NO760084L - - Google Patents

Description

Process for the preparation of A^-3-substituted cephalosporanic acid derivatives.

The present invention relates to a method for the production of A3 -3-substituted cephalosporanic acid derivatives with formula

and their pharmaceutically acceptable salts, wherein R denotes where X is amino, hydroxy, carboxyl or C-^-C^-alkanoyloxy, and Q denotes hydrogen, hydroxyl, chlorine or .methoxy, where X<1> denotes oxygen, sulfur or a chemical bond, y is 0 - 35z is 1 or 2 and Q<1> is hydrogen, nitro, chlorine, trifluoromethyl or fluorine; where Q<2> denotes hydrogen. where each group Z represents hydrogen or, together with the carbon atoms to which they are attached, terminates a benzene ring,

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where each of the groups R and R denotes hydrogen or methyl, 2-sydnon-3-(C^-C^)-alkanoyl-,

(C1-Clo)alkanoyl,•

(C1-Clo)alkenoyl,

(C₁-C₂)alkyl-X-(C₁-C₂)alkanoyl, where X denotes oxygen or sulphur,

(C-5-Cy)alkenyl-X-(C 1 -C 4 )alkanoyl, where X denotes oxygen or sulphur,

(C^.C.-^haloalkanoyl, where halogen denotes fluorine, chlorine or bromine, or^

(C2_C10 )cyanoalkanoyl,

R' denotes cyan or -GY where Y denotes (C-L-C-L0)alkyl, acetyl or propenyl, and R" is selected from hydrogen, (C2-C8)t-alkyl, nitrobenzyl or methoxybenzyl.

The procedure for the preparation of the above compounds is characterized by the corresponding Ap■-compound<r>with formula:

where R' and R" have the above meaning, and R'" denotes

phenoxyacetyl, thiophenacetyl or phenylacetyl,

is reacted with an oxidizing agent to produce the corresponding b. J-sulfoxide which is then reduced to said A^-cephalosporin ester,

after which the R"' group is optionally cleaved from the 7-position and the 7-position is then acylated to an R-group ■as stated above, and that one optionally prepares desired salts of these compounds.

The semi-synthetic preparation of 7-acylamido-desacetoxycephalosporin antibiotics from starting materials of

the penicillin type has relatively recently become important because of Morin. and Jackson's invention (US Patent 3-275,626) relating to a process for converting penicillin sulfoxide esters to des-acetoxycephalosporaninic acid esters. Cephalosporin compounds derived from penicillins by this method have the general formula:

where R is the remainder of the acylamido group in the 7-position, and R"'" is hydrogen, a salt-forming cation, an ester group or an anionic charge when C00- forms a salt with a cation either outside or inside the molecule.

In an attempt to improve the properties and expand the application areas of these penicillin-derived semi-synthetic 'cephalosporin substances', attempts have been made to change the 3-methyl group in the above-mentioned A<3->desacetoxycephalosporins to a group which gives the resulting cephalosporin compounds increased antibiotic activity against one or more gram-positive or gram-negative microorganisms. However, it has so far not been possible to directly convert an A<3->desacetoxycephalosporin into a 3-methyl-functionalized A<3->cephalosporin with particularly good yield. There is consequently a need for an alternative method for the production of the more effective A<3->3~methyl-functionalized cephalosporin antibiotics, which have so far only been prepared from the fermentation-derived cephalosporin C, and the 7-aminocephalosporanic acid (7-ACA ) prepared from this compound.

For the preparation of starting materials for the present method, (a) N-bromosucciimide is reacted with a 4 2-cephalosporin ester compound with the formula:

where R is an amino-protecting group and R-, the rest of an ester group, and where the reaction takes place in an essentially anhydrous organic solvent at temperatures varying from 0°C to hook flow temperatures, preferably from approx. 40 - 100°C, thereby producing the corresponding 3-bromomethyl-A 2-cephalosporin ester intermediate, after which one (b) reacts said 3-bromomethyl-A -cephalosporin ester with a nucleophilic compound to thereby replace the bromo atom in the 3- the bromomethyl compound with the nucleophilic group, and thus obtain a 3-"functionalized-methyl"-A-cephalosporin ester starting material. The term "nucleophilic substance" as used here is understood to mean a substance which provides a negatively charged group or a neutral molecule with an unshared electron pair and which enters into a nucleophilic substitution reaction with said 3-bromomethyl-A p-cephalosporin master, whereby a 3-(nucleophilic methyl)-A 2-cephalosporin ester is produced. Countless examples of nucleophilic substances which can be used to obtain cephalosporin antibiotics with the nucleophilic group on the methyl group in the 3-position are already known from the patent literature in connection with cephalosporin antibiotics. Starting materials for the preparation according to the present method have the general formula: where R and R^ are as indicated above, while Y is the nucleophilic or "functional" group derived from the nucleophilic reagent used in step (b) of the above method. Purely expediently, these starting substances can be termed 3-(nucleo-phile-methyl)-A 2-cephalosporin esters. A p-7-acylamido-3-(nucleophilic-methyl)-cephalosporin ester starting materials according to the present method can be used for the production of A<3->cephalosporin antibiotics which can be used for the treatment of diseases caused by gram-positive and gram- negative microorganisms. The 7-acylamido-3-(nucleophilic-methyl)-3-cephem-4-carboxylate ester starting material is converted to the corresponding A3 ^-ester by (1) oxidation of the A 2-sulfide ester product to the corresponding A<3->sulfoxide ester with a peracid, .(2) reduction of the A<3->sulfoxide ester with a reducing agent such as e.g. sodium bisulphite or sodium dithionite in the presence of an activator such as e.g. acetyl chloride in an organic solvent such as •acetic acid or dimethylformamide, whereby the A<3->sulfide ester is produced, and (3) then de-esterify the A<3->sulfide ester if this is desired, to the antibiotically active A<3-> sulfuric acid. If desired, mixtures of the active A 3 -cephalosporic • acid antibiotic and the inactive A 2 -cephalosponic acid, as well as pharmaceutically acceptable salts thereof, can be used for certain antibiotic purposes, e.g. as a topical antibiotic for open wounds, for veterinary purposes in cases where the mixture can be applied to the wound or made into an ointment to prevent the growth of various gram-positive or gram-negative microorganisms,

The A p-7-(protected amino)-3-(nucleophilic-methyl)-sulfide starting material can be used as intermediates for the preparation of A<3->cephalosporin antibiotics with any desired 7-acylamido group known to give cephalosporins antibiotic activity. These starting materials are treated to remove the 7-protecting group and thereby obtain the A 2-7-amino-3_'(nucleophilic methyl)-sulfide ester, which can then be acylated, depending on desire, e.g. with thiophene-2-acetic acid acid chloride or a mixed anhydride,. thereby producing the corresponding A 2'-7-(thienyl-acetamido)-3_(nucleophilic methyl)-cephalosporin ester, which can then be isomerized to the corresponding A -7-(thienylacetamido)-3_

(nucleophilic methyl)-cephalosporin esters by an oxidation-reduction reaction, as described above, and de-esterified,

e.g. by a treatment with trifluoroacetic acid alone, or together with zinc in formic or acetic acid, in order to thereby produce the active cephalosporin actibiotic. Alternatively, the A-cephalosporin ester can first be converted to an A-^-ester by the above oxidation-reduction method, after which this ester is cleaved to an A3_Y_amino_3_(nucleophilic methyl)-cephalosporin ester which can then be N-acylated and then de-esterified to the A <3->cephalosporin. An example of a compound that can be produced by such a method is cephalothin, a widely used and commercially available cephalosporin actibiotic which has so far only been possible to produce by fermentation-derived cephalosporin C and 7-ACA produced from this compound.

