Cephalosporin sulfonate esters

(54) Title: Process for the preparation of 3-cephem-3-sulfonate ester compounds

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The present invention relates to the preparation of 3-cephem-3-sulfonate ester compounds. These compounds are valuable as antibacterial agents. They have the following general formula I:

<img file="SE414769B_D0001.tif" />

where! R represents tenyl or des-amino-benzyl, yl and represents θθ-C ^ alkyl, halophenyl or C C-Cj alkyl fertyl. The invention also encompasses the preparation of the pharmaceutically acceptable, non-toxic salts of these compounds.

In the above definition of the compounds in question, by alkyl is meant the straight and branched alkyl hydrocarbon groups such as e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, n-amyl, isoamyl or n-hexyl. By the term halogen is meant in the present specification

74Θ3306-9 fluorine, chlorine, bromine and iodine.

The sulfonate esters of the invention are prepared by producing a 3^-hydroxy-3-cephem compound of the formula

<img file="SE414769B_D0002.tif" />

wherein R has the above meanings and R 2 represents a carboxylic acid protecting, ester-forming group, reacted with a sulfonyl halide of the formula₂-sx wherein R 2 has the above meanings and X represents halogen and removing the carboxylic acid protecting, ester-forming group to form the corresponding acid.

The reaction can be carried out at a temperature between about -5 and + 35 ° C in an aprotic solvent in the presence of a hydrogen halide acceptor.

Examples of sulfonyl halides which may be used are methanesulfonyl chloride, methanesulfonyl bromide, ethanesulfonyl chloride, n-butane sulfonyl chlorine n-hexanesulfonyl bromide, p-chlorobenzenesulfonyl chloride, p-fluorobenzenesulfonyl chloride, p-toluenesulfonide, p-toluenesulfonide

Aprotic solvents which may be used are ether solvents such as, for example, tetrahydrofuran, dioxane or the dimethyl ether of ethylene glycol. A preferred solvent, which may be used, is dimethyl ether amide.

The reaction is carried out in the presence of a hydrogen halide acceptor, for example a non-reactive tertiary amine, such as triethylamine or pyridine, or also an alkylene oxide, for example propylene or butylene oxide. The preferred hydrogen halide acceptor is propylene oxide. The tertiary amine acceptors have a certain tendency to cause isomerization of

The 3-cefem compound to a 2-cefem compound. When using an alkylene oxide, such isomerization is minimized when most sulfonyl halides are used.

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The reaction is carried out by adding a stoichiometric amount of the sulfonyl halide or a small excess of the sulfonyl halide to a solution of the 3-hydroxy-3-cephem ester in the aprotic solvent containing a least stoichiometric amount of the hydrogen halide acceptor. The reaction mixture is stirred at a temperature, which is preferably 10-25%, for 3 to 12 hours. The sulfonate ester product is recovered from the reaction mixture by extraction with an organic solvent such as ethyl acetate or methylene chloride, and is recovered from the extract.

When the R substituent of the starting material is β6-aminobenzyl, the oC-amino group of the phenylglycyl side chain can be protected during the sulfonyl ester formation by a number of different amino protecting groups. Thus, for example, urethane protecting groups such as t-butyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl or p-nitrobenzyloxycarbonyl can be used; enarin-protecting groups formed with ethyl acetoacetate, or acetylacetone; trityl group and other amino group protecting groups. After the sulfonylation reaction, such protecting groups are removed. The C C-carboxylic acid protecting group is also removed, giving the antibiotic-active compound 3-sulfonyloxy-3-cephem-3-carboxylic acid.

The above description of the preparation of the sulfonate esters is illustrated in the following reaction scheme.

hrs

<img file="SE414769B_D0003.tif" />

COOR.

<img file="SE414769B_D0004.tif" />

the ester group is removed. 7403306-9 v

<img file="SE414769B_D0005.tif" />

wherein R = and R₂ has the above meanings.

In one embodiment of the invention, p-nitrobenzyl-7- (N-butyloxycarbonyl-D-phenylglycylamido) -β-hydroxy-3-cephem-4-carboxylate is reacted with methanesulfonyl chloride in dimethylacetamide in the presence of propylene oxide at about 5 ° C. to give 7- (N-butyloxycarbonyl-D-phenylglycylamido) -3-methylsulfonyloxy-3-cephem-4-carboxylic acid p-nitrobenzyl ester. The product is hydrogenated over pre-reduced palladium on carbon as a catalyst in an inert solvent for removal of the p-nitrobenzyl ester group, whereupon the esterified product is reacted with p-toluenesulfonic acid in acetonitrile and to remove the butyloxycarbonyl group. 7- (D-phenylglycylamido) -3-methylsulfonyloxy-3-cephem-4-carboxylic acid.

