. The substituent A can also be azido, halogen, cyano, alkoxy, aryloxy, aralkyloxy, heterocycloxy, mercapto, alkylthio, arylthio, aralkylthio, heterocyclothio, amino, alkylamino, alkanoylamino, hydroxyphenyl, acylthio, acyloxy, sulfaraoyloxy and the like. The heterocyclic groups can be 5- or 6-membered heterocyclic rings which can contain one or more nitrogen, oxygen or sulfur atoms, such as (1-methyl-1,2,3,4-tetrazolyl). The acyl group can be a lower alkanoyl group with 2 to 6 carbon atoms, a thiocarbamoyloxy group or an N-alkyl derivative or Ν, Ν- ^ dialkyl derivative of a carbamoyloxy

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or thiocarbamoyloxy group. The alkyl group of the above Substituents contains 1 to 6 carbon atoms and can in turn be replaced by radicals such as alkoxy, halogen, amino, cyano, Carboxy, sulfo and the like.

The acyl substituents B ^f and R 'in the general formulas I, II and III are preferably carboxylic acid residues. When the "compounds are made from certain microorganisms such as S. clavuligerus, S. lipmanii, or S. lactamdurans, B ^{f is} the aminoadipoyl residue. B 'can also be any of the other acyl groups conventionally used in cephalosporin chemistry. B 'can be replaced by any commonly used R' group. Both B ^f and R 'can be represented by the general formula R ₁₁ R ₁₀ CHCO, in which R ₁₀ and R _{11 have} the following meanings and because of their antibiotic activity R ₁₀ is hydrogen, halogen, amino, guanidino, phosphono, hydroxy, tetrazolyl, carboxy, sulfo or sulfamino. R ₁₁ denotes phenyl, substituted phenyl, a monocyclic heterocyclic 5- or - . - 6-membered ring, the contains one or more oxygen, sulfur or nitrogen atoms in the ring, a substituted heterocyclic radical, phenylthio, a heterocyclic or substituted he terocyclic thio group or cyano. The substituents can be halogen, (egg arboxymethyl, guanidino, guanidinomethyl, carboxaminomethyl, aminomethyl, nitro, methoxy or methyl. Examples of preferred acyl groups B ¹ or R ¹ are phenacetyl, 3-bromophenylacetyl, p-aminomethylphenylacetyl, 4-carboxymethylphenylacetyl, 4-carboxymethylphenylacetyl, 4-carboxamidomethyl , 2-purylacetyl, 5-nitrofurylacetyl, 3-furylacetyl, 2-thienylaeetyl, 5-chlorothienylacetyl, 5-methoxythienylacetyl, a-guanidino-2-thienylacetyl, 3-thienyl-. Acetyl, 4-methylthienylacetyl, 3-isothiazetyl, acetoly 4-methoxyisothiazolylacetyl, 4-isothiazolylacetyl, 3-methylisothiazolylacetyl, 5-isothiazolylacetyl, 3-chloroisothiazolylacetyl, 3-methyl-1,2,5-oxadiazolylacetyl, 1,2,5-thiadiazolyl

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4-acetyl, 3-methyl-1 ^^ - thiadiazolyl - ^ - acetyl, 3-chloro-1,2,5-thiadiazolyl-4-acetyl, 3-methoxy-1,2,5-thiadiazolyl-4-acetyl, phenylthioacetyl, 4-pyridylthioacetyl, cyanoacetyl, tetrazolylacetyl, oc-ITu.orphehyl acetyl, D-phenylglycyl, 3-hydroxy-D-phenylglycyl, 2-thienylglycyl, 3-thienylglycyl, phenylmalonyl, 3-chlorophenylmalonyl, 2-thienylmalonyl, 3-thienylmalonyl, a-phosphonophenylacetyl, a-sulfaminophenylacetyl, oc-hydroxyphenylacetyl, oc-tetrazolylphenylacetyl and α-sulfophenylacetyl.

The reaction .1 -> - II shown schematically above is an equilibrium reaction. In order to increase the yield of the desired end product III, the aylating agent (which contains the group R ') is used in excess. The diacyl compound II formed as an intermediate product has to be cleaved in order to remove the group B 'and the end product. III to get. This split can take place in a number of ways. First, if the reaction time is simply increased, spontaneous cleavage takes place (in the presence of a molecular shot of the R * halide, which acts as an acylating agent). Second, when the molecular sieve contains water, that water acts as a "cleaving agent" and the acylamido product is recovered in high yield. Both of these methods can be termed "passive" in the sense that there is no need to add a special "cleaving agent" to the reaction mixture .

A third method of cleavage is the addition of benzyl alcohol, an alkanol or a lower alkyl thiol with 1 to 6 carbon atoms. According to a fourth method, hydrochloric acid can be added as a splitting agent.

According to a preferred embodiment of the invention, leave cephalosporins, such as those obtained by fermentation with various species of Streptomyces, convert into derivatives that instead of the aminoadipoyl

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group have another acyl group without first splitting off this group and the resulting intermediate. Then acylate the 7-amino compound again. The general procedure is as shown in the following flow diagram:

COOH (CH (CH ₂ ) ^ CN. NH "

COOR "O CH (CH ₂ ) ₃ CN NHR"

VIII

X) OR "

VI

In the general formulas in the flow diagram above, R ₁ denotes hydrogen or methoxy, A denotes hydrogen or a substituent which is not influenced in the reactions described or which can be converted back into the original substituent by splitting off a protecting group, the protecting group preferably being a Is acetoxy or carbamoyloxy, R ¹ is an acyl group according to De-

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finition, R "is hydrogen or a protective substituent and Rp is a protective substituent, the two protective substituents can easily be split off by known methods.

According to the flow diagram above, the cephalosporin compound IV or the derivative thereof in which the amino substituent and / or the carboxyl group is optionally protected are (V), in the presence of the molecular sieve with an acylating agent to the diacylated intermediate (VI or VII) implemented. The aminoadipoyl radical of the latter is then selectively cleaved off, so that the new acylated Cephalosporin compound (VIII) or, if R "is hydrogen, a salt thereof is formed.

Although the invention can be practiced without the amino and carboxyl groups of the starting cephalosporin compound (VII) to protect, the process is preferably carried out in general so that first the amino, group and the carboxyl group protects, because in this case you get the highest yields of the desired new Cephalosporin compounds obtained.