The α-desacetoxy cephalosporin ester starting material can be prepared from a number of different penicillins or cephalosporins in a known manner. They are produced by base treatment of the corresponding 3-methyl-A<3->cephem-4-carboxylate ester which, e.g. described in Example 1 of US Patent 3,275,626 to Morin and Jackson. They can also be prepared by hydrogenating a cephalosporin C derived cephalosporin ester to produce the corresponding A<3->desacetoxycephalosporin ester and then treating this with a base such as e.g. pyridine in the cold (0.- 10°C) to isomerize the A3 ^-double bond to the A 2-double bond position. Attempts to brominate the allylic 3-methyl group in the A<3->desacetoxycephalo-■ sporonatesters did not turn out to be successful.

The amino-protecting group represented by R in the above-mentioned formula II can be any group known to

protect the nitrogen atom to which it is attached from attack by . N-Bromosuccinimide. If the nitrogen is in the free amino state, an excess of the bromination reactant will be required to achieve the purpose of the first step. The R group can e.g. be an acyl group. Many acyl groups that are well suited for this purpose are already known from the literature in connection with penicillins and cephalosporin antibiotics. Some of these acyl groups can be subjected to bromination, and in such cases it is. necessary to use a molar excess of N-bromo" succinimide to brominate all the 3-methyl groups in the A 2-desacetoxy-cephalosporonate ester.. If one uses, for example, thienylacetyl or' furylacetyl as the acyl-protecting group, then can the ring systems in these groups be brominated in the first step of the present method,

but this is not harmful, especially in those cases where the 7-acyl group is later to be cleaved off to produce the corresponding 7-aminocephalosporin ester derivative. However, the preferred amino protecting group is an acyl group of the formula:

where m is a number from 0-3 inclusive, n is a number from 1-2 inclusive, X is oxygen or sulfur or a chemical bond, and such acyl groups substituted in the phenyl carbon atoms with fluorine-chloro-, nitro- or trifluoromethyl groups. The acyl group can also have methyl groups instead of one or two of the hydrogen atoms on the carbon atoms in the m or n groups. A couple of representative examples of such preferred acyl groups are:

phenylacetyl,

phenoxyacetyl,

phenylmercaptoacetyl,

benzyloxyacetyl,

benzylmercaptopropionyl,

phenylpropionyl,

3- fluorophenoxyacetyl,

phenyl-α,α-dimethylacetyl,

4- nitrophenylmercaptoacetyl,

.-.Numerous other compounds which form amino-protecting acyl groups and which can be used in the R position are known from previously known literature, e.g. those described in US Patents 2,479-295 to 2,479-297 and 2,562,407 to 2,562,411 and 2,623-876.

The R^ symbol represents the residue of an ester-forming alcohol. The alcohol used to form these esters should be one that can be removed by known methods such as e.g. by means of dilute aqueous bases or by means of trifluoroacetic acid, or by hydrogenation in the presence of palladium or rhodium catalysts on suitable supports such as carbon, barium sulphate or aluminum oxide, so that the cephalosporin is not degraded. The preferred ester groups are those indicated above.

Certain A<3->cephalosporin esters such as acetoxymethyl-cephalotin esters of the type described in US patent 3,488,729 can be used as such for antibiotic therapy to combat . bacterial infections. For most purposes, however, it is preferred to remove the protecting ester group by known methods in order to thereby prepare the A<3->cephalosporanic acid or a zwitterion derivative. The resulting cephalosporanic acid or zwitterion can be used as such or converted to pharmaceutically acceptable salts, e.g. the alkali metal salt (e.g. the sodium or potassium salt) or soluble or insoluble amine salt forms, all depending on the type of treatment the salt is to be used for. Insoluble salts with 1,4-bis(aminomethyl)cyclohexane are useful for a "depot-type" cephalosporin delivery whereby a slow absorption into the blood of a<y>cephalosporin antibiotic is obtained. In such a form, the cephalosporin is usually administered by intramuscular injection in doses varying from 0.5 to 1 gram. Water-soluble salts of cephalosporin antibiotics such as

sodium, monoethanol or diethanolamine cephalosporin salts can

administered parenterally to provide total daily doses

varying from 1 - 6 g of active cephalosporin antibiotics per day to a patient with a body weight of approx. 70 kg.

The invention also provides connections with the formula:

where R is selected from the group consisting of hydrogen, where X is amino, hydroxy, -C00H or C-^-C^-alkanoyloxy, and Q denotes hydrogen, hydroxyl, chlorine or methoxy, or:

where X<1> is oxygen or sulfur or a chemical bond, ,y is a

numbers from 0 - 3 and z is 1 or 2, and Q"'' denotes hydrogen, nitro, chlorine, trifluoromethyl or fluorine, .

2 where Q is hydrogen, where X is sulfur and where each Z separately denotes hydrogen, or taken together with the carbon atoms to which they are attached, can complete a benzo ring.

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where each R and R are hydrogen or methyl,

2-sydnon-3-C1-C-j-alkanoyl-,

C<->^<-C>^Q-alkanoyl-,

O^-C^Q-alkenoyl, C-^-Cy-alkyl-X-C-j^-C-j-alkanoyl where X is oxygen or sulfur, C-^-C^-alkenyl-X-C-i^-C^-alkanoyl where X is oxygen or sulphur,<C>^<-C>^Q-haloalkanoyl where the halogen can be fluorine, chlorine or bromine, as well as C^-C^Q-cyanoalkanoyl,

•R' is cyano (-CN) or -OY, where Y is

C-^-C-^Q-alkyl, acetyl or propenyl and wherein R" is selected from the group consisting of

-hydrogen,..

- a zwitterionic charge or a salt with a pharmaceutical acceptable cation or anion, and C 1 -C 8 -t alkyl, e.g. t-butyl, t-pentyl and t-hexyl,

- methoxybenzyl,

- nitrobenzyl.

