IE41622B1 - Cephalosporin compounds - Google Patents

Abstract

1457237 Cephem compounds RECHERCHE ET INDUSTRIE THERAPEUTIQUES 21 May 1975 [22 May 1974 2 Jan 1975] 21816/75 Heading C2C The invention comprises compounds of formula and their non-toxic pharmaceutically acceptable salts. In examples, these compounds are prepared by converting benzyl or tert-butyl thienylacetate to the corresponding malonate monoester, converting this to monobenzyl (or tert - butyl)mono(2,5 - dioxopyrrolidin - 1 - yl)- thienylmalonate, reacting the latter with the corresponding amino / methyltetrazolylthiomethyl cephen compound, and finally hydrolysing the thus-formed side-chain monoester of I. Thereapeutic compositions having antibacterial activity comprise compounds of the above formula and may be administered by injection.

Description

This invention relates to cephalosporin compounds with «-carboxy-n-thienylacetamido group at position 7 and 1-methyltetrazol-5-ylthiomethyl group at position 3 of the cephem nucleus.

These compounds are thus the 2-thienyl and 3thienyl derivatives and it has been found that the 3-thienyl derivative shows better stability than the 2-thienyl derivative. These compounds have antibacterial activity. A few examples of substituted «-carboxy acetamido groups have been used at position 7 of the cephalosporin nucleus in the search for improved antibiotics. For example, rt-carboxy-«-thienylacetamido cephalosporanic acid has been disclosed in Belgian patent N° 794,844 and in South African patent n° 68/1208, respectively, but cephalosporins with «-carboxy-a-thienylacetamido group at position 7 and 1-methyltetrazol-5-ylthiomethyl group at position 3 have not been previously described.

. The compounds of this invention have the following structural formula I; COOH CH3 The side chain at position 7 presents an asymmetrical carbon atom and the corresponding cephalosporins may thus exist in the form of DL, D and L isomers each of which being part of the invention.

Also within the scope of the invention are the non-toxic pharmaceutically acceptable salts of the acidic compounds defined by the formula given above.

Many salts and methods of preparation are known within the art. These include alkali metals, such as sodium or potassium, and ammonium salts including organic amines such as triethylamine.

The 7-aminocephalosporanic acids and derivatives and methods for their preparation are well known in the cephalosporin art. General methods to prepare these compounds are in the book Cephalosporins and Penicillins Chemistry and Biology, ed. Flynn, Academic Press, New York, 1972. Methods to prepare the 7-amino-3-heterocyclicthiomethyl compounds are given in U.S. patent N° 3,516,997· The compounds of the invention are prepared by hydrolyzing the acylation product of the 7~amino-3-(lmethyltetrazol-5-ylthiomethyl)-3-cephem~4-carboxylic acid with an activated derivative of the monotert-butyl-(or benzyl)2(3)-thienyl malonic acid ester, e.g. N-^-tert-but-for benzyl)-oxycarbonyl-n:-/2(3)-thienyl/-acetoxy^-succinimide.

The 2(3)-thienyl malonic acid ester is obtained from 2(3)-thiophene acetic acid according to any method known to the art. The whole process is represented in the following schema I. - 3 I SCHEMA I CHg-COOH esterification carboxylation activation acylation hydrolysis CH2-COC1 RCHg-COOR v CH„ 3H2sJ ) - 4 41622 In schema I, the starting 2(3)-thiophene acetic acids can be prepared according to the procedure described by P. Cagniant in Bull. Soc. Chim. France p. 847-53 (1949) and in C.R. Acad. Sci., Paris Ser. C 264 (1), p. 112-14 (1967), respectively, and common methods of carboxylation as well as to activate a carboxyl group such as mixed anhydride, acid halide or activated ester are known to the art and may be used.

Alternatively, the 3-thiophene acetic acid can also be prepared according to the procedure described in Belgian patent N° 777,570.

Also a coupling reagent such as dicyclohexylcarbodiimide or NjN'-carbonyldiimidazole may be used to acylate the 7-amino-3-(1-methyltetrazol-5-ylthiomethyl)-3cephem-4-carboxylic acid.

Hydrolysis can be performed by any method known to the art for hydrolyzing labile organic radicals such as tert-butyl or benzyl, e.g. by trifluoroacetic acid at 0° C or by re-chymotrypsin, respectively.