To explain this preferred method according to the invention serve the following flow chart:

NH O "

I ^ Il "

CH (CH _n ) -CN COOH

COOH

IX

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^R 2

NH

CH _(CH2) 3C N j

COOH

⁰ H

COOH

NH O

CH (CH ₂ ) ₃ CN-j COXR,

XI

^R 2 O

NH j _R ,

CH (CH ₂ J ₃ CN-COXR ₄

-N. ^ L-CH ₂ A ¹

COXR,

XII

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R, H • 1>

η:; .s

R'N-ι Y

COXR

XIII

R ₁ H

X ι

• Ν -. Y

, COOH

XIV

In the above flow diagram, R is hydrogen or methoxy, A ^{f is} carbamoyloxy or acetoxy and R _{2 is} a protecting group. The radical COXR means a protected carboxyl or thiocarboxyl group (X = O or S; R. = protecting group), and R 'means an acyl group.

According to this method, the amino group of the Ausgangscephalosporinverbindung (IX) is first prepared by reacting with a suitable reactant which protects the 5'-amino blocked (R ₂₎ * I ⁿ this sense, the amino-protecting by amino groups, such as acyl, aroyl , Alkoxycarbonyl, alkylsulfonyl, arylsulfonyl and the like, blocked by known methods. Special groups that are suitable for protecting the amino group are trichloroethoxycarbonyl, tert-butoxycarbonyl, benzoylmethoxycarbonyl, trimethylsilyl, p-merhoxybenzyloxy, 2-nitrophenylsulfenyl, 2,4-dinitrophenylsulfenyl, chloroacetyl, p-nitrophenylthio,

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p-nitrobenzenesulfonyl, p-toluenesulfonyl, methanesulfonyl, Benzoyl, p-chlorobenzoyl, p-nitrobenzoyl, toluoyl, and the like; preferably one uses in general the p-toluenesulfonyl- or the benzoyl derivative, which is conveniently prepared by adding the gephalosporin compound with lets p-toluenesulfonyl chloride or benzoyl chloride react, keeping the pH of the mixture on the alkaline side, i.e. between 9 and 10. '"

In general, it is preferred to carry out the reactions described above with a gephalosporin compound in which the Carboxyl groups of the aminoadipoyl side chain and in position no. 4 are also protected (XI), because one with such Derivatives get the best yields. While the protecting group from the carboxyl group on the aminoadipoyl side chain does not necessarily need to be specially cleaved off because the side chain is cleaved off anyway the protective group in position no. 4 is preferably one that can be easily split off, to get the free acid without splitting the ß-lactam group, since the cephalosporin compounds usually in Form of salts such as the alkali salt or an amine salt can be used. Suitable protecting groups for this purpose are known. Examples of suitable derivatives are esters of alcohols, phenols, mercaptans and thiophenols, where the group -COXR, which represents esters. In this general -Formula, X means oxygen or sulfur and R, the remainder an alcohol or a thiol such as methyl, ethyl, tert-butyl, a substituted alkyl group such as phthalimidomethyl, Succinimidomethyl, phenacyl, a substituted phenacyl group, e.g. p-bromophenacyl, a β-substituted one Ethyl group, such as 2,2,2-trichloroethyl, 2-methylthioethyl, 2- (p-Me thylphenyl) ethyl, 2- (p-Me thylphenyl) sulfonylethyl, 2-methylaminoethyl, 2-chloro (or bromine) ethyl, benzyl, a substituted benzyl group such as p-nitrobenzyl, p-methoxybenzyl, 3,5-dinitrobenzyl, 2,4,6-trimethylbenzyl, 3,5-diehlor-4-hydroxybenzyl . and the like, a benzhydryl group

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or a substituted benzhydryl group, such as p-methoxybenzhydryl, an acyloxyalkyl group such as acetoxymethyl, pivaloyloxymethyl, an alkoxy group such as methoxymethyl, or a monocyclic aryl group such as phenyl or substituted phenyl such as p-nitrophenyl or 3,5-dinitrophenyl. The for Protection of the carboxyl group the most advantageous group is the methoxymethyl group, in which case X is oxygen. These protective groups for the carboxy substituents can be easily removed produce according to known methods.

Then the protected cephalosporin compound with an acylating agent in the presence of the molecular sieve to the Imide or diacylated product (XII) implemented. As an acylating agent you can use an acid halide (chloride or bromide) or a functional equivalent of such as an acid anhydride, a mercaptide, a mixed acid anhydride with another carboxylic acid, use an activated ester of the carboxylic acid such as the p-nitrophenyl ester, and the like.

The acylating agents preferred in the process according to the invention are those of carboxylic acids. The preferred acyl groups R ^f in the above flow diagram are those of the general formula

IU-CH-C
2 ι

X '

in which X is hydrogen, halogen, amino, azido, guanidino, phosphono, Hydroxy, tetrazolyl, carboxy, sulfo or sulfamino, IU phenyl, substituted phenyl, a monocyclic one heterocyclic 5- or 6-membered ring, the one or more Contains oxygen, sulfur or nitrogen atoms in the ring, a substituted heterocyclic group, phenylthio, Phenyloxy, a heterocyclic or substituted heterocyclic thio group., Lower alkyl (with 1 to 6 carbon atoms) or cyano and the substituents of

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G group IU halogen, carboxymethyl, guanidino, guanidinomethyl, Carboxamidomethyl, aminomethyl, nitro, methoxy or methyl could be.

If the acylating agent has groups such as the amino or Carboxyl group, these groups can be protected during the acylation reaction and the protecting groups later be split off by known methods. Otherwise, the acylating agent may have a substituent such as an azido group which can later be reduced to an amino substituent by known methods.

Particularly preferred acylating agents are those having an acetyl group or a substituted acetyl group, such as Phenylaeetyl, Ihieny! acetyl (2- and 3-thienylacetyl), Purylacetyl, (2- and 3-Furylaeetyl), a-Hydroxyphenylacetyl, Phenoxyacetyl, a-formyl.oxyphenylacetyl, 1-tetrazolylacetyl, α-aminophenylacetyl, phenylthioacetyl, α-azidophenylacetyl and others such as the acylated cephalosporin compounds thus obtained have increased antibiotic activity.

The acylating agent is used in a molecular excess over the starting cephalosporin; preferably works one with twice to 6 times the amount of acylating agent, based on the cephalosporin.