Some of the above-mentioned 7-acylamido-3-cyano-methyl- and 3-oxymethyl-ether-A3-cephem-4-carboxylate esters can be prepared by oxidizing the corresponding A -compound to the A^-sulfoxide, and then reducing this to The A<3->cephalosporin ester compound. p-Methoxy-benzyl-7-phenoxyacetamido-3-cyanomethyl-A<3->cephem-4-carboxylate can e.g. is prepared by oxidizing p-methoxybenz-z'yl-7-phenoxy-acetamido-3-cyanomethyl-A-cephem-4-carboxylate to the corresponding 1-oxide (sulfoxide), e.g. with m-chloroperbenzoic acid in a suitable solvent and then reducing the sulfoxide with sodium dithionite or a similar reducing agent in the presence of acetyl chloride.

7-Amino-3-cyanomethyl- and 3-oxymethyl ether-A<3->cephem-4-carboxylate esters, above designated as nucleus-type compounds are prepared by cleaving the 7-acyl group or another 7-amino-blocking group which was present during the aforementioned oxidation and reduction. The p-nitro-benzyl-7-amino-3-ethoxymethyl-α'J-cef em-4-carboxyl can e.g. is prepared by reacting p-nitrobenzyl-7-phenoxyacetamido-3-ethoxymethyl-α<3->cephem-4-carboxylate with PCl^ in the presence of a tertiary amine, followed by treatment with an alkanol such as . methanol, and then with water to carry out the cleavage.

The remaining 7-acylamido-3-cyanomethyl and 3-oxymethyl ether-A<3->cephem-4-carboxylate compounds are prepared by acylating the 7-amino-3-cyanomethyl or 3-oxymethyl ether-A^-z. compounds, either in ester or the acid form with the desired acyl group according to known methods for the acylation of cephalosporin compounds.

7-amino-3-cyanomethyl- and 3-oxymethylether-A<3->cephem-4-carboxylic acids and 7-acylamido-3-cyanomethyl- and 3-oxymethylether-A.<3->cephem-4-carboxylic acids are prepared by deesterifying the esters by known methods or by methods described here, either before or after the 7-amino group is re-acylated.

The invention is illustrated by the following detailed examples which describe the method for converting the α2-functionalized cephalosporin ester products into the corresponding α2-cephalosporanic acids which are active antibiotics.

Example 1

m-chloroperbenzoic acid oxidation of a mixture of 4 2-A.<3->p-methoxybenzyl esters of cephalosporanic acid with formula:

in chloroform gave a high yield of the corresponding p-methoxybenzyl-3-acetoxymethyl-7-(2'-phenoxyacetamido)-α<3->cephem-4-carboxylate-1-oxide, m.p. 161-163°C (from methanol).

Reduction of p-methoxybenzyl-3-acetoxymethyl-7-phenoxyacetamido-A<3->cephem-4-carboxylate-1-oxide.

A solution of 500 mg of the sulfoxide, 4-methoxybenzyl-3-acetoxymethyl-7-(2'-phenoxyacetamido)-A-cephem-4-carboxylate-1-oxide in 40 cm<3>dimethylformamide (DMP) was added to 10 cm <3>acetyl chloride and then 3 g of sodium dithionite.

An exothermic reaction was obtained and after stirring for 4 hours at room temperature to ensure a complete reaction, the mixture was cooled and diluted with benzene and then an aqueous sodium bicarbonate solution was added. After the vigorous evolution of gas had stopped, more benzene was added, and the organic layer was separated and washed with aqueous sodium bicarbonate solution and then with sodium chloride solution. dried over magnesium sulfate, filtered and evaporated to 635 mg of a brown oil.

This product was purified on a column of silica gel 15% H 2 <0. 253 g of an oil were obtained whose NMR, infrared spectrum (I.R.) and ultraviolet spectra (U.V.) were the same as for p-methoxybenzyl-7-(2'-phenoxyacetamido)-A<3->cephem-4-carbpxylate prepared from the fermentation-derived 7-aminocephalo-sporaninic acid (7-ACA). This oily ester could be crystallized from ether to 195 mg if m.p. was ll8-119°C and if sm-.-p. was not lowered by a mixture with known samples of the same compound.

Cleavage of the p-methoxybenzyl ester to cef(V).

To a solution of 105 mg of p-methoxybenzyl-7-(phenoxy-acetamido)-3-acetoxymethyl-A3-cefe'm-4-carboxylate ester and 22 mg of anisole in 10 cn<r>dry benzene was added 0.5 crrr of trifluoroacetic acid . The mixture was stirred for 2 hours at room temperature under nitrogen and then evaporated to dryness. The residue was dissolved in ethyl acetate and extracted three times with aqueous sodium bicarbonate solution.

The ethyl acetate solution was then evaporated to 55 mg of

the neutral product.

The bicarbonate extract was cooled, ethyl acetate added and adjusted to pH 2.4 with 20% HCl. The ethyl acetate layer was separated and the aqueous portion extracted once more with ethyl acetate. The

combined organic layers were washed twice with sodium chloride solution, dried, filtered and evaporated to 86 mg of a colorless oil which was worked up at the same Rf as 7-(phenoxyacetamido)-3-acetoxymethyl-A-3^-cef em-4 -carboxylic acid in a thin layer kr ornat ogram. The chloroform solution of this oil deposited a solid which was removed by filtration. The filtrate was evaporated to 65 mg of product which proved to be 7-(phenoxyacetamido)-3-acetoxymethyl-A<3->

cephem-4-carboxylic acid, as its NMR spectrum was identical to that obtained from a known sample of this compound, a known antibiotic, previously prepared only from the fermentation derivative 7-ACA.

Example 2

A solution of 4-methoxybenzyl-7-(phenoxyacetamido)-. 3-Bromomethyl-α-cephem-4-carboxylate in dry acetonitrile was mixed with 1.1 molar equivalents of dry sodium nitrite, after which the mixture was stirred for 13 hours at room temperature to prepare 4-methoxybenzyl-7-(phenoxyacetamido)- 3-(Nitromethyl)-A - cephem-4-carboxylate. This A 2-cephalosporin ester was converted to the corresponding A-3^-cephalosporanic acid derivative which showed antibiotic activity by a bioautogram against S. lutan. NMR of this product showed a 3'-methylene group.

Example 3

Sulfoxide formation.

A cooled solution of 215 mg of 4-methoxybenzyl-3-methoxymethyl-7-phenoxyacetamido-A 2-cephem-4-carboxylate ester produced according to the present invention in 5 cm<3>chloroform was added to a solution of 85 mg of m-chloroperbenzoic acid (88 J ?. pure) in chloroform. The mixture was stirred and slowly heated over 1 hour. The chloroform was washed with sodium bicarbonate solution, sodium chloride solution, dried over magnesium sulfate, filtered and evaporated to dryness. The solid residue was crystallized from benzene containing methylene chloride to give 42 mg of 4-methoxybenzyl-3-methoxymethyl-7-phenoxyacetamido-A<3->cephem-4-carboxylate-1-oxide, m.p. 183 - 185°C, and this compound gave a satisfactory elemental analysis.

Further experiments have shown that hydroxylic solvents such as Isopropanol or t-butanol gives better yields than chloroform in this reaction.

Reduction of sulfoxide.