The compounds of this invention have broadspectrum antibacterial activity with minimum inhibitory concentrations (MIC) ranging from 0.2 to >200 jug/ml. when determined by standard agar inclusion methods.

Table 1 shows MlC's for the compounds of the invention, i.e. DL-7-/it“car,:iOxy~«_(2-thienyl)-acetamidq/3_(l_methyltetrazol-5-ylthiornethyl)-3-cephem-4-carboxylic acid disodium salt or (RIT 3733) and DL-7-/rc-carboxy-o!-(3thienyl)-acetamido/-3-(l -methyltetrazol-5-ylthiomethyl)-3cephem-4-carboxylic acid disodium salt (RIT 3838) against representative Gram-positive and Gram-negative bacteria - 5 41633 by comparison of MIC's for 7-/rt-(2-thienyl)-acetamidq/-3(1-methyltetrazoi-5-yl-thiomethyl)-3-cephem-4-carboxylic acid (RIT 3734), for DL-7-/a-(3-thienyl)-acetamido/-3-(lmethyltetrazoi-5-ylthiomethyl)-3-cephem-4-carboxylic acid (RIT 3839), for 7-/rt-carboxy-a(2-thienyl)-acetamido/cephalosporanic acid (RIT 3759), for a cephalosporin compound known for its particularly high activity (cefazolin) and for 6-(re-carbqxy-phenylacetamido) penicillanic acid (carbenicillin). The figures demontrate the specially high activity of the products of the invention against Serratia marc, and Proteus morganii.

TABLE 1 Compound RIT 3838 RIT 3733 RIT 3839 RIT 3734 RIT 3759 Cefa- zolin Carbe- nicil- lin staph. Aureus ATCC 6538P 4 <0.25 <0.25 <0.25 <0.25 <0.25 1 staph. Aureus HH 127 4 1.6 <0.25 ND ND <0.25 1 staph. Aureus SK 23390 16 0.4 <0.25 ND ND <0.25 8 E. coli ATCC 10536 0.5 0.5 1 <0.25 2 1 >256 Kleb. pneumo. SK 4200 2 2 2 <0.25 4 0.5 4 Kleb. pneumo. SK 1200 <0.25 0.5 <0.25 ND ND <0.25 256 Salmonella RIT 23 2 2 <0.25 0.5 4 <0.25 ND Shigella RIT 26 <0.25 <0.25 <0.25 <0.25 1 <0.25 1 Pseudo.aeruginosa RIT 15 >256 >256 >256 >256 >256 >256 256 Serratia marc. RIT 302 16 32 >256 >256 ND >256 >256 Serratia marc. RIT 303 0.5 1 32 32 ND 256 2 Entero.aerogenes RIT 229 1 3.1 16 ND ND 64 4 Entero.aerogenes RIT 296 0.5 0.5 ND ND ND 128 ND Entero.cloacae RIT 225 <0.25 1 32 ND 16 >256 0.5 Proteus morganii RIT 223 <0.25 0.5 128 ND ND 256 0.5 Proteus morganii RIT 12 <0.25 <0.25 128 32 8 256 <0.25 Proteus mirabilis RIT 11 <0.25 0.5 <0.25 1 4 <0.25 <0.25 ND = not determined.

Upon in vivo subcutaneous administration the compound also shows significant activity.

These results are summarized in Table 2.

TABLE 2 Compound ED _0 (mg/kg) in vivo Enterobacter cloacae RIT272 Serratia marc RIT 270 Proteus, morganiι RIT 309 Proteus morganii RIT446 RIT 3838 44 35.7 40 41 RIT 3733 77-9 36.6 54 34.08 Cefazolin >120 >120 >120 >120 Carbenicillin 109 >120 80 >120 This invention also provides pharmaceutical compositions comprising a com· pound of Formula I and a pharmaceutically acceptable carrier therefor.

The compounds of this invention are formulated and administered by injection in the same manner as other cephalosporins in dosages of From 250 mg to 1 g. The daily dosage, which may be divided, may range from 1 to 5 g and is dependent on the age and weight of the subject and on the infection being treated. The dosage can be determined by those skilled in the art based on the data disclosed herein and experience with known cephalosporins.

The following examples illustrate the invention but are not to be construed as limiting the scope thereof.