The most diverse ones can be used as molecular sieves in the trade Use available substances. A synthetic zeolite with a regular crystal structure is preferably used and even pore size. The most easily available molecular sieves, namely types JA »4A, 5A. and 13X, are all useful in the method according to the invention. These molecular sieves have the following properties:

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Type formula - Pore diameter 3A K Na ₃ E (AlO _{2 I 1 2} (SiO ₂ )] · 27Η ₂ Ο 3Α 4A Na ₁₂ [(AlO ₂ I ₁₂ (SiO ₂ )] · 27Η ₂ Ο 4Λ 5A Ca ₄₅ Na ₃ KAlO ₂ ) ₁₂ ] · 30Η ₂ Ο 5Α 13X Na ₈₆ [(AlO ₂ I ₈₆ £ XUd- 0 XU ₇ O ■ 1OA

The molecular sieves are available in an essentially anhydrous form; they can be used in this form or dehydrated prior to use by heating to high temperatures (about 500 ° O or above) to a water content of O - 2 fo , or they can also be used when they are up to about 30 fo water of hydration (to Weight basis) included. The hydrated molecular sieves are produced by allowing the original molecular sieve to stand in a chamber or environment of high humidity, or by slurrying the molecular sieve in water and then adjusting the desired moisture content by drying in vacuo or drying at room temperature or at an elevated temperature.

Generally this drying takes about 2 to 5 hours; however, the length of time is not decisive. The water content can be determined according to the well-known Karl Fischer method or determined by other methods.

The conversion of the protected cephalosporin compound (XI) into the imide or diacylated product (XII) is preferably carried out by intimately mixing the cephalosporin compound with the acylating agent in a suitable solvent in the presence of the molecular sieve. The temperature at which this reaction is carried out is not particularly critical; Temperatures of about -20 to +100 C are generally satisfactory. However, since the implementation is temperature dependent seems to be and runs faster at higher temperatures, it is preferably carried out at temperatu-

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ren from about 50 to 90 C. As a solvent for the Reaction mixture are those that do not contain reactive hydrogen, such as chloroform, acetonitrile, Methylene chloride, dioxane, benzene, halobenzenes, carbon tetrachloride, 1,2-dichloroethane and diethyl ether. It is essential to keep the slurry agitated by stirring or "otherwise" during the reaction.

The amount of molecular sieve required for the exchange reaction karm, to be adapted to the working conditions. in the in general, it is preferable to work with approximately the same amounts by weight Starting material and molecular sieve; one can however; also with weight ratios of starting material to molecular sieve work from 1: 0.5 to 1: 2.

As already mentioned, the original acyl group is after split off a number of different methods. In some cases, e.g. when the molecular sieve contains 10 to 30% water, Simply letting the reaction mixture stand is sufficient for a period of 30 minutes to 30 hours. After a shorter "aging" period, you can get an alkanol, a lower one Add alkylthiol or benzyl alcohol. The alkanol or the lower alkylthiol can have 1 to 6 carbon atoms, and preference is given to using methanol, ethanol, isopropanol or tert-butanol. Hydrochloric acid can also be added to bring about the split. The acylation reaction occurs depending on the conditions under which it is carried out becomes, also to some "spontaneous" cleavage of the aminoadipoyl group, because the reaction is an equilibrium reaction. Prolonged heating of the reaction mixture leads also for splitting off the aminoadipoyl group and for BiI-cang of the desired 7-aylated cephalosporin compound, especially when the molecular sieve contains more than 10% water. ·

. ORIGINAL INSPECTED

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The protective group is split off from the carboxyl function according to known methods. For example, the methoxymethyl group is generated using hydrochloric acid at 0 to 10 C, the trichloroethoxycarbonyl group by reaction with zinc and acetic acid and the tert-butoxycarbonyl group and the benzhydryl group split off by reaction with trifluoroacetic acid. Other methods of splitting off are just as easy carry out.

Example 1 '

3-Carbamoyloxymethyl-7-ynethoxy-7ß-thienylacetamido-3-cephem-4-carboxylic acid

Level A: 7ß- (D-5-Tosylaroino-5-carboxyvaleramido) -3-

carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid

45.0 ml of an aqueous solution containing 49.5.mg of the monosodium salt of 7β- (D-5-amino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid per ml are mixed with 450 ml of acetone and 450 ml of water. After hiring the pH of the mixture with 50 percent sodium hydroxide solution to 9.5 to 9.6, 19 g of tosyl chloride is added to 100 ml of acetone partly to. The pH value is kept at 9.5 to 9.6 by the frequent addition of sodium hydroxide solution. After 15 to 20 minutes the pH remains constant and the sulfonylation is allowed to proceed for a total of 1 hour. During the entire reaction time the solution is at temperatures of 20 to 23 ° C.

The solution is then cooled in an ice bath and the pH value is reduced to 7 by adding ice-cold 1: 1 HCl. The solution is extracted with ethyl acetate and the ethyl acetate layer in turn extracted with 100 ml of 5 percent sodium chloride solution. The organic layer is discarded and the aqueous layers are restored along with 500 ml of ethyl acetate adjusted to pH 2.5, whereupon the layers separates. The aqueous layer is then used three more times Extracted 500 ml of ethyl acetate each time. The ethyl acetate layer will

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washed with 100 ml of saturated saline solution. The extracts are dried with sodium sulfate and the solvent evaporated to a small volume (temperature 30 ° C).

The concentrated solution is dissolved in 200 ml of isopropanol heated to 40-45 ° C. and mixed with 5.8 ml of acetic acid and 21.6 ml of dicyclohexylamine offset.

This slurry is allowed to cool slowly and overnight stand at room temperature. The product is filtered off, washed with 100 ml of isopropanol and overnight at room temperature dried under high vacuum.

The product obtained is 44.5 g of dicyclohexylamine salt 7ß- (D-5-tosylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl-

7-methoxy-3-cephem-4-carboxylic acid;

UV: (pH 7.0 buffer). . .

A max. 2620 E # 94.7 ■ \

Equivalent weight (by titration with HClO.) 481.5 (theoretically 481.5). .

Elemental analysis

Calculated for

₇₄ N ₆ O ₁₁ S ₂ : C = 58.60%; H = 7.74 # 5 H = 8.72 #;

found: C = 58.29 ^ j H = 7.29 #; N = .8.73 $>.