The starting compounds used for the preparation of the compounds of the invention are prepared by producing a 7-acylamido-3-exomethyleneefam-4-carboxylic acid ester or^-amino-3-exomethylenespham-4-carboxylic acid ester is reacted with ozone in an inert solvent at a temperature between -80 ° C and 0 ° C to form the ozone derivative of the β-exomethylene double bond. The ozonide obtained as an intermediate, which is not isolated, is decomposed by reacting in situ with a mild reducing agent, such as sodium bisulfite or preferably sulfur dioxide, to form the corresponding 3-hydroxy-5-cephem-4-carboxylic acid ester.

The ozonolysis of a 7-amino-3-exomethylenespham-4-carboxylic acid ester or a 7-acylamido-3^ex ° methylenespham-4-carboxylic acid ester of the formula III below is carried out by passing ozone through a solution of the 3-xomethylenesame ester in an inert solvent at a temperature of about -80 ° C. The exomethylene double bond reacts with ozone, thus forming in situ an ozonide as an intermediate. This ozonide is decomposed in the manner described below to form the 3-hydroxy 3-cefemester of formula IV.

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RN

C

<img file="SE414769B_D0006.tif" />

l = CH 2 (III)

RN

COOR

<img file="SE414769B_D0007.tif" />

<img file="SE414769B_D0008.tif" />

OH

COORi (IV)

In the above formulas, R represents hydrogen or an acyl group derived from a carboxylic acid, which acyl group is non-oxidizable under the ozonolysis conditions described. R represents an ester forming group, preferably such an ester forming group which can be easily removed under hydrogenolysis or acid hydrolysis or base hydrolysis conditions.

Ozone gas is produced by an ozone generator of the type commonly used in chemical synthesis and analysis work to produce ozone by electrically discharging the oxygen.

Such an ozone generator is, for example, that manufactured by Welsbaok Corporation. The ozone is generated in a stream of oxygen, which is then passed directly into the reaction vessel. The percentage amount of ozone in the oxygen stream may vary as desired, for example, by varying the rate at which oxygen is brought to the stream through the ozonizer and also by varying the intensity of the electric discharge. The percentage amount of ozone in the oxygen stream can be determined iodometrically; by titrating with sodium thiosulfate the amount of iodine produced from a standard solution of potassium chloride by means of ozone obtained from the generator. The percentage of ozone in the oxygen stream is not critical; however, in practicing the ozone oolysis method according to the invention, it is convenient to make an estimate of the amount of ozone flowing into the reaction mixture, for in this way it is possible to determine the time at which the desired reaction should be completed, and thus can be completed.

7403306-9 formation of over-oxidation products is kept to a minimum.

Alternatively, the ozonolysis reaction can be continuously observed chromatographically. For example, a small sample of the reaction mixture is taken, decomposes the ozonide, and an estimate of the amount of unreacted starting material and J -> cephem.

Thus, inert solvents which can be used in ozone analysis are solvents in which the 3-exomethylene cephamesters are at least partially soluble and which are not reactive to ozone under the conditions described. Commonly used organic solvents such as methanol, ethanol, ethyl acetate, methyl acetate and methylene chloride are satisfactory.

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

The preferred temperature for ozone analysis is about -80 ° C to 50 ° C.

When ozone formation is complete - as determined by one or the other of the methods described above - excess ozone may be driven off, which may be present in the reaction mixture by bubbling nitrogen or oxygen through the mixture.

After the possible ozone excess has been removed, the ozone is decomposed by adding a mild reducing agent to the reaction mixture; this is selected from the following: sodium bisulfite, sulfur dioxide, trimethyl phosphite. Thus, the 3-hydroxy-3-cephem-4-carboxylic acid ester is obtained. The decomposition is carried out by adding an excess of the reducing agent, and then stirring the reaction mixture at a temperature of approx. -8o to 0 ° C until the reaction mixture in the potassium iodine test-¹and starch produces negative results.

A preferred reagent for decomposing the ozonide obtained as an intermediate is gaseous sulfur dioxide. This reagent is preferred because it is completely volatilized from the reaction mixture in the subsequent work-up and consequently does not cause any complication in the reaction product recovery.

The 7-acylamido-3-hydroxy-5-cephem-4-carboxylic acid esters are recovered from the reaction mixture by first evaporating it to dryness, then extracting the product from the residue. Alternatively, N-acylated 3-hydroxy-3-cephemesters can be recovered from the organic phase of the mixture obtained by decomposition, by separating this organic liquid phase from insoluble matter; After washing and drying, the organic layer is evaporated to give the 3-hydroxy ester.

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The 3-hydroxy nucleus ester, a 7-amino-3-hydroxy-3-cephem-4-carboxylic acid ester, can best be isolated in the form of a salt, for example hydrochloride or the hydrobromide salt.

When an ester of 7-amino-2-exomethylenespham-4-carboxylic acid (formula III, R = H) is ozonized, it is preferred to use a salt of this nucleus, for example the hydrochloride or p-toluenesulfonate salt.