A solution of 1.028 g (0.002 mol) of 4-methoxybenzyl-3-methoxymethyl-7-phenoxyacetamido-A<3->cephem-4-carboxylate-1-oxide 3 '3 1 75 cm-' dry dimethylformamide was added 15 cur of acetyl chloride - and then 6 g of sodium dithionite (Na2S20^). After stirring at room temperature for 4 hours, the resulting mixture was cooled, after which benzene and aqueous sodium bicarbonate solution were added. After the decomposition of the excess acetyl chloride was complete, water was added, after which the mixture was extracted twice with benzene. The combined benzene extracts were washed with aqueous sodium bicarbonate solution and then with sodium chloride solution, dried over magnesium sulfate, filtered and evaporated to give 1.32 g of a dark brown semi-solid 4-methoxybenzyl-3-methoxymethyl-7-phenoxyacetamido-A <3->cephem-4-carboxylate.

This crude product of A<3->cephalosporin ester was purified by column chromatography and eluted from silica gel containing 15% water, using benzene 5% ethyl acetate as eluate. 300 mg of 4-methoxybenzyl-3-methoxymethyl-7-phenoxyacetamido-A3 3 -cephem-4-carboxylate were obtained, m.p. 116-117.5 oC after recrystallization from ethyl ether. The given structure was verified by NMR and elemental analysis.

Ester removal.

A solution of 174 mg of 4-methoxybenzyl-3-methoxy-methyl-7-phenoxyacetamido-A<3->cephem-4-carboxylate and 119 mg of anisole in 25 cm<3>dry benzene was added to 125 cm<3>trifluoroacetic acid . After stirring for 2 hours at room temperature, the reaction mixture was evaporated, the residue dissolved in ethyl acetate and the resulting ethyl acetate mixture, extracted three times with aqueous sodium bicarbonate solution. The aqueous bicarbonate extracts were cooled, triturated with ethyl acetate and acidified to pH 2.8. From the ethyl acetate layer, after evaporation, 140 mg of a foam was obtained which gave 86 mg of 3-methoxymethyl-7-phenoxyacetamido-A<3->cephem-4-carboxylic acid, m.p. 135-137°C, after recrystallization from ether. This acid gave an antibiotic active zone on a bioautograph on its paper chromatogram.The spot for the derivative moved somewhat slower than that for desacetoxycef (V)...

Example 4

4-Methoxybenzy1-3-cyanomethyl-7-phenoxyacetamido-A3-2§£§mliyl^å2^2!S§y.i§t-1-oxide_1

A solution of 372 mg of 4-methoxybenzyl-3-cyanomethyl-7-phenoxyacetamido-A-cephem-4-carboxylate in 300 cnr of isopropyl alcohol was added to a solution of 133 mg of metachloroperbenzoic acid in isopropyl alcohol. The mixture was stirred overnight at room temperature. The precipitated sulfoxide, 4-methoxybenzyl-3-cyanomethyl-7-phenoxyacetamido-α<3->cephem-4-carboxylate-1-oxide, (210 mg - mp 204-207°C) was collected by filtration. The filtrate was evaporated to dryness. The residue was dissolved in ethyl acetate, washed with aqueous sodium bicarbonate solution, aqueous sodium chloride solution, dried over magnesium sulfate, filtered and then evaporated to 160 mg of an oil from which a further 45 mg of the A<3->sulfoxide ester was prepared. The structure of 4-methoxybenzyl-3-cyanomethyl-7-phenoxyacetamido-α<3->cephem-4-carboxylate-1-oxide was verified. by spectroscopic methods.

1-Methoxybenzyl-3-cyanome.ty 1-7-phenoxyacetamido-A■_ 7.=ceTem-4-carbox^late

A solution of 400 mg of 4-methoxybenzyl-3-cyanomethyl-7-phenoxyacetamido-A<3->cephem-4-carboxylate-1-oxide, prepared as described above, in 25 cm<3> of dry dimethylformamide, was added 1, 6 g sodium dithionite, (Nap^O^). The mixture was cooled in an ice-water bath, after which 4.5 cm<3>acetyl chloride was added. The mixture was stirred in a cold bath for 1 hour and then poured into a cold aqueous sodium bicarbonate solution-benzene mixture. After a complete decomposition of the excess acetyl chloride, more benzene was added and the mixture was washed with an aqueous sodium bicarbonate and then with aqueous sodium chloride solution, dried over magnesium sulfate, filtered and evaporated to give 620 mg of a crude product of 4-methoxybenzyl-3-cyanomethyl- 7-Phenoxyacetamido-A 3-cephem-4-carboxylate.

This crude product was applied to a column containing

40 g silica gel - 15% water. The pure 4-methoxybenzyl-3-cyanomethyl-7-phenoxyacetamido-A<3->cephem-4-carboxylate was eluted with

a benzene - 4% ethyl acetate mixture and crystallized from the ethyl is. The product had a m.p. at 145-147°C. The structure was confirmed by spectroscopic methods.

4-Methoxybenzyl-3-cyanomethyl-7-phenoxyacetamido-A3-cephem-4-carboxylate was treated with a molar excess of trifluoroacetic acid in benzene solution at room temperature for 2 hours, whereby the corresponding 3_cyanomethyl-7-phenoxyacetamido-A3-cephem was produced -4-carboxylic acid, which, among other things, is antibiotically active against Bacillus subtilis, which could be shown by a bioautograph of the compound's paper chromatogram.

Example 5«

Oxidation on.

Using t-butyl-7-phenoxyacetamido-3-isopropoxy-methyl-Δ o -cephem-4-carboxylate ester as an illustration, this example shows how 3-oxyether-Δ 2 -cephalosporin esters described above can be oxidized to the corresponding A<3->cephem-1-oxide esters, are reduced to the corresponding A-3 ^-cephem esters and then converted to A-^-cephemic acid antibiotics.

t-But.yl-7-phenoxyacetamido-3-isopropoxymethyl-A 2- • cephem-4-carboxylate ester (2.32 g) prepared as described above was dissolved in a mixture of methylene chloride and 50 ml of isopropanol and stirred at ice temperature while a solution of 1.00 g of m-chloroperbenzoic acid in 50 ml of methylene chloride was added dropwise.. After the reaction was complete, the solvent was removed under reduced pressure.. The residue was dissolved in ethyl acetate, washed with aqueous 5% sodium bicarbonate solution, dried over sodium sulfate and evaporated to dryness, yielding 2.16 g of a crude product of sulfoxide ester t-butyl-7-phenoxyacetamido-3-isopropoxymethyl-N-cephem-4-carboxylate-1-oxide. This crude product was chromatographed on 200 g silica gel - 15% water to 1.10 g of the pure sulfoxide ester in 40% ethyl acetate - 60% benzene mixture. The structure of the pure sulfoxide ester was verified by spectroscopic examination. Reduction on.