Stage B: dirnethoxymethyl ester of 7ß- (D-5-tosylamino-5-

carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid

20 g of the tosyl salt obtained in stage A are in a Three-necked flask entered. After adding 200 ml of methylene Chloride becomes the slurry in an ice bath under nitrogen cooled to 0 ° C. In the course of 90 minutes you sit down to that Add 4.1 ml of chloromethyl methyl ether in 30 ml of methylene chloride to the reaction mixture while stirring well and cooling in an ice bath. When the addition has lasted an hour, a solution is set of 1.58 ml of collidine in 5 ml of methylene chloride.

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After the addition, the mixture is stirred for a further 2 hours, filtered and the filter cake is washed with dry methylene chloride. After extraction with aqueous phosphoric acid, sodium chloride solution, sodium bicarbonate solution and again sodium chloride solution, the filter cake is washed with methylene chloride. The organic layer is dried, filtered, concentrated to a small volume and allowed to crystallize. The product, the dimethoxymethyl ester of 7β- (D-5-tosylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-5-cephem-4-carboxylic acid, precipitates in an amount of 9.6 g (yield 83.5 f °) . Ultraviolet analysis as well as thin layer chromatography determine that the product contains only a single component.

Step C: 3-carbamoyloxymethyl-7-methoxy-7β-thienylacetamido-3-cephem-4-carboxylic acid

To a slurry of 6.9 g of the tosyl methoxymethyl ester obtained in stage B and 7.5 g of powdered molecular sieve 4A (particle size 600 mesh; hydrated to 17 - 2 $ water) in 85 ml of 1,2-dichloroethane is set with stirring 5 ml of distilled 2-thienylacetyl chloride to. The slurry is heated to 65 ° C. for 16 hours with stirring and under nitrogen. . ■

The reaction is monitored by thin layer chromatography. After the specified period of time, the main component of the reaction mixture is the desired intermediate. After addition of 0.8 ml of methanol, the slurry is allowed to stand for 2 hours. At this point it is 4-methoxymethyl ester of the desired product is the predominant compound in the slurry.

The ester is hydrolyzed by cooling the solution to 25 G, filtering and washing with cold methanol. The filtrate and the washes are combined and cooled to 0 ° C. A cold solution of 20.8 ml at 0 ° C is used

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trated hydrochloric acid and 23.6 ml of methanol ■ to the solution heated to 15 ⁰ C and stirred for a further 2 hours and 40 minutes at 15 ⁰ C. At this time, TLC analysis is performed. The pH of the mixture is first adjusted to 2 "to 2.5 by passing gaseous ethylene oxide through and then to 5" to 6 by adding solid sodium hydroxide. The mixture is filtered and the dichloroethane layer is separated off. The cold aqueous layer contains the sodium salt of the product. This layer is purified by column chromatography on "IEA-68" resin in the filtrate form, eluting with 0.02 molar phosphate buffer solution of pH 7.0. The final yield of product is 1.74 g; thin layer chromatography reveals only a single spot. The angle of rotation is 192 °. The product is identified by NMR analysis. The product obtained is the desired 3-carbamoylox; p] ethyl-7-methoxy-7β-thienylacetamido-3-cephem-4-carboxylic acid; P. 165-167 ° C.

The monosodium salt used as the starting material in this example of 7β- (D-5-amino-5-carboxyvaleramido) -3-darbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid is produced as follows: -

Preparation of the monosodium salt of 7ß- (D-5-amino-5-

carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-

4-carboxylic acid

Modified ermentation procedure -

Step 1; Slanted tubes

A freeze-dried tube containing a culture of Streptomyces lactamdurans (NERL 3802) is opened aseptically and. the organism on a medium of the following composition:

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Medium XI:

1 fo molasses (Blackstrap) 1 fo yeast (national brewer's) 2.5 fo agar (Difco), pH 7.0 Top up with water.

The slant tubes are incubated for 7 days at 28 ° C. If If the slanted tubes are stored in the cold, they will last longer than 13 weeks.

Stage 2: Sowing stages: Two-stage sowing system

First sowing: The first sowing is carried out by directly inoculating 40 ml of 1 percent dried primary yeast NF (pH 7.0) (obtained from Yeast Product Corporation) in a 250 ml Erlenmeyer flask equipped with deflectors with the inclined tube of level 1. The Flasks are then shaken for 2 to 3 days at 28 ° C. in a shaker at 220 rpm with an amplitude of 5 cm.

Second sowing: A 2.5 percent inoculum from the first sowing stage is added to a flask containing 2 percent yeast autolysate (Fleischmann S-150) at a pH of 7jO. The culture at this stage is characteristically bright and the incubation, carried out as in the first stage, is not extended beyond 48 hours.

Level 3: production medium

The production medium contains 30 g of soluble sludge components per liter of distilled water and 7.5 g of dried primary yeast N.F. and 0.25 YoI. $ Antifoam agents ("Mobilpar-S", emulsified petroleum product). The medium is adjusted to a pH of 7.0 with a little concentrated sodium hydroxide solution, spread over Erlenmeyer flasks and add 15 or 20 minutes 121 ° C treated in an autoclave. After cooling, the medium receives $ 2.59 inoculum from the seed obtained in stage 2. the Incubation time can vary from about 50 to 100 hours;

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however, an incubation time of about 72 hours is preferred. The volume of production medium in each flask can vary in the range of 30 to 50 ml; routinely, however, one works with 40 ml. The amount of the inoculated material: can vary from 1 to 5 ° ; in practice, however, one generally works with $ 2-5 .'-- ■ -

Stage 4: Analysis

When the fermentation is over, the cells are centrifuged off, and the fermentation liquid is diluted with phosphate buffer solution (pH 7.0). The concentration of 7β- (D-5-amino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem * -4-carboxylic acid in the fermentation liquid is made according to the conventional biological disc method certainly. The analysis organism is Vibrio percolans (ATCC 8461). Filter paper disks are thinned into the Fermentation liquids immersed and placed on the surface of agar-containing Petri dishes, which with the Analytical organism Vibrio percolans (ATCC 8461) were inoculated. Disks are also placed on the Petri dishes, which have previously been immersed in standard solutions containing known concentrations of 7β- (D-5-amino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid contain. The disks are incubated overnight at 28 ° C, whereupon the diameters of the zones of inhibition are recorded. The product concentration in the fermented liquid will be calculated by interpolating from a üTormkurve, which the Indicates inhibition zone diameter as a function of known concentrations of standard solutions of the product. To This method is calculated to be Streptomyces lactamdurans NRRL-3802 78.6 γ / ml 7β- (D-5-amino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid produced in the modified fermentation process.