The starting materials for the preparation of the 3-exomethyleneefamesters are prepared by reacting a 7-acylamidocephalosporanoic acid with a sulfur-containing nucleophilic agent in accordance with known methods and for producing nucleophilic-substituted-7 -methyl-3 "Cefem-4-carboxylic acid. The 3-position thio-substituent cefem product is then reduced with hydrogen in the presence of Raneyniokel or with zinc / formic acid in the presence of dimethylformamide to form the 3-exomethylene acetic acid. For example, 7-phenylacetamidocephalosporanoic acid can be quenched to react with potassium ethyl xanthate to give 7-phenylacetamido-3 “-toxionocarbonylthiomethyl-3-ephem-4-carboxylic acid, which upon reduction with zinc / formic acid in the presence of DMP gives 7-phenyl 4-carboxylic acid of the formula

<img file="SE414769B_D0009.tif" />

<img file="SE414769B_D0010.tif" />

Similarly, the 3-exomethylenespha nucleus of the formula

<img file="SE414769B_D0011.tif" />

is prepared by reacting a 7-acylamido-3-exomethylene-cefam-4-carboxylic acid ester with phosphorus pentachloride (PCLL) in methylene chloride in the presence of pyridine to form the imino chloride as an intermediate. This imino chloride is reacted in cold with methanol to give the imino ether. This imino ether can be readily hydrolyzed to give 7-amino-3-exomethylenespham-4-carboxylic acid ester hydrochloride. The ester group is then removed, thus obtaining 3-x x °ⁿ’^etylencefamkärnan.

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An ester of the 3-exomethylenespham nucleus can be acylated with the desired derivative of a carbonic acid to form the 7-acylamido-3-exomethylenespham-4-carboxylic acid ester and the acylated ester can be ozonized to the 3-hydroxy ester starting material of formula II. Alternatively, an ester of the 3-hydroxy-7-amino-3-cephem core can be acylated under non-anhydrous conditions to give the 7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid ester. The acylation of these core esters is carried out under the conditions used for the acylation of 7-aminocephalosporanoic acid. However, when acylating a 7-amino-3-hydroxy-3-cefemester, it is convenient to carry out the acylation reaction in an aqueous medium, for example in aqueous acetone or aqueous acetonitrile.

7-aeylamido-3-alkyl or phenylsulfonyloxy-β-cephem-4-carbox, the acetic esters are intermediates in the preparation of the antibiotic free acid forms of the compounds in question. Ester-forming groups as defined above are all known groups, those which are very often used to protect the C ^-carboxylic acid group of the cephalosporin molecule while conducting reactions to which other groups in the molecule are to react. These ester-forming spheres can be easily removed to form the free acid, the removal being accomplished by known reduction or hydrolysis methods. For example, the p-nitrobenzyl ester group is removed by catalytic hydrogenolysis over palladium on carbon; the diphenylmethyl group (benzhydryl) is removed with trifluoroacetic acid in anisole at about 10 ° C; The p-methoxybenzyl group is removed with trifluoroacetic acid at about 10 ° C / J. Org. Chem., 36, 1259 (1971) 7 in the 2,2,2-trichloroethyl group is removed with zinc and acid / J. Am. Chem. Soc. 88, 852 (1966/7; the benzyl ester group is removed by catalytic hydrogenolysis over a palladium catalyst / J. Org. Chem., 27 &lt;1381 (1962/7 and the tert.butyl group is removed in the manner described by J. Org. Chem. , 31, 444 (1966).

In the preparation of the 3-sulfonate esters of the invention, it is sometimes the case that a smaller amount of the corresponding 2-cephem isomers is formed. Preparation of the tosylate esters is generally accompanied by more isomerization to the 2-cephem isomer than preparation of the alkyl sulfonate esters. When the amount of 2-cefem-3-sulfonate ester formed in the sulfonylation reaction is significant, the mixture of the 2- and 3-cefem-sulfonates can be chromatographically separated over silica gel. However, it is preferred to oxidize the isomer mixture with a peracid, such as chloroperbenzoic acid or peracetic acid, to give the corresponding sulfoxides. As one skilled in the art knows, the sulfoxide formation of a 2-cephem is isomerized to the 3-cephem isomer / µ.^amChem. Soc.

2S, 2430 (1970/7. Then the 3-cephem-3-sulfonate ester sulfoxide is reduced

7403306-9 to the sulfide, for example by means of a phosphorus halide such as phosphorus trichloride. Accordingly, any optional 2-cephem isomer product in the desired 3-cephem sulfonate ester can be converted to the 3-cephem isomer, which is the desired compound.

The 7-acylamido-3-cefem-3-sulfonate esters of formula I, (the free acid) are antibiotic agents suitable for controlling such infections caused by Gram-positive and Gram-negative bacteria and also penicillin-resistant staphylococci. The antibacterial activity of these sulfonate esters is illustrated by the following test data for in vitro experiments with representative compounds of the invention. Table I below lists the minimum concentration (MIC) required for representative compounds against Gram-negative bacteria. The data in question were obtained by the standard Gradient-Plate method.