A solution of 0.891 g of sulfoxide ester-t-butyl-7-phenoxyacetamido-3-isopropoxymethyl-A<3->cephem-4-carboxylate-1-oxide, 0.71 g of stannous chloride, 0.5 ml of acetyl chloride, 15 ml of acetonitrile and 5 ml of dimethylformamide was stirred at 25°C for 2 hours. The solvents were removed under reduced pressure and the residue was suspended in ethyl acetate. This suspension was washed twice with aqueous 3'% hydrochloric acid solution, aqueous 5% sodium bicarbonate solution and then with water, dried over sodium sulfate, evaporated to dryness, yielding 0.78 g of the reduced (sulfide) ester, t-butyl -7-Phenoxyacetamido-3-isopropoxymethyl-A<3->cephem-4-carboxylate. Spectral analysis verified the structure. De-esterification.

A solution of 0.156 g of t-butyl-7-phenoxyacetamido-3-isopropoxymethyl-A<3->cephem-4-carboxylate ester in 7 ml of 98% formic acid and water was stirred at 25°C for 28 hours to ensure a complete reaction. The reaction mixture was poured into water and extracted with ethyl acetate. The ethyl acetate solution was washed with water and then extracted with aqueous 5% sodium bicarbonate solution. The resulting solution was acidified with aqueous 3% hydrochloric acid and extracted with ethyl acetate. The resulting ethyl acetate solution was dried over sodium sulfate and evaporated to dryness, yielding 0.088 of the free acid, 7-phenoxyacetamido-3-isopropoxymethyl-α<3->cephem-4-carboxylic acid which is, among other things, an active antibiotic against a group of penicillin-sensitive and resistant staphylococci in an in vitro test.

3-oxymethyl ether-A p-cephalos<p>ormester starting materials according to the present invention can be easily converted into the corresponding 3-oxymethyl ether-A3 ^-cephalosporem. acid antibiotic by the above-mentioned oxidation-reduction-deesterification. These compounds can also be used as intermediates for the preparation of 7-amino-3-oxymethyl ether-3-cephalosporin ester nuclei by cleaving the 7-amino-blocking group or the acyl group by known methods. The latter esters can then be acylated,

by known methods with any desired acylating agent or with groups known to contribute to the formation of antibiotic

■active A.3-cef alosporaninic acid compounds. When carrying out such 7-amino-acylation, the ester group can be removed from the carboxyl group by known methods. Alternatively, the 7-amino-3-oxymethyl ester-A^-cephalosporin ester core can first be deesterified and

then acylated with the desired acyl group to thereby obtain the desired 7-acylamido-3-oxymethyl ether-A<3->cephem-4-carboxylic acid antibiotic.

In a similar way, 3-cyanomethyl-A 2-cephalosponne ester starting materials can be easily converted into the corresponding 3-cyanomethyl-A<3->cephalosporin acid antibiotics according to the present invention by the above-mentioned oxidation-reduction-deesterification. These compounds can also be used as intermediates for the production of 7-amino-3-cyanomethyl-A<3->cephalosporin ester nuclei by cleaving the 7-amino-blocking or 7-acyl group by known methods. The latter esters can also be acylated by known methods with the desired acylating agent, as described above, in a process for preparing 7-acyl-amido-3-cyanomethyl-α<3->cephem-4-carboxylic acid antibiotics.

3-thiomethyl ether-A p-cephalosporin ester starting materials can be converted into A<3->cephalosporin ester compounds by the above-mentioned oxidation-reduction. If desired, the 7-acyl group in these compounds can be cleaved off using known methods, and the resulting esters can be re-acylated with any desired acyl group, after which the ester groups can be removed by known methods in order to obtain effective A3-cephalosporin antibodies -^-biotics.

Example 6

This and the following examples illustrate the acylation of a typical 7-amino-A<3->cephem ester with several different acylating agents as well as the hydrolysis of the ester groups in the preparation of antibiotically active cephalosporin compounds.

Acylation.

In this specific example, the p-methoxybenzyl-7-amino-3-methoxymethyl-α<3->cephem-4-carboxylate nucleus was first prepared by adding 4-methoxybenzyl-7-phenoxyacetamido-3-methoxymethyl-α 2-cephem -4-carboxylate was subjected to the above-mentioned oxidation-reduction process. The 7-phenoxyacetyl group was then cleaved off by methods as mentioned above, after which the p-methoxybenzyl-7-amino-3-methoxymethyl-A<3->cephem-4-carboxylate ester nucleus was obtained as the tosylate (p-toluenesulfonic acid) salt.

A mixture of 268 mg (0.4 mmol) of the tosylate salt of p-methoxybenzyl-7-amino-3-methoxymethyl-3-cephem-4-carboxylate and 336 mg (2.0 mmol) of sodium bicarbonate was stirred in 25 ml of acetone under cooling in an ice bath. After addition of 130 mg (0.62 mmol) of 2-benzothiophene acetyl chloride, the reaction mixture was stirred for 1 hour in the cold (0-5°C), followed by a further two hours at room temperature. The reaction mixture was then diluted with 100 ml of water. The precipitated solid was collected, washed with water and vacuum dried at 45°C to give 250 mg (93% yield) of the product, m.p. 182 - 185°C. NMR spectrum and microanalysis confirmed the structure of this compound. It was found by thin-layer chromatography (TLC) to contain only one product, namely p-methoxybenzyl-7-(2'-benzothienylacetamido)-3-methoxymethyl-A3-cephem-4-carboxylate. The following acylations were also performed.

Hydrolysis.

p-Methoxybenzyl-7-(4'-nitr of enylacetamido)-3-methoxymethyl-A3-cephem-4-carboxylate ester from the above table was used to illustrate this step.

A solution of 120 mg (0.23 mmol) of p-methoxybenzyl-7-(4-nitrophenylacetamido)-3-methoxymethyl-3-cephem-4-carboxylate, 0.4 ml of trifluoroacetic acid and 20 drops of anisole in 10 ml of benzene was set aside at room temperature for 1.5 hours. 100 ml of pH 7 phosphate buffer was then added to the reaction mixture. After separation of the layers, the aqueous layer was washed with water and then with saturated NaCl solution, dried over MgSO 4 and then concentrated. The residue was dissolved in 15 ml of ethanol after which 0.4 ml of a 1 N solution of sodium acetate in methanol was added. After 2 hours at 0°C, the solid was collected, washed with ethanol and dried in vacuo at 45°C to give 76 mg (83% yield) of a crystalline product namely 7-(4'-nitrophenylacetamido-3- Methoxymethyl-A<3->cephem-4-carboxylic acid... NMR and UV spectra confirmed the structure of this compound. The microanalysis was also in agreement with the calculated values. An overview of the physical/chemical data obtained for this salt of the above and of other acids with the formula given below, are indicated in the following table.

The above-mentioned new sodium 7-acylamido-3-alkoxymethyl-A<3->cephem-4-carboxylic acid salts are antibiotically active against a number of gram-positive and gram-negative microorganisms.