- 21 50-9823 / 0947

15 393Y

Level 5: Isolation

The filtered fermentation liquid is diluted with Hydrochloric acid adjusted to pH 7.0, and 2900 ml at a rate of 10 ml / min through a Poured column containing 100 g of strongly basic anion exchange resin with a polystyrene-divinylbenzene structure ("Dowex 1 χ 2" in the chloride form). The solvent used is collected in fractions of 500 ml each. The Harü pillar is washed out with water and with 3 percent ammonium chloride solution eluted in 90 percent methanol. The eluate is collected in fractions of 100 ml. The activity the political groups is monitored; the most active fractions are combined with one another and after adjusting the pH with dilute sodium hydroxide solution to 7.2 to 8.0 at one speed from 14 ml / min to 100 g of a strongly basic anion exchange resin with a polystyrene-divinylbenzene structure adsorbed ("Dowex 1 χ 2" in the chloride form). The column is washed with water and with 5 percent aqueous Saline eluted. The concentrate is diluted to 500 ml with dilute hydrochloric acid with a pH of 8.8> brought to a 2.0 and at a speed from 2.5 ml / min to 25 ml of a strongly acidic cation exchange resin s of the sulfonate type with a polystyrene-divinylbenzene structure ("Dowex 50 χ 2" in the hydrogen ion form) adsorbed. The column is washed with 25 ml of water and then eluted with 2 percent pyridine until the pH of the column effluent rises to 7.0 (54 ml). The eluate thus obtained is adjusted to a pH of -8.0 with dilute sodium hydroxide solution adjusted and concentrated in vacuo to drive off the pyridine. The monosodium salt of 7β- (D-5-amino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid is obtained in this way.

Elemental analysis

Calculated for C ^ H ₂ - ^ N, SOgNa:

C = 41.0 #; H = 4.5 #; N = £ 12.0; S = 6.8 #; found: C = 39.31 #; H = 4.76 #; N = 11.16 96; S = 6.46 fo.

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Example 2 ^c ** '2 * 58528

3-carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylic acid

Level A: Dime.thoxymethyl ester of 7ß - ^ / Jb-5-tosylamino - 5-

carboxyvaleryl) -phenylacetylamiao7-3-carbamoyloxyraetllyl-7-πlethoxy-3-gepl·leIΠ-4-carboxylic acid

A solution of 9 »3g (10 mmol) dimethoxymethyl ester of 7ß- (D-5-tosylamino-5-" carboxyvaleraniido) -3-carbamoyloxyinetliyl-7-methoxy-3-cephem-4-carboxylic acid, 10.8 g of powdered molecular sieve 12A . (20 - hydrated 2 fi water) and 5.3 ml (40 mmol) of phenylacetyl ml in 50 of acetonitrile is heated for 20 hours at 40 ° C the mixture is then cooled to room temperature and filtered the Piltrat is evaporated to dryness and. The insoluble residue, which contains the dimethoxymethyl ester of 7β- / jD-5-tosylamino-5-carboxyvaleryl) -phenylacetylainino7-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid, becomes without

Purification used in the next stage.

Step B: 3-Carbaraoyloxymethyl-7-methoxy-7β-phenylacetamide-3-cephem-4-carboxylic acid

The product of stage A is dissolved in 50 ml of Ί, 2-dichloroethane gel. After adding 1j, 0 ml of methanol, the solution is stirred for 1 hour. The methoxymethyl ester is hydrolyzed by adding a 0 ° C. 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 by äum procedure of Example 1 and purified. 3-Carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylic acid is obtained; P. 159-161 ° C. The UV and the NMR spectrum agree with the structure indicated.

example 3

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

The dimethoxymethyl ester of 7J- (D-5-l ¹ osylamino-5-carboxy-

- 23 -

503823 / 0947--

15 393Γ ^

valeraiiiido) -3-carbanioyloxymethyl-7-inethoxy-3-cephein-4-carTDonsäure with 2-Purylacetylchlorid in the presence of 12g hydrated molecular sieves 4A (to a water content of 15 - reacted for 2 fo hydrated) according to the process just described "The. The side chain and the ester protective group are also split off by the process described. The product obtained is 3-carbamoyloxymethyl-7-methoxy-7β- (2-furylacetamido) -3-cephem-4-carboxylic acid; ϊ ^1. 156-161 C; UV ( pH 7.0 buffer) λ max. 265 πιμ £ 7200; the results of the IR and NMR analysis agree with the given structure.

3-Carbamoyloxymethyl-7-methoxy-7β-thiophenoxyacetamido-3-cephem-4-carboxylic acid is prepared in the same way by using phenylthioacetyl chloride instead of the 2 -] urylacetyl chloride used. Characteristics of the product: mp 119-123 ° C; UV (pH 7.0 buffer) λ max. 247 ΐημ £ 10400; the NMR spectrum agrees the structure.

Example 4

3-acetoxymethyl-7ß- (2-thienylacetamidο) -3-cephem-4-carboxylic acid

Stage A: 7β- (I> -5-trichloroethoxycarbonylamino-5-carboxyvaler amido) -3-acetylmethyl-3-cephem-4-carboxylic acid

To a solution of 2.5 g (0.53 mol) of 7β- (D-5-amino-5-carboxyvaleramido) -3-acetoxymethyl-3-cephem-4-carboxylic acid in 13 ml of acetone and 40 ml of 10 percent aqueous dipotassium phosphate solution, 3.35 g (0.159 mol) of trichloroethoxycarbonyl chloride are added added dropwise. The pH is maintained during the addition the solution by gradually adding 17 percent aqueous sodium hydroxide solution in the range from 8.5 to 9.0. After 30 minutes the mixture is washed with ethyl acetate and the aqueous layer with concentrated hydrochloric acid to a pH of 2.5 acidified. The precipitate is extracted with ethyl acetate, the solution is dried over sodium sulfate, filtered and the solvent evaporated. This gives 2.7 g of 7ß- (D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido) -3-acetylmethyl-

- 24 503823/0947

15 393Y
3-cephem-4-carboxylic acid.