Table I

Antibiotic activity of 3-cefem-3^_sulfonatesterföreningar; impact on Gram-negative bacteria

Test compound 17

Test organism For minimum inhibition required (yUg / ml)

2 5 4 5 6

<td>Shigella sp.</td><td> 10,7</td><td> 7,0</td><td> 6,5</td><td> 19,5</td><td> 16,5</td><td> 13,6</td>

<td>Escherichia coli</td><td> 11,0</td><td> 13,1</td><td> 8,8</td><td> 16,3</td><td> 24</td><td> 19,5</td>

<td>Klebsiella pneumoniae</td><td> 6,5</td><td>7, O</td><td> 5,5</td><td> 9,3</td><td> 16,3</td><td> 5,5</td>

<td>Aerobacter aerogenes</td><td> 3,5</td><td> 3,5</td><td> 6,0</td><td> 16,5</td><td> 12,0</td><td> 8,8</td>

<td>Salmonella heidelberg</td><td> 2,0</td><td> 4,0</td><td> 6,0</td><td> 125</td><td> 13,5</td><td> 7,0</td>

<td>Pseudomonas aeruginosa</td><td> )200</td><td> )200</td><td> )200</td><td> &gt;200</td><td> )200</td><td> )200</td>

<td>Serratia marcescens</td><td> 110</td><td> 120</td><td> 180</td><td> &gt;200</td><td> &gt;200</td><td> 100</td>

1/

Test compounds 1-6 are as follows:

1: 7^-/ 2- (2-thienyl) -acetamidoJ7^_3-methylsulfonyloxy-3-cephem-4-carboxylic acid. 2: 7-Z2- (2-thienyl) -acetamido7-3-ethylsulfonyloxy-3-cephem-4-carboxylic acid. 3: 7- [2- (2-thienyl) -acetamido] -3-n-butylsulfonyloxy-3-cephem-4-carboxylic acid 4: 7-Z, 2- (2-thienyl) -acetamide] 7-3- (p -toluenesulfonyl (xi) -3 "cefem-4-carboxylic acid.

5: 7- / 2- (2-thienyl) acetamido / 3- (4-fluorobenzenesulfonyloxy) -3-cephem-4-carboxylic acid.

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6: 7 ~ (D-Phenylglycylamido) -3-methylsulfonyloxy-3-cephem-4-carboxylic acid.

In the same testing procedure, it is found that the cephalosporin sulfonate esters inhibit the growth of clinical isolates of penicillin-resistant staphylococci.

The 7-acylamido-3 “cefem-3-sulfonate esters of formula I form pharmaceutically acceptable salts with inorganic and organic bases. For example, the sodium, potassium and calcium salts can be prepared with sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate or calcium hydroxide. Amine salts are formed with amines such as benzylamine, 2-aminoethanol, diethanolamine or diisopropylamine. Compounds of formula I wherein RC (O) - represents the phenylglycyl side chain, form acid addition salts with appropriate mineral acids and organic acids. For example, the hydrochloride, hydrobromide and sulfate salts can be formed with hydrochloric, hydrobromic and sulfuric acids. Similarly, the p-toluenesulfonate salt of the amine can be prepared with p-toluenesulfonic acid. Such salts of the C ^ carboxyl group and an amino group of the C ^:

The invention is further illustrated in the examples below which indicate the preparation of the starting materials and the preparation of the compounds according to the invention.

A. Preparation of starting material

Example 1. p-nitrobenzyl-7-amino-3-methylenespham-4-carboxylate hydrochloride.

To a solution of 965 mg (2 mmol) of p-nitrobenzyl-7-phenoxyacetamido-3-methylenespham-4-carboxylate in 10 ml of methylene chloride was added 175 mg of dry pyridine and 460 mg of phosphorus pentachloride, and the mixture was stirred for 6 hours at room temperature. One ml of isobutanol was added to the mixture, which was then allowed to stand overnight at 0 ° C. The reaction product, p-nitrobenzyl-7-amino-3-methylenespham-4-carboxylate hydrochloride, which was obtained in the form of a crystalline precipitate, filtered off to give 430 mg (58 L, yield).

Elementary analysis for ^G15^hrs16^N3°5^SC1 in

Calculated: C, 46.69; H, 4.18; N, 10.89

Found: C, 46.40; H, 4.20; N, 10.62 i

IR (nujol mold)

Carbonyl absorption at 5.65 (β-lactam) and 5.75 (ester) µm

I t

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NMR (DMSO dg) signals at 6.34 (2d, 2H, Cg-Hg),

4.98 (d, 1H, Cg-H); 4-, 7-4.4 (m, 6H, C ^-H, ester-CHg, C₄-CHg and Cy-H); and 2.4-1.6 (m, 4H, aromatic H) +.

Example 2. p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride.

A solution of 4 g of p-nitrobenzyl-7-amino-3-methylenespham-4-carboxylate hydrochloride in 620 ml of methanol was cooled in a bath of dry oil and acetone, and through the cold solution ozone was bubbled for about 20 minutes.

The residual ozone was evaporated from the reaction mixture by passing nitrogen through the solution and 10 g of sodium bisulfite was added.