Example 7

t-Butyl-7-amino-3-cyanomethyl-A3-cef em-4-carboxylate-p-toluenesulfonate

A solution of 2.15 g (5 mmol) of t-butyl-7-phenoxy-acetamido-3-cyanomethyl-A 3 -cephem-4-carboxylate in 100 cnr 3 of dry benzene was added (590 mg, 1.5 equivalent) dry pyridine and (1.56 g, 1.5 equiv) phosphorus pentachloride. The mixture was up-. heated below N? for 2 hours at 56°C. The benzene was removed, after which 100 cm 3 of ice-cold methanol was added. After standing at room temperature overnight, the methanol was removed under reduced pressure, 75 cm<3>THP added and, after cooling, 75 cm3 of cold pH 4.5 buffer solution. After a delay of 20 min. THF was removed under reduced pressure after which ethyl acetate and some water were added. The pH of the mixture was adjusted to 6.5 with sodium bicarbonate. The organic layers were separated, washed twice with NaCl solution, dried over MgSO 4 , filtered and evaporated to a smaller volume. A solution of this t-butyl-7-amino-3-cyanomethyl-A-<3>^-cephem-4-carboxylate in 50 cm<3>ethyl acetate was added to a solution of 951 mg of p-toluenesulfonic acid in 50 cm <3>ethyl acetate. The p-toluenesulfonate salt was crystallized overnight and then collected by filtration. It weighed 856 mg and melted at 177°C (decomposition). The structure was confirmed by I.R., U.V., N.M.R. and elementary analysis.

Example 8

t-butyl^-amino^-methoxymethyl-A3^-cef ém-4- carboxylate-p-toluenesulfonate

A solution of 364 mg (0.84 mmol) of oily t-butyl-7-phenoxyacetamido-3-methoxymethyl-A<3->cephem-4-carboxylate dissolved in 1 30 cm<3> of dry benzene was added to 83 mg of dry pyridine and 218 mg of phosphorus pentachloride (1.25 equivalent). The mixture was heated in

2 hours below at 56-58°C. The benzene was removed and 35 cm3 of ice-cold methanol was added. The solution was left for 24 hours at room temperature. The methanol was removed. under reduced pressure and 20 cm<3>THF. was added. After cooling, 20 cm<3>pH 4.5 buffer was added and the mixture was left for 20 min. at room temperature. After the THF was removed under reduced pressure, dilute NaCl solution and ethyl acetate were added, after which the pH was adjusted to 6.5 with solid NaHCO 3 . The organic layer was removed, washed twice with saturated NaCl solution, dried over MgSO 4 , filtered and evaporated to a small volume. A solution of this t-butyl-7-amino-3-methoxymethyl-α<3->cephem-4-carboxylate in 20 cm<3>ethyl acetate was added to a solution of 160 mg of p-toluenesulfonic acid monohydrate. The p-toluenesulfonate salt of t-butyl-7-amino-3-methoxymethyl-A<3->cephem-4-carboxylate crystallized spontaneously and weighed 177 mg, (45% yield). The solid salt was recrystallized from isopropyl alcohol to give a purer sample, m.p. 183-187°C, and the structure was confirmed by I.R., U.V., N.M.R. and elementary analysis.

Example 9

t-buty1-7-(2'-thienylacetamido)-3-cyanomethyl-■ Ar- 7.- cef em- 4- carboxy lat-

A cooled mixture of 467.5 mg (lmmol) t-butyl-7-amino-3-cyanomethyl-Ar■ 7'.-cef em-4-carboxylate-p-toluenesulfonate, (420 mg, 5 mmol) solid NaHCO , and 30 cm<3> of dry acetone was added to 48 mg (3 mmol) of distilled thiophene-2-acetyl chloride in 10 cm of dry acetone. The reaction mixture was stirred for 1 hour in the cold (0-5°C) and 3 hours at room temperature. The acetone was removed under reduced pressure, whereupon benzene was added. This mixture was then washed successively with sodium bicarbonate solution, cold 5% HGT solution, five times with NaHCO 3 solution and then with a NaCl solution, dried over MgSO 4 , filtered and evaporated to a product weighing 481 mg. This product was crystallized from carbon tetrachloride to give 271 mg (66% yield) of t-butyl 7-(2'-thienyl)acetamido-3-cyanomethyl-A-'-cephem-4-carboxylate, m.p. 164-166°C. The structure was confirmed by I.R., U.V., N.M.R. and elementary analysis.

Example 10

7-(2'-thienyl)acetamido-3-cyanomethyl-A-cef em-4- carboxylic acid

A solution of 455 mg of t-butyl-7-thiophenacetamido-3-cyanomethyl-α 3 -cephem-4-carboxylate in 40 cm 3 of 98-100% formic acid was stirred under N 2 at room temperature for 2.5 hours. The formic acid was then removed under reduced pressure. Ethyl acetate and water were added - and the pH was adjusted to approx. 7.5 with sodium bicarbonate. The aqueous layer was added with ethyl acetate and the mixture cooled and acidified to pH 2.8 with 20% HCl. The organic layer was removed, washed two . times with saturated NaCl solution, dried over MgSO 4 , filtered and evaporated to give 234 mg of a golden foam.

This foam was dissolved in boiling ethyl ether (10 mg of insoluble matter). After evaporation to a small volume, the desired acid was crystallized, namely 7-(2'-thienyl)-acetamido-3-cyanomethyl-A3-cephem-4-carboxylic acid, the yield was 97 mg, and the substance melted at 114- 117°C. The structure was confirmed by I.R., U.V.,.and N.M.R. It produced an active, antibiotic zone on a bioautograph in a paper chromatogram that developed somewhat faster than for sodium cephalothin.

Example 11

p-Methoxybenzyl-7-amino-3-methoxymethyl-A"'<i>cephem- 4- carboxylate- p-toluenesulfonate- salt A solution of 996 mg (2 mmol) p-methoxybenzyl-7-phenoxyacetamido-3-methoxymethyl -A'i<->cephem-4-carboxylate and 221.5 mg of dry pyridine (1.4 equivalent) in 75 cm3 of dry benzene was added 601 mg (40% excess) of phosphorus pentachloride. The mixture was heated under N~ for two hours at 50° C. The benzene was removed by evaporation under reduced pressure, after which 100 cm-' of ice-cold methanol was added. After standing overnight at room temperature, the methanol was removed by evaporation under reduced pressure. The residue was added to .40 cm<3> THF, and after cooling, 40 cm<3>water was added. The homogeneous mixture was allowed to stand for 25 min., THF removed under reduced pressure, some ethyl acetate and water were then added, after which the pH was adjusted to 6.5 with solid NaHCO^ The organic layer was removed, washed twice with saturated NaCl solution, dried with MgSO 4 , filtered and evaporated to a small volume. In a solution of this p-methoxy benzyl-7-amino-3-methoxymethyl-fem-4-carboxylate in a total of 80 cm<3>ethyl acetate was added to a solution of 380 mg (2 mmol) p-toluenesulfonic acid monohydrate in 10 cirr 3 ethyl acetate. Within a couple of minutes, the p-toluenesulfonate salt was crystallized. After 3 hours the crystals were collected by filtration. Weight: 781 mg (73 yield), m.p. l60-164°C. The structure was confirmed by I.R., U.V., N.M.R. and

elementary analysis.