Stage B-: D.ibenzhydryl ester of 7- (D-5-trichloroethoxycarbonylamino-5-car "boxyvaleramido) -3-acetoxyniethyl-3-cep] ieni- 4-carboxylic acid

A solution of 7β- (D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido) -3-acetylmethyl-3-Pliem-4-carboxylic acid in 30 ml of ethyl acetate is mixed with 2.0 g of diphenyldiazomethane in 25 ml of ether offset. The mixture is stirred overnight and so is the solvent aborted. 4.0 g of crude product are obtained. The raw product is purified by chromatography on silica gel while eluting with chloroform. This gives practically 2.3 g pure .Dibenzhydrylester of 7- (D-5-TrichloräthoxycarbonylaminO-5-carboxyvaleramido) -3-acetoxymethyl-3-cephem-4-carboxylic acid »

NMR: (solvent - CDCl ₅ ) p = 2.0 (methyl, s), 4.9 (10-H ₂ , quartet), 3.2 (2-H ₂ , quartet), 4.95 (6-H , d), 5.92 (7-H), 7.0 (benzhydryl-. protons, 2s).

Stage C; Dibenzhydryl ester of 7- / TD-5-trichloroethoxy-

carbonylamino'-5-carboxyvaleryl) -2-thienylacetylamino / ^r -3-acetoxymethyl-3-cephem-4-carboxylic acid

A mixture of 2.0 g (0.02 mol) of dibenzhydryl ester of 7β- (D-5-triehloroethoxycarbonylamino-5-carboxyvaleramido) -3-acetoxymethyl-3-cephem-4-carboxylic acid, 11.0 g of molecular sieve 5A (on a Water content of 23-2 <fo hydrated), 1.31 g (0.0815 mol) of 2-thienylacetyl chloride and 6 ml of methylene chloride are heated to 40-45 ° C. in an oil bath under nitrogen for 20 hours. The reaction mixture is then poured into 100 ml of hexane and filtered. After evaporation of the solvent, the dibenzhydryl ester of 7- / J "D-5-trichloroethoxycarbonylamino-5-carboxyvaleryl) -2-thienylacetylamino7-3-acetoxymethyl-3-cephem-4-carboxylic acid remains.

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15 393Y.

Stage P: Benzhydryl ester of 3-acetoxymethyl-7- (2-thienyl- - acetamido) -3-cephem-4-carboxylic acid

The dibenzhydryl ester of 7 - </ jD-5-Trichloräthoxycarbonylamino-5-carboxyvaleryl) -3-tMenylacetylaiiiino / ^r -3-acetoxymethyl-3-cephem-4-carboxylic acid is dissolved in 10 ml of ethyl acetate and the solution with a mixture of 10 ml 90 percent aqueous acetic acid and 1.0 g zinc dust were added. The mixture is stirred for 2 hours at room temperature, whereupon the zinc is filtered off. The reaction mixture is washed successively with 2 parts of water, a cold sodium bicarbonate solution and then with 15.0 ml of saturated sodium chloride solution. The ethyl acetate solution is dried over sodium sulfate, filtered and the solvent is driven off. This gives 1.9 g of crude product which, by chromatography on silica gel using a mixture of 50 parts of chloroform and 1 part of ethyl acetate for elution, is converted into 0.380 g of product which, after recrystallization from ethyl acetate, is 141.5-143 ° C melts.

UV: (CH ₅ OH) X max. 263 <£ 7580

Elemental analysis

Calculated for

C ₂₉ H ₂₆ N ₂ O ₆ S ₂ : C β 61.91 ^; H = 4.66 fa N = 4.98 #;

found: C = 62.14 ^; H = 4.84 #; N = 4.92 #.

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

A cold solution of 100 mg of benzhydryl ester of 3-acetoxymethyl-7- (2-thienylacetamido) -3-cephem-4-carboxylic acid in 1.0 ml of anisole and 0.5 ml of trifluoroacetic acid, the mixture is stirred at 0 ° C. for 35 minutes. After adding 50 ml of carbon tetrachloride the reaction mixture is evaporated to dryness. The residue is stirred with hexane. The hexane is decanted off and the residue dissolved in 10 ml of ethyl acetate, the solution concentrated to 1 ml and treated with diethyl ether, whereby a Precipitation forms. The precipitate is recrystallized from a mixture of diethyl ether and ethyl acetate, and one

- 26 509823/0947

15 393Y. ^.

receives 0.025 g of 3- (acetoxynietliyl) -7- (2-tiaienylacetan) ido) -3-eephem ~ 4-carboxylic acid; P. 164 ° 0. The mixed melting point with an authentic sample is 163 C.

Example 5

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

Stage-A: Disodium salt of 7ß- (D-5-benzoylamino-5-carboxyvaleramido) -3-earbamoyloxymethyl-7-methoxy-3- cephem-4-carboxylic acid '_ __. ■

500 ml of an aqueous solution of 48.5 mmol of the monosodium salt of 7β- (D-5-amino-5-earboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid are adjusted to a pH of 9.5 with 50 percent sodium hydroxide solution. the solution is stirred with 15 ml (128 mmol) of benzoyl chloride offset. The pH is kept at 9.5 over the course of 30 minutes by adding sodium hydroxide solution as required.

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

The aqueous layer is cooled to 0 ° C. and 200 ml of isopropanol and 300 ml of ethyl acetate are added with stirring. then the pH is adjusted to 2.0 with hydrochloric acid. The organic Layer is discarded and the aqueous layer extracted three times with ethyl acetate. The combined extracts are washed with sodium chloride solution, dried over sodium sulfate and concentrated in vacuo to give 43.0 g of a dark oil. .

The oil is dissolved in 200 ml of ethanol and a solution of 30 g of the sodium salt of 2-ethylhexanoic acid is added to the solution. The slurry is cooled to 0 ° C., filtered and the filter residue is washed with ethanol and dried in vacuo. 28.8 g of disodium 7ß- (D-5-benzoylamino-

- 27 -

-. - 509823/0947

15 393Y, jyt

5-carboxyvaleramido) -3-carbanioyloxymethyl-7-me thoxy-3-cephem-4-carboxylate (yield 102 fo), which, according to a chroroatographischen comparison with the pure substance has a purity of 67 i>.