The reaction mixture was stirred for one hour at ice bath temperature, after which the reaction gave negative results in the potassium iodine starch test.

The mixture was evaporated in vacuo to give the reaction product as an amorphous yellow residue. This residue was crystallized in acetone to give 3.4 grams of p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride as a crystalline acetone solvate.

IR (ground with rujol)

Carbonyl absorption band at 5.6θ (β-lactam) and 6.04 (ester carbonyl with hydrogen bonds to the 3-hydroxy group) yum.

NMR (DMSO dg) signals at 7 »92 (s, 3H, 1/2 mol acetone),

6.22. (2d, 2H, Cg-Hg), 5.07 (d, 1H, Cg-H), 4.8-4.5 (m, 3H, esterCHg and Cy-H), 2.4-1.6 ( m, 4H, aromatic H) T.

Example 3. p-Nitrobenzyl 7- / 2- (2-thienyl) -acetamido7-3-hydroxy-3-cephem-4-carboxylate.

To a solution of 1.55 grams of p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride in 3θ ml of acetone, containing 364 mg (0.5 ml, 3.6 mmol) of triethylamine, 962 mg of urea was added. While stirring at room temperature, a solution of 730 mg (4.4 mmol) of 2-thiophenacetyl chloride in 20 ml of acetone was added dropwise to the mixture. After 2.5 hours, the reaction mixture was filtered and evaporated. The residue was dissolved in ethyl acetate, and the solution was washed with water, 50 µg sodium bicarbonate solution, 5% hydrochloric acid, and saturated sodium chloride solution in turn. The washed solution was dried and then concentrated by evaporation in vacuo. There was obtained 1.2 grams of the reaction product in the form of a crystalline residue. The product was recrystallized from ethyl acetate to give pure p-nitrobenzyl-7- (2-thienyl) acetamido7-3-hydroxy-3-cephem-4-carboxylate having the following spectral properties:

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IR (nujol mold):

Absorption peaks at 3.0 (amide-NH), 5.68 (/ 9-lactam carbonyl) and 6.1 (amide, and ester with hydrogen bond to the 3-hydroxy group) 41Π1.

NMR (CDCl3 / DMSO d_g): signals at 6.54 (2d, 2H, C)₂-HRS₂), 6, 16 (s, 2H, side chain CH₂), 4.90 (d, 1H, C)_g-H), 4.60 (d, 2H ester-CH₂), 4.43 (kv, 1H, C)₇-H), 3.1-1.6 (m, 7H, aromatic H), and

1.30 (d, 1H, amide-NH)

B. Preparation of sulfonate esters

Example 4. p-Nitrobenzyl 7-Z 2 - (2-thienyl) -acetamido / -3-methylsulfonyloxy-3-cephem-4-carboxylate.

To a solution of 4.75 grams (10 mmol) of p-nitrobenzyl-7- [2- (2-thienyl) -acetamido7-3-hydroxy-3-cephem-4-carboxylate in 50 ml of dry dimethylacetamide was added 2 ml of propylene oxide . To the solution was added with stirring 1 equivalent of methanesulfonyl chloride, and stirring was continued for 3 hours. Then, the reaction mixture was taken up in ethyl acetate, and the solution was washed with a saturated sodium chloride solution. The washed organic phase was evaporated in vacuo to dryness. There was thus obtained the reaction product mixture which remained. This reaction product was purified by preparative thin layer chromatography on silica gel using 65 L of ethyl acetate / hexane.

In microanalysis, the purified product showed the following values ​​for the individual elements:

Calculated for ^C21^19^3*³9^S3

Calculated: C, 45.56; H, 3.46; N, 7.59; S, 17.38

Found: C, 45.74; H, 3.56; N, 7.30; S, 17.06

The NMR spectrum and the infrared absorption spectrum confirmed the structure of the product formed.

NMR (DMSO dg) T-value δ 3.47 (s, 3H, methyl); 3.80 (broad s, 2H, side chain CH₂); 3.91 (Kv, 2H, C)₂-HRS₂); 5.29 (d, 1H, Cg-H); 5.46 (broad s, 2H, ester-CH₂ ); 5.84 (kv, 1H, C1H); 6.86-7.44 (m, 3H, thiophene); and 7.98 (kv, 4H, phenyl).

IR (ground mass) 1785, 1350 and 1158 cm^-1

UV (ethanol) λ max 264 np.

The above product, 2 grams, was dissolved in a solvent mixture of 15 ml of methanol and 20 ml of tetrahydrofuran, and 3 grams of previously reduced catalyst, 5 $ palladium on carbon, were added; (the catalyst had

7403306-9 was reduced in advance in 15 ml of methanol for 1 hour before use). The mixture was hydrogenated 1.5 hours; during this time hydrogen had been absorbed to a theoretical extent.