Example 12

t-butyl-7-(2'-sydnon-3-acetamido)-3-methoxymethyl-A3 - cef em- 4- carboxylate [

The ibsyl salt of t-butyl-7-amino-3-methoxymethyl-A 3-cephem-4-carboxylate (236 mg, 0.5 mmol) was stirred in a water/ethyl acetate mixture. The pH was adjusted to 7 with Na 2 HPO 3 . The ethyl acetate layer was shaken with saturated sodium chloride solution, dried with magnesium sulfate and then evaporated. The residue was dissolved in 10 ml of tetrahydrofuran (THF) and then added a t-butyl mixed anhydride of 2-sydnon-3-acetic acid prepared at -5°C by adding 50 mg (0.07 ml) of triethylamine and 0.06 ml of pivaloyl chloride to 80 mg of 2-sydnone-3-acetic acid in 10 ml of dry tetrahydrofuran. After stirring in an ice bath for 1 hour and then 2 hours, at room temperature, the reaction mixture was diluted with 100 ml of water and extracted with ethyl acetate. The ethyl acetate solution was shaken at pH 8 with phosphate buffer, dried with magnesium sulfate and concentrated. The residue was stirred with ethyl ether, collected, washed with ether and vacuum dried to give 95 mg of the above ester. An NMR spectrum confirmed

the structure. Example 13

7-(2'-sydnon-3'-acetamido)-3-methoxymethyl-A. - cephem-4-carboxylic acid

An 86 mg portion of t-butyl 7-(2'-sydnon-3-acet-amido)-3-methoxymethyl-A 3-cephem-4-carboxylate was dissolved in 2 ml of 98% formic acid and stirred at room temperature for 2 hours. The solution was evaporated to dryness. The resulting semi-solid residue was mixed with ethyl ester and the solid was then collected and vacuum-dried, yielding 52 mg of 7-(2'-sydnon-3'-acetamido)-3-methoxymethyl-A3-cephem- 4-carboxylic acid. Elemental microanalyses and an ultraviolet spectrum were consistent with the structure.

Example 14

t-butyl-7-(2'-carboxy-2'-phenylacetamido)-, 3~ methoxymethyl- A3- cephem- 4- carboxyl

A solution of .328 mg (1.4 mmol) of mono-t-butyl ester of phenylmalonic acid and 0.21 ml (156 mg, 1.54 mmol) of triethylamine in 30 ml of THF was cooled to -5°C, after which one added

■168 mg (0.168 ml) (1.4 mmol) of pivaloyl chloride to produce the t-butyl mixed anhydride. After 20 min. a solution of 342 mg (1.4 mmol) of 7-amino-3T-methoxymethyl-A<3->cephem-4-carboxylic acid (7-AMCA) was prepared by hydrolyzing t-butyl-7-amino-3-methoxymethyl-A ^-cephem-4-carboxylate ester in formic acid, in a mixture of 10 ml water - 10 ml THF, containing an equivalent amount of triethylamine, added to the mixed anhydride solution. After the mixture was stirred for 1 hour at 0°C and 2 hours at room temperature, the reaction solution was cooled, ethyl acetate was added, and the pH was adjusted to 2.0 mpd 6-N HCl. The ethyl acetate solution was dried and concentrated. The residue was taken up in ethyl ether, then reprecipitated with a commercially available petroleum hydrocarbon fraction, mostly hexane, bp 60-68°C), yielding 88 mg of t-butyl-7-(2'-carboxy-2' -phenylacetamido)-3-methoxy-methyl-A<3->cephem-4-carboxylate..

Example 15

The t-butyl-7-(2'-carboxy-2'-phenylacetamido)-3-methoxy-methyl-A3-aephem-4-carboxylate ester from Example 14 was stirred in 1 ml of cold trifluoroacetic acid. After 15 minutes, the solution was evaporated. The residue was stirred in 5 ml of a 1:1 v/v mixture of

petroleum hydrocarbons (boiling point 60-68°C); ethyl ether for 1 hour, then collected and vacuum dried, yielding 56.1 mg of 7-(2'-carboxy-2'-phenylacetamido)-3-methoxymethyl-α<3->cephem-4-carboxylic acid. Elemental analyzes and U.V. spectrum was in accordance with this structure.

The above-mentioned new cephalosporins gave characteristic R^ values by paper chromatography and were tested in vitro microbiological samples where all compounds showed excellent activity against several strains of staphylococci. After an oral administration, the derivatives in mice showed a very effective protection against fatal streptococcal infections.

Example 16

7-amino-3-methoxymethyl-A<3->cephem-4-carboxylic acid.

268 mg (1 mmol) of the p-toluenesulfonate salt of p-methoxybenzyl-7-amino-3-methoxymethyl-A3-cephem-4-carboxylate was stirred in water and then disodium orthophosphate was added to pH 7 while

to the solution was added ethyl acetate. The ethyl acetate solution was dried over magnesium sulfate and evaporated to dryness. The oily residue was dissolved in 25 ml of benzene containing 40 drops of anisole and 0.8 ml of trifluoroacetic acid. After the mixture had been stirred for 2 hours at room temperature, the benzene was removed, after which the residue was dissolved in 10 ml of water, with 10 ml of ethyl acetate added. The pH was adjusted to 3.6 by an addition of triethylamine. After the mixture was cooled overnight, the precipitated solid was collected, washed with water and ethyl acetate and then vacuum dried at 45°C to give 75 mg of 7-amino-3-methoxymethyl-A<3->cephem-4- carboxyl.ure. The structure was confirmed by I .R. , U.V. and N.M.R. spectra.

A similar procedure was used to prepare 7-amino-3-ethoxymethyl-α<3->cephem-4-carboxylic acid from its p-methoxy-benzyl ester. Example 17