Stage B: Dimethoxymethyl ester of 7ß- (D-5-benzoylamino-5-

carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy- 3-cephem-4-carboxylic acid

To a slurry of 20 g of disodium 7β- (D-5-benzoylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylate in 200 ml of acetonitrile at 0 ° C

16 ml of 6 molar chloromethyl methyl ether in the course of 90 minutes added dropwise. After 1 hour of addition, 6 ml of S-collidine are added. The slurry is at for an additional 2 hours Stirred at 0 ° C. Then the mixture is washed with 500 ml of methylene chloride. diluted and twice with dilute phosphoric acid, once with dilute sodium bicarbonate solution and once with 5 percent Washed saline. The aqueous layers are in turn washed with 50 ml of methylene chloride. The organic Phase is dried over sodium sulfate and on in vacuo 100 ml concentrated.

The solution is poured through 200 ml of silica gel G, which is washed with 200 ml of methylene chloride and then with 800 ml Ethyl acetate is eluted. The ethyl acetate eluates are concentrated in vacuo to give 18.5 g of a yellow oil.

The crude product is recrystallized from 50 ml of ethyl acetate, and 10.0 g of dimethoxymethyl ester of 7β- (D-5-benzoylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid are obtained; Yield $ 67>.

Level 0; Sodium 7 ^ (2-thienylacetamido) -7-methoxy-3- carbamoyloxymethyl-3-cephem-4-carboxylate

A slurry of 192 mg (0.3 mmol) of dimethoxymethyl ester of 7ß- (D-5-benzoylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid, 225 mg powdered

. - 28 509823/0947

15 393Ϊ. · ■■. ■■■. _A%

Linde molecular sieve 4A, which contains 10 - 2 / »water, 0.3 ml of 2-thienylacetyl chloride and 4 ml of dichloroethane is Heated to reflux temperature for 4 hours with vigorous stirring. The mixture is cooled to 65 ° C. over the course of 2 hours with 10 ml of a 0.05 molar solution of tert-butanol added in dichloroethane and then kept at 65 ° C. for 1 hour. The reaction mixture is cooled to 0 ° G and filtered and the filter residue was washed with 5 ml of methanol. The FiI kicked is cooled to 0 ° C. with stirring, and 1.4 ml of a mixture of equal parts of hydrochloric acid and methanol are added and the mixture thus obtained was stirred at 15 ° C. for 3 hours. The mixture is dissolved in 10 ml of water containing 1.6 g of sodium bicarbonate, poured. The organic phases are discarded. The analysis of the aqueous phases shows a 65 percent product yield of 3-sodium 7- (2-thienylacet'amido) -7-methoxy-3-carbamoyloxymethyl-3-cephem-4-oarboxylate.

Level D:

In an analogous manner, p-chlorobenzoyl chloride, p-mrobenzoyl chloride or toluoyl chloride are used in stage A. In these cases, the final yield of the desired product sodium 7- (2-thienylacetamido) -7-methoxy-3-carbamoyloxymethyl-3-eephem-4-carboxylate 70 $, 68 $> or 72 f °.

Example 6

Sodium 7- (2-thienylacetamido) -7-methoxy-3-carbamoyloxy methyl-3-cephem-4-carboxylate

A. Mixture of 2.76 g (4 mmol) of dimethoxymethyl ester of 7β- (D ~ 5-tosylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7a-methoxy-3-cephem-4- "carboxylic acid, 3 g of dry Linde molecular sieve 4A which contains less than 2 $> water, and 2 ml (16 mmol) of thienylacetyl chloride in 34 ml of dichloroethane is stirred for 5 hours at reflux temperature. The addition of 0.38 ml (4 mmol) of tert-butanol is stirred for a further 2 hours. A further 0.095 ml (1 mmol) of tert-butanol are then added

- 29 509823/0947

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and the mixture is stirred for a further 1/2 hour at the reflux temperature. The reaction mixture is cooled to 0 to 5 ° C. in an ice water bath. The molecular sieve is filtered off with suction and washed with 40 ml of ice-cold methanol. The filtrate and the washing liquids are combined and cooled to ^{0 ° G.} After adding an ice-cold solution of 8.3 ml of concentrated hydrochloric acid and 9> 5 ml of methyl alcohol, the solution is warmed to 15 ° C. and stirred at this temperature for 2 hours and 40 minutes. As soon as the hydrolysis is complete, the reaction is stopped by adding a suspension interrupted by 22 g of sodium bicarbonate in 120 ml of water at 0 to 5 ° C. The two-phase solution is stirred for 10 minutes. The heavy salt which formed precipitate is filtered off and washed with a 5 percent sodium chloride solution containing 0.5 i ° sodium bicarbonate. The dichloroethane layer is separated off and extracted twice with 20 ml each of a solution of 0.5 i ° NaHCD. * + 5 NaCl. The aqueous fractions are combined and washed with 20 ml of dichloroethane. The bicarbonate solution, when analyzed by liquid chromatography, reveals 73 sodium 7- (2-thienylacetamido) -7-methoxy-3-carbamoyloxymethyl-3-cephem-4-carboxylate and 2.1 <fo unchanged starting material. .

Examples 7-20

Sodium 7- (2 ~ thienylacetamido) -7a-methoxy-3-carbamoyloxy methyl-3-cephem-4-carboxylate .

Following the general procedure of Example 6, 4 mmol (2.8 g) dimethoxymethyl ester of 7ß- (D-5-tosylamino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7a-methoxy-3-cephem-4-carboxylic acid with the amounts given in the table below of the given reactants among those given Reaction conditions implemented. In each case the total reaction time is 16 hours. In all cases it will Linde molecular sieve 4A is used, which has been heated to 700 ° C. before the reaction and a weight loss on drying of $ 3 results. According to the calculation, the molecule

509823/0947

15 393Y

Larger sieve is essentially anhydrous in that it contains less than 2 percent by weight of water. The reaction temperature is 67 ⁰ O in all cases.

- 31 _ 509823 / 0947-

Thienylacetyl

At- molecular chloride,
game sieve 4A, g mmol

16

16

. 4 *

10
11
12th
13th

1.5

16

Imide cleavage middle Lot,
mmol Time ^a , H End product
yield, i ° Starting
material
yield, fo 5 393Y HCl 0.4
0.8
0.8 3
1 69 4.5 ' Isopropanol 3
1
1 3
■ 1
1 68 6.2 It Il
It ti 2.4
2
2 3
1
1 67 3.1 Il Il
It It 5
1
5
5 3
2
2
2 62
48 ■ 10.7
6.2 0
V *
ψ tert-butanol VJlVJl 3
2: 70 5.8 It 'it VJlVJl 2
1 • 69.5 3.6

a) Period of time between the individual additions of the imide cleavage agent.