The catalyst was filtered off and the filtrate was evaporated to dryness in vacuo on a rotary evaporator. The residue was dissolved in 20 ml of ethyl acetate, and 20 ml of cold water was added. The pH of the solution was adjusted to pH 7 with a solution of sodium bicarbonate, and the organic layer was separated. Ethyl acetate was layered over the aqueous phase and the pH was adjusted with 2.0 N hydrochloric acid to 2.0. The organic layer was separated and combined with an ethyl acetate extract of the acidified aqueous layer. The combined liquid formed from the extract and organic layer was dried over magnesium sulfate and evaporated to dryness. There was thus obtained the esterified product, 7- [2- (2-thienyl) -acetamido / -3-methylsulfonyloxy-3-cephem-4-carboxylic acid.

NMR (acetone dg) δ values: 3.33 (s, 3H, methyl); 3.50-4.00 (m, 4H, 20H)₂); 5.10 (d, 1H, Og-H); 5.88 (d, 1H, C, H); 6.80-7.40 (m, 3H, thiophene).

IR (KBr) 1795, 1175 cm¹

UV (ethanol) ^Amax 265 njp (allocated).

Electrometric titration (80 / aqueous methyl cellulose) pK cl

3,9.

Example 5. 7- [2- (2-Thienyl) -acetamido] -3-ethylsulfonyloxy-3-cephem-4-carboxylic acid.

Using the same procedure described in Example 4, 4.7 grams (10 mmol) of p-nitrobenzyl-7-Z2- (2-thienyl) -acetamido7-3-hydroxy-3-cephem-4-carboxylate was reacted for about 3 hours. in 50 ml of dimethylacetamide containing about 2 ml of propylene oxide, with a stoichiometric amount of ethanesulfonyl chloride. The reaction mixture was dissolved in ethyl acetate, and the solution was washed with a saturated solution of sodium chloride. The organic phase was dried and evaporated to dryness to give the reaction product. This was purified by preparative thin layer chromatography on silica gel using 65% ethyl acetate / hexane for the development. The purified product, p-nitrobenzyl-7- [2- (2-thienyl) -acetamido / -3-ethylsulfonyloxy-3-cephem-4-carboxylate, gave the following values ​​in microanalysis for the composition of^C22^hrs21^N3°9^S3^:

Calculated: 0, 46.55; H, 3.73; N, 7.40; S, 16.95

Found: C, 46.32; H, 3.48. N, 7.5; S, 16.07

IR (chloroform) 1800, 1358, 1163 cm¹

UV (ethanol) \ nax 265 nju.

NMR (CDCl 3) values: 1.47 (t, 3 +, methyl); 3.40 (kv, 2H, CH₂);

3.73 (kv, 2H, C)₂-CH₂); 3.88 (s, 2H, side chain CH₂); 5.13 (d, 1H,

C₆-H); 5.20 (s, 2H, ester-CH₂); 5.91 (kv, 1H, C)_?-HRS); 6.85 (d, 2H,

NH); 7.00-7.44 (m, 3H, thiophene); 7.96 (kv, 4H, phenyl).

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The above product was hydrogenated to remove the p-nitrobenzyl ester group by the hydrogenation procedure described in Example 4. There was obtained 7 - [(2-thienyl) * acetamido] -3-ethylsulfonyloxy-3-cephem-4-carboxylic acid.

NMR (CLClj + acetone-dg) values: 1.45: (t, 3Ά, methyl) ·; 3,4l.

(kv, 2H, CH 3); 3.6? (Kv, -2H, ring-CHg); 3.84 (s, 2H, side chain, -CH 2); 5.12 (d, 1H, Cg-H); 5.83 (kv, 1H, C)₇-HRS); 6.88-7.32 &lt;jn, 3H, ten fen), '----- ° f

IR (ground mass) 17θ7, 1350 and 1170 cm

Electrometric titration in 80 $ aqueous methylellosolve: pK

4,15.

Example 6. 7- / 2- (2-Thienyl) -acetamido / -3-butylsulfonyloxy-3-cephem-4-carboxylic acid.

In the same manner as in the esterification process of Example 4, 4.7 grams of p-nitrobenzyl 7- / 2- (2-thienyl) -acetamido / -3-hydroxy-3-cephem-4-carboxylate were reacted with a stoichiometric amount of n-butylsulfonyl chloride in dimethylacetamide, containing propylene oxide, to give 2.1 grams of the 3-n-butylsulfonyloxide derivative.

Elementary analysis, percent for ^C24^hrs25^N3°9^S3^:

Calculated: C, 48.39; H, 4.23; N, 7.05

Found: C, 47.94; H, 4.27; N, 6.31

NMR (DMSO d values: 0.87 (t, 3H, methyl);

1.10-1.90 (m, 4H, 2 CH 3); 2.45-2.87 (m, 2H, CH₂); 3.71 (kv, 2H, ring CHg); 3.79 (_s, 2H, side chain CH 2); 5.28 (d, 1H, Cg-H);

5.42 (broad s, 2H, ester-CH 3); 5.82 (Q. 1H, cy-H); 6.50-6.97 (m, 3H, thiophene); 8.07 (sq. 4H, phenyl).

IR (CHCl3) 1801, 1355, 1170¹.