7-(D-2-amino-2-phenylacetamido)-3-methoxy-methyl-3-cephem-4-carboxylic acid

A solution of 185 mg (0.74 mmol) of D-2-t-butoxy-carboxamido-2-phenylacetic acid in 15 ml of tetrahydrofuran (THF) was stirred and cooled to -5°C. After addition of 82 mg (0.81 mmol) of triethylamine and 110 mg (0.81 mmol) of isobutyl chloroformate, the reaction mixture was stirred for 20 minutes. To the resulting mixed anhydride solution was added a solution of 180 mg (0.74 mmol) of 7-amiho-3-methoxymethyl-3-cephem-4-carboxylic acid (7-AMCA) in 5 mL of water, 5 mL of THF, and 0.103 mL of triethylamine. . After the mixture was stirred for 1 hour in an ice bath and 2 hours at room temperature, ethyl acetate was added, cooled, after which the pH was adjusted to 2.0 with 6N HCl. The ethyl acetate layer was dried with MgSO 4 and evaporated to dryness. The residue was dissolved in ethyl ether. After crystallization began, the flask was cooled for several days. The solid was collected and vacuum dried, yielding 95.5 mg of 7-(D-2-t-butoxycarboxamido-2-phenylacetamido)-3-methoxymethyl 3-cephem-4-carboxylic acid (t-BOC compound). A new yield was obtained by diluting the above filtrate with a petroleum fraction (mostly hexane), boiling point 60-68°C. A cold (0°C) solution of 150 mg of the t-BOC compound in 2 ml of trifluoroacetic acid was stirred for 5 min. and then evaporated to dryness. The residue was dissolved in 2 ml of water. To this solution was added 25% "Amberlite LA-1" liquid anion exchange resin (acetate form) in methyl isobutyl ketone (MIBK) to pH 4.-5. The aqueous layer was retreated with liquid anion exchange resin and then washed with MIBK. The aqueous layer was then evaporated to approx. 0.5 ml and diluted with acetonitrile. The precipitated gel showed slow crystallization. The solid crystals were collected, washed with acetonitrile and vacuum dried, yielding 42 mg of 7-(D-2<1->amino-2'-phenylacet-amido )-3-methoxymethyl-A<3->cephem-4-carboxylic acid . The structure was confirmed by U.V. and NMR spectra as well as by microanalyses. Examples 18-21

.7-[d-2'-amino-2<1->(3"-hydroxyphenyl)-acetamido]-5- methoxymethyl- A3- cephem- 4- carboxylic acid

The acylation of 7-AMCA with D-2-t-butoxycarboxamido-2-(3-hydroxyphenyl)acetic acid was carried out using the same mixed anhydride method as in the aforementioned examples. The removal

of the t-BOC group and the isolation of the 7-[d-2'-amino-2'-.(3"-hydroxy-phenyl)acetamido]-3-methoxymethyl-A3-cephem-4-carboxylic acid as the zwitterion product was carried out as follows: A solution of 513 mg of the t-BOC-protected compound was prepared in 6 ml of cold (0°C) trifluoroacetic acid. After 5 min, the excess of trifluoroacetic acid was evaporated. The residue was dissolved in 2 ml of water with a overlying layer of 10 mL of MIBK. The pH was adjusted to 5 by the addition of tributylamine. The aqueous layer was separated, washed with MIBK, and evaporated to about 0.5 mL. The solution was diluted with 5 mL of acetonitrile and then cooled. After several hours they were

precipitated crystals collected, washed with acetonitrile, and vacuum dried at 50°C, giving 249 >1 mg of the product. In the following tables, phenylglycine derivatives prepared in the same way are indicated. as in example 18.

Example 22

A series of acylations was carried out in the following ways:

A solution of 11 mg of 7-AMCA and 11 mg of NaHCO-^ was prepared in 5.5 ml of water. Portions (0.75 ml) of this solution were stirred in an ice bath and portions of acid chloride solutions of the indicated R group (containing 1.5 mg of acid chloride) were added. After stirring for 30 min. the acetone was evaporated and each solution diluted with H 2 O to 1.5 ml. These solutions were then tested against a wide spectrum of microorganisms. It was found that the reaction solutions could also be tested directly without removing acetone. The compounds were also subjected to paper chromatography and bioautograms.

The table below indicates antibiotic data obtained for the solutions, the side chains used as well as R^ values obtained from said bioautograms.

Footnotes.

1. is defined as the ratio between the distance of the respective point (bioautogram) from the starting point and the distance between the point of the standard antibiotic sodium cephalothin and its starting point. 2. The P^ ratio was determined from the bioautograph on the paper chromatogram of the sample cephalosporin compound relative to a standard compound which was Keflin (sodium cephalothin, Lilly) designated by "K", a commercially available cephalosporin antibiotic.

The test methodology itself was generally that described by Bird and Pugh in Antibiotics and Chemotherapy, Vol. IV, No. 7, July 1954, except that Whatman No. 4 paper, buffered to pH 4.6 with 0.1 molar sodium acetate, . and in that the liquid of the chromatography chamber consisted of a mixture of 600 parts of methyl ethyl ketone and 400 parts of water, and that the developing solution consisted of a mixture containing 92 parts of methyl ethyl ketone and 8 parts of water. Each chromatogram was run for from 2 1/2 to 3 1/2 hours.

The paper chromatography technique itself is described in general terms in a book, namely 'Paper Chromatography, third edition, published by I.M. Hais et al. (1963)>Academic Press, New York and London1, page 155.

3 • S . A. = Staphylococcus aureus

XL2 = B. 'subtilis

X186 = Sarcina sp.

X85 = Saccharomyces pastorianum

X.45 - Proteus vulgaris

Xl42 = Salmonella gallinarum

Xl6l = Escherichia coli

X48 = Pseudomonas aeruginosa

If the zone size on the agar disc was greater than

8 mm, then this was an indication that the test connection was

antibiotically active against the test organism in this qualitative sample.

Claims (1)

Process for the preparation of A <3-> '3-substituted cephalosporanic acid derivatives of formula and their pharmaceutically acceptable salts, wherein R represents hydrogen where X is amino, hydroxy, carboxyl or C 1 -C 2 -alkanoyloxy, and Q denotes hydrogen, hydroxyl, chlorine or methoxy, where X"*" denotes oxygen, sulfur or a chemical bond, y is 0-3, z is 1 or 2 and Q<1> is hydrogen, nitro,. chlorine, trifluoromethyl or fluorine; 2 where Q denotes hydrogen, where each group Z represents hydrogen or, together with the carbon atoms to which they are attached, terminates a benzene ring, where each of the groups R 1 and R 2 denotes hydrogen or methyl, 2-sydnon-3~(C1~C^)alkanoyl-j ( <C>1 -C1(-) )alkanoyl, . (C-j^-C-^q )alkenoyl, (Cj-C^, )alkyl-X-(C-j^-C^ )alkanoyl, where X denotes oxygen or sulphur, (C^-C^, )alkenyl-X-(C-L-C^)alkanoyl3 where X denotes oxygen or sulphur, (C2-C10)haloalkanoyl, where halogen denotes fluorine, chlorine or bromine, or (C8-C-8)cyanoalkanoyl, R' denotes cyan or -OY where Y denotes (C^-C^ )alkyl, acetyl or propenyl, and R" is selected from hydrogen, (C 1 -C 8 )-t-alkyl, nitrobenzyl or methoxybenzyl, characterized in that corresponding A 2 -compounds with formula: where R' and R" have the above meanings, and R".' denotes phenoxyacetyl, thiophenacetyl or phenylacetyl, is reacted with an oxidizing agent to produce the corresponding A^3 -sulfoxide, which is then reduced to said A3-cephalosporin ester, after which the R"' group is optionally cleaved from the 7-position and the 7-position is then acylated to an R -group as indicated above, and that one optionally prepares desired salts of these compounds.