In the examples, the indicated Rp values have been determined by thin layer chromatography on SELECTA TLC Plastic Sheets F 1500 LS 254 Silica gel (a product Of K. SCHLEICHER *· -ι & SCHULL, W. Germany) and the H nmr spectra have been recorded on a Perkin Elmer R-l2 (60 Me), the chemical shift being expressed in ppm, using tetramethylsilane or sodium-2,230 dimethyl-2-silapentane-5-sulfonate as internal reference (S=O.Oppt EXAMPLE 1 A solution of 18.3 g of 2-thiophene acetyl chloride (prepared according to the procedure described by P. Cagniant in Bull. Soc. Chim. France 847-53, 1949), in 9.25 g of tertbutyl alcohol and 50 ml. of diethyl ether is heated to reflux and 15.2 ml. of dimethylaniline dissolved in 25 ml. of diethyl ether is added dropwise. The mixture is refluxed for 5 hours and filtered.' The filtrate is washed 3 times with 20 ml.

N HgSO^, once with water, twice with 20 ml. N NaHCO^ solution, once with water, and then dried over anhydrous Na2S0^ and distilled under reduced pressure (87°C/l.2mm) to yield tertbutyl 2-thienylacetate.

Rp = 0.83 (+ 0.10) in the system benzene/acetone (8/2), U.V. detection. nmr (CC14) : 7.19 dd (1H) (J 3.5 c./sec.), 6.95 d (211) (J 3.5 c./sec.), 3.67 s (2H), 1 .45 5 (911).

EXAMPLE 2 To a solution of 11.8 ml. of diisopropylamine in 55 ml. of tetrahydrofuran maintained under dry nitrogen atmosphere there is added 36 ml. of a 15 % solution of n-butyllithium in hexane, the reaction medium being maintained between -5 and -10° C. To the obtained solution there is added under dry nitrogen atmosphere 15 g of tert-butyl 2-thienylacetate as prepared in example 1, the mixture being stirred for 15 minutes at -10° C, and then poured onto solid carbon dioxide and stirred for one hour.

The solvent is evaporated and the residue is dissolved in 25 ml. of water and extracted with 75 ml. diethyl ether. The organic layer is evaporated under reduced pressure for recovering unreacted tert-butyl 2-thienylacetate. The aqueous layer is acidified up to pH 2.5 with 12 N HCl and extracted with 2 portions of 50 ml. of ether. The combined ethereal solutions are washed with water, dried over anhydrous Na2S04 and evaporated under reduced pressure to yield crystallized mono tert-butyl 2-thienylmalonate.

Rp = 0.30 (+ 0.10) in the system benzene/acetone (8/2), detection with U.V. or bromocresol green. nmr (CC14) : 9.67 s (1H), 7.30 d (1H) (J 5.0 c./sec.), 7.05 m (2H), 4.80 s (1H), 1.47 s (9H).

EXAMPLE 3 To a cooled (-5° C) solution of 3.65 8 of tert-butyl 2-thienylmalonate (as prepared in example 2) dissolved in 30 ml. of tetrahydrofuran there is added 1.84 g of N-hydroxysuccinimide and 3.75 g of dicyclohexylcarbodiimide. The mixture is stirred for 4 hours from -10 up to 5° C and then filtered. The filtrate is evaporated to dryness and the residue is treated with 50 ml. of tetrahydrofuran and 50 ml. of ether. The mixture is filtered and the filtrate is evaporated under reduced pressure. The residue is crystallized from diisopropyl ether to yield Ν-/άtert-butoxycarbonyl-a-(2-thienyl)-acetoxy/-succinimide.

Melting point : 89-90° C. Rf = 0.48 (+ 0.10) in the system benzene/acetone (8/2), detection with U.V. or bromocresol green. nmr (CDClj) : 7-46-6.9 m (3H), 5.16 s (1H), 2.80 s (4H), 1.53 s (9H); nmr (acetone-dg) 7.64-6.94 m (3H), 5.46 s (1H), 2.88 s (4H), 1.52 s (9H).