The p-nitrobenzyl ester group is removed from the above-mentioned n-butylsulfonyl derivative by the same hydrogenolysis method described in Example 4 to give 7- / 2- (2-thienyl) -acetamido / -3-n-butylsulfonyloxy-3-oemem-4-carboxylic acid.

NMR (CDCl 3) δ values: 0.98 (t, 3H, methyl); 1.10-2.00 (m,

4H, 20H₂); 2.65-3.00 (m, 2H, CH 3); 3.60-4.10 (m, 4H, Cg Joch Side Chain CHg); 5.09 (d, 1H, Cg-H); 5.78 (kv, 1H, C_?-HRS); 6.85-7.38 (m, 3H, thiophene).

IR (CHCl 3) 1787, 1336 cm¹

Electrometric titration in 80% aqueous methylellosolve pK_A 4.75 Example 7. 7- [2- (2-Thienyl) -acetamido] -3- (p-toluenesulfonyloxy) -3-cephem-4-carboxylic acid.

To a solution of 9.5 grams (20 mmol) of p-nitrobenzyl-7- [2- (2-thienyl) -acetamido] -3-hydroxy-3-cephem-4-carboxylate in 30 ml of DMAC and 30 ml of 9 pylene oxide, kept at ice bath temperature, were added 4.2 grams (22 mmol,

1.1 equivalents of p-toluenesulfonyl chloride. The reaction mixture was stirred at ice bath temperature for about 15 hours and then at room temperature for about 3 hours. Then, the reaction mixture was evaporated to remove excess propylene oxide, and the concentrate was dissolved in ethyl acetate. The solution was washed with saturated sodium chloride solution and dried. Evaporation of the dried solution under reduced pressure gave the crude tosylate ester as a dry residue. The residue was dissolved in ethyl acetate and chromatographed over water-deactivated silica gel (Woelm silica gel, 10 # water quenched) packed in a glass column. The column was eluted with 45% v / v hexane in ethyl acetate. Four fractions were collected with a volume of about 100 ml. Fractions 2 and 3 were combined and evaporated under reduced pressure to give 4.75 grams of p-nitrobenzyl-7- [2- (2-thienyl) -acetamido7-3- (p-toluenesulfonyloxy) -3-cephem-4-carboxylate , mixed with the corresponding 2-cephem isomer.

To a solution of 1.26 grams of the isomer mixture in 20 ml of methylene chloride kept at ice bath temperature was added a solution of 0.4 grams of m-chloroperbenzoic acid in 20 ml of methylene chloride. The mixture was stirred for 40 minutes and evaporated to dryness. The residue was triturated with isopropanol, filtered, washed with ether and dried. There was obtained 1.1 grams of product, p-nitrobenzyl-7-_z/ 2- (2-thienyl) acetamido7-3- (p-toluenesulfonyloxy) -3-cephem-4-carboxylate sulfoxide. The following^C27^hrs23^N3°10^S3 ^BeraKnown elemental analysis values ​​were obtained for the sulfoxide product:

Calculated: C, 50.23; H, 3.59; N, 6.51

Found: C, 49.98; H, 3.30; The N, 6.53 x 3 cephemsulfoxide was reduced to the 3

To a solution of 1.0 grams of the sulfoxide in 25 ml of acetonitrile, containing 5 ml of DMF, kept at ice-bath temperature, was added 0.157 grams of phosphorus tribromide with stirring. The reaction mixture was stirred for 1 hour in cold. Ethyl acetate and a saturated sodium chloride solution were added to the mixture. The product was extracted with the ethyl acetate, and the organic layer was washed 3 times with saturated sodium chloride solution. The organic phase was dried and evaporated to give 1.1 grams of the product, p-nitrobenzyl-7- / 2- (2-thienyl) acetamido-3- (p-toluenesulfonyloxyl) -3-cephem-4-carboxylate.

The above product was hydrogenated over previously reduced palladium on carbon as a catalyst in methanol-THF according to the procedure described in Example 4. The resulting free carboxyl was obtained.

7403306-9 the acid, namely 7- [§ (2-thienyl) -acetamido-3- (p-toluenesulfonyloxy) -3-cephem-4-carboxylic acid.

NMR (CDCl 3) Swords: 2.47 (s, 3H, methyl); 3.40-4.10 (m, 4H,

Cg-joeh side chain-CH₂); 5.05 (d, 2H, Cg-H); 5.80 (kv, 1H, C1H);

6.85-7.38 (m, 3H, thiophene); 7.61 (kv, 4H, phenyl).

IR (CHCl3) 1790, 1380, 1170 cm¹

Electrometric titration (80 # aqueous methyl cellulose solvent) _PK_AM.

UF (ethanol) ^Amax 265 now (ledge)

Example 8. 7- / 2- (2-Thienyl) -ethetamido-3- (4-fluorobenzenesulfonyloxy) -3-cephem-4-carboxylic acid.