"b) Addition takes place after cleavage with isopropanol for 3 hours, and reaction is carried out
continued for an additional 16 hours before the final imide cleavage.

c) After the macylation and before the addition of alcohol, a further 1.5 g
Molecular sieve added.

-P-CTl CD

Continuation of the table

I. at Molecular Thienyl
acetyl Imide cleavage middle Lot, Time ^a , h End product Starting ) 393 " ¹ ₉ Vj4 game sieve 4A, g chloride, - ■ mmol - yield, ok material κ; V 14th 0 ^d mmol tert-butanol - 1.5 5.2 yield, $> I. 15th 3 16 ti It 10 1.5 64 0.4 * 16 3 It tt ti 10 Il 58 4.1 17th It ti tt It It Il 28 0.5 cn
O 18th It It It It 5 2 64.5 1.1 i £ t
OO 19th 2 ^e ti 5 5 67 5.2 κ » 12th 5. 8.2 dd dd. 2 O 20th ₂ e 5 1 68.5 to • 24 5 3.1

d) transacylation time 1 1/2 h. \ ·

e) After the macylation, a further 1 g of molecular sieve are added.

CD. OT

15 393Y.

Examples 21-25 2 ^ 56528

The general procedure of Example 6 is followed "up to 20, but at different reaction temperatures, as can be seen in the table below. The reaction mixture consists of 4 mmol (2.8 g) dimethoxymethyl ester of 7ß- (D-5-tosylamino-5-carboxyvaleramido) -3-carbamoyloxymethYl-7a-methoxy-3-cephem-4-carboxylic acid, 3 g dried Linde molecular sieve 4A (water content less than 2 (percent by weight) and 16 mmol of thienylacetyl chloride in 34 ml. dichloroethane.

- 34 -509823/0947

. During- transacylation- ^T ® ^m P · »

game duration, h C

21 25 55

22 16 67 w 23 7 78

»24 5 86

^^ 25 5 86

Imide cleavage middle Lot,
mmol Time 5
O End product
yield, $ Starting
material-
yield, f t
9 • Isopropanol I.
VJIVJl 8th,
3, 25th 7.2 tt η in in 3
2 70 5.8 If tt 4th 2 5 71.5 5.2 ti tt 4- 1, 67 4.1 ti tt 4th 2 73 2.1

cn σ) cn

15 393Y

In all cases the end product is sodium 7- (2-thienylacetamido) -7a-methoxy-3-carlDanioyloxyinetliyl-3-cepheiD-4-carboxylate.

Example 26

7- (2-thienylacetamido) -7-methoxy-3-carbamoylox; y-methyl-3- eephem-4-carboxylic acid

1 g of sodium 7β- (D-5-amino-5-carboxyvaleramido) -3-carbamoyloxyinethyl-7-methoxy-3-cepheni-4-carboxylate is placed in a reaction vessel which contains 20 ml of dichloroethane. Is reacted 1 g of Linde 4A molecular sieve in powder form to a Vassergehalt of $ 15> and 8 mmol (1 ml) thienylacetyl. The slurry is refluxed for 8 hours. After filtering off, the molecular sieve is washed with methanol. The methanol washes from the molecular sieve wash are analyzed and contain a 25 percent yield of the desired product 7-thienylacetamido-7α-methoxy-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid as determined by thin layer chromatography and liquid chromatography. The Dichloräthanschicht the reaction mixture is extracted with 5 percent sodium bicarbonate solution, and it is proved by thin layer chromatography and Flüssigkeitsehromatographie that it contains additional $ 5> final product. Thin layer chromatography of the organic layer at this point shows the presence of the mixed anhydride; after evaporation of the solvent, the mixed anhydride is hydrolyzed in 50 ml of a 50 percent solution of acetone in water in the presence of 10 mole percent pyridine. Thin-layer chromatography and liquid chromatography show that a further 13 # end product has been formed in the form of the free acid. The total yield is 156 mg of acid = £ 23.

- 36 509823 / U947

Claims (12)

Claims 1. Process for the preparation of compounds of the general formula R, H R ¹ N COOR " in which R-] is hydrogen or methoxy, A ^{1 is} carbamoyloxy or lower alkanoyloxy and R ^{1 is} an acyl group of the general formula X. where X is hydrogen, hydroxy, amino, a protected amino group or a protected hydroxyl group or carboxyl and R _{5 is} phenyl, 2-! dhienyl, 3-thienyl, 2-luryl, 3-puryl, 1-tetrazyl, C 1-4 .-nied.Alkylphenyl, halophenyl, hydroxyphenyl, C. -C . -nied.Alkyloxyphenyl mean, while R "means an easily split off protective group, characterized in that a compound of the general formula COOR " - 37 - S098-23 / 0947 5 393Y in which B is a protected amino adipoyl group or an acyl group R '"which is different from the desired substituent differs in the end product, with an acylating agent of the general formula 0 R _x CH-COl J ι X ' in which R, and X have the above meanings, in the presence a molecular sieve as a catalyst and then splitting off the B substituent. 2. The method according to claim 1, characterized in that the molecular sieve is a synthetic zeolite of the type 3A, 4A, 5A or 13X used. 3. The method according to claim 2, characterized in that the catalyst used is a molecular sieve which contains about 0 to 30 percent by weight of water of hydration. 4. The method according to claim 3, characterized in that the molecular sieve in about 0.5 to 2 times the amount by weight of the source material. 5. The method according to claim 4, characterized in that the molecular sieve in about the same or a slightly larger amount (on a weight basis) than the starting material applies. 6. The method according to claim 5 »characterized in that one carries out the reaction in one against the reactants inert solvent carries out. 7. The method according to claim 6, characterized in that the reaction mixture is constantly in motion during the reaction holds. - 38 509823/0947 15 393Y ~ '' Ά · 8. The method according to claim 71, characterized in that the splitting off of the substituent B is brought about by extending the reaction time to about 30 minutes to 30 hours. 9. The method according to claim 8, characterized in that the cleavage is carried out with the addition of a lower alkanol or lower alkylthiol after completion of the extended Reaction time- carries out. 10. The method according to claim 1, characterized in that R.J means the methoxy group. 11. The method according to claim 10, characterized in that " B denotes an aminoadipoyl group which has protective groups on the amino function and on the carboxyl function. 12. The method according to claim 11, characterized in that R · denotes the 2-thienylacetyl radical. - 39 - 5Ö9823 / Ö947