By the same procedure for sulfonation and deesterification as in Example 4, p-nitrobenzyl 7- / 2- (2-thienyl) -acetamido7-3-hydroxy-3-oemem-4-carboxylate was reacted with 4-fluorobenzenesulfonyl chloride in DMAC in the presence of propylene oxide; the resulting sulfonation product, p-nitrobenzyl-7- [2- (2-thienyl) -acetamido] -3- (4-fluorobenzenesulfonyloxy) -3-cephem-4-carboxylate, was hydrogenated in methanol-THF over a previously reduced catalyst of $ 5 palladium on carbon to give the product, i. 7- / 2- (2-thienyl) acetamido / 3- (4-fluorbensensulfonyloxi) -3-cephem-4-carboxylic acid.

In microanalysis, the following values ​​for C för HHg HN ^O ^S ^F were obtained:

Calculated: C, 45.78; H, 3.03; N, 5.62; F, 3.81

Found: C, 46.04; H, 3.51; N, 5.55; F, 3.89

NMR (CDCl 3) δ values: 3.48-4.10 (broad m, 4H, Cg-Jach side chain CHg); 5.05 (d, 1H, Cg-H); 5.77 (kv, 1H, C)_?-HRS); 6.80-7.48 and 7.77-8.16 (broad m, 7H, thiophene and phenyl-H).

IR (CHCl3) 1792, 1385 and 1160 cm¹

Electrometric titration (80 <f> aqueous methylcellulose) pK_A 4,25.

Example 9. 7- (D-Phenylglycylamido) -3-methylsulfonyloxy-3-cephem-4-carboxylic acid.

To a solution of 11.1 grams of p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride in 500 ml of tetrahydrofuran was added 15.1 grams of sodium bisulfite. The mixture was stirred for one hour at room temperature, and then 6.4 grams of N- (t-butyloxycarbonyl) phenylglycine and 6.25 grams of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) were added. The reaction mixture was stirred at room temperature for 7 hours, after which the mixture was evaporated to remove tetrahydrofuran. The concentrate was dissolved in ethyl acetate, and the solution was washed successively

7403306-9 a solution of sodium bicarbonate, dilute hydrochloric acid and saturated sodium chloride solution, and then dried. The dried solution was evaporated to dryness to give 11.14 grams of p-nitrobenzyl-7- (N-tbutyloxycarbonyl-D-enylglycylamino) -3-hydroxy-3-cephem-4-carboxylate.

To a solution of 11.14 grams of the above product in 50 ml of DMAC containing 25 ml of propylene oxide was added at room temperature 1.47 ml of methanesulfonyl chloride. After the mixture was stirred for about 3 hours, an additional 1.47 ml of methanesulfonyl chloride was added and the mixture was stirred for an additional 15 hours. The reaction mixture was diluted with ethyl acetate, and the solution was extracted four times with saturated sodium chloride solution. The washed organic phase was dried and evaporated to dryness to give the crude reaction product p-nitrobenzyl-7- (Nt-butyloxycarbonyl-D-phenylglycylamido) -3-methylsulfonyloxy-3-cephem-4-carboxylate₁The product was purified by dissolving in methylene chloride and precipitated from the solution by dilution with hexane.

D. The purified product, 8.09 grams, was filtered and dried.

The p-nitrobenzyl ester group was removed by hydrogenating the product over a previously reduced 5% palladium on carbon in accordance with the procedure described in Example 4. This gave 4.21 grams of the free acid.

The resulting free acid, 1.545 grams, was dissolved in 3 nil of dry acetonitrile, after which 1.7 grams of p-toluenesulfonic acid were added. The reaction mixture was stirred overnight at room temperature. Water was added to the mixture and its pH was adjusted to 5.0 with sodium bicarbonate solution. Then the mixture was evaporated to remove acetonitrile, and the aqueous residue was filtered. The pH of the filtrate was adjusted to pH 4.0, after which the filtrate was freeze-dried. The freeze-dried mixture was torn with acetone and filtered. The solid was dissolved in 15 ml of water and about 5 ml of acetone was added to the solution at ice bath temperature. The product, 7- (D-phenylglycylamido) -3-methylsulfonyloxy) -3-cephem-4-carboxylic acid, crystallized from the cold solution and filtered, washed with cold water and acetone, dried to give 143 mg of the product. :

Clementary analysis for c., &lt;H-, "N-O" S_n:

bi {3 {ά

Calculated: C, 44.92; H, 4.01; N, 9.83. Found: C, 44.13; H, 4.24; N, 9.26

Electrometric titration in 80 $ aqueous methyl cellulose solvent: pK_A 3.6 and 6.75

IR (ground mass) 1780, 1360, and 1178 cm

UV (buffer for pH 6) Xmax 261 nju. (£ = 8400)

7403306-9

NMR (DMSO d_g) Δ values: 3.28 (s, 3H, methyl); 3.55 (sq. 2H, C₂-CH₂); 4.92-5.1 (m, 2H, C)_g-H and side chain CH); 5.68 (Q. 1H, C₇-HRS); 7.48 (m, 5H, phenyl).