EXAMPLE 4 To 13.02 g of 1-methyl-5-mercapto-1,2,3,4-tetrazole prepared according to the procedure described by Stolid R. et al. in J. Prakt. Chem. /2/, 124, 261-79 (1930) and dissolved in 800 ml. of a 0.2 M phosphate buffer solution (pH 6.5-6.6), there is added 20.4 g of 7-aminocephalosporanic acid (7-ACA). The mixture is brought to pH 6.3-6.4 by _ addition, of 21.5 ml. of triethylamine, heated at 70° C for 80 minutes, at once cooled to 20° C, acidified to pH 3.5 by addition of 65 ml. of 2N HCl and then stirred for 15 minutes.

The obtained precipitate is filtered, washed with 200 ml. of water, 200 ml. of methanol and 200 ml. of diethyl ether and dried under reduced pressure to yield a crude product which is dissolved in 300 ml. of 2N HCl, treated with*DARCO G60 (a product manufactured and sold by Atlas Chem. Ind., New Murphy Road, Wilmington, DEL 19899) and filtered.

Crystallization occurs from the filtrate brought to pH 3.7 by. addition of 45 ml. of concentrated ammonium hydroxide.

The crystallization medium is stirred for 30 minutes at 25° C and filtered under reduced pressure.

The precipitate is washed with. 200 ml. of water, 200ml. of methanol and 200 ml. of diethylether and dried at 40° C over ?2θ5 N^der reduced pressure to yield 7-amino-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4carboxylic acid.

Rp = 0.44 (+ 0.10) in the system acetonitrilewater (80/20), detection with U.V. or bromocresol green. nmr (D20 - NagCOg) 5.48 d (1H) (j 4.7 c./sec.), .08 d (IH) (J 4.7 c./sec.), 4.40 and 4.06 2d (2 x 1H)(j 13 c./ sec.),3.95 s(3H), 3.86 and 3.43 2d (2 x 1H) (j 18 c./sec.).

EXAMPLE 5 To a cooled mixture of 1.00 g of 7-amino-3(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid (as prepared in example 4) and 0.84 gr. of triethylamine in 30 ml. of acetonitrile there is added 1.02 g of N-/n-tert-butoxycarbonyl-a-(2-thienyl)-acetoxy/succinimide - 10 * DARCO is a trade mark (as prepared in example 3).

The mixture is stirred for one hour at 25° C, the solvent is then removed under reduced pressure and 100 ml. of water/ethyl acetate (1/1) is added thereto. The pH of the solution is brought to 7.0-7.5 and the organic layer is eliminated.

Ethyl acetate (50 ml.) is added thereto and the mixture is brought to pH 2-3 with N HCl. After separation, the aqueous layer is reextracted twice with ethyl acetate (50 ml.).

The ethyl acetate layer is washed with water, dried over anhydrous sodium sulfate, treated with DARCO G60 and evaporated under reduced pressure. The residue is taken up in ethyl acetate 15 (5 ml.) and precipitated in 25 ml. of diisopropyl ether to yield DL-7-/ft~tert-butoxycarbonyl-a-(2-thienyl)-acetamido/-3-(l-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.

Rp =10.62 (+ 0.10) in the system methylisobutylketone/methanol/formic acid (δθ/18/2), detection with U.V. or 2u bromocresol green. if nmr (DMSO-dg) : 7.57 m (1H), 7.11 m (2H), 5.75 m (1H), 5.18 m (1H + 1H), 4-37 s (2H), 4.01 s (3H), 3.73 m (2H), 1.44 s (9H); nmr (acetone-dg) 7.38 d (1H) (j 5-0 c./sec.), 7.04 m (2H), 5.77 q (1H) (j 4.5 c./sec.), 5.13 m (1H + 1H), 4.28 s (2H), 3.96 s (3H), 3.76 m (2H), 1.45 s (9H).

EXAMPLE 6 A mixture of 0.675 g of DL-7-/a-tert-butoxycarbonyln-(2-thienyl)-acetamido/-3-(1-methyltetrazol-5-ylthiomethyl)3-cephem-4-carboxylic acid (as prepared according in example 5), ml. of trifluoroacetic acid and 0.7 ml. of thiophenol is stirred for one hour at 0°. Most of the solvent is removed under reduced pressure and the residue is poured into ether. The obtained crude product is dissolved in ethyl acetate and treated with DARCO G60.. The mixture is filtered and poured into diisopropyl ether to yield a precipitate of DL-7-/a-carboxy-ec-(2-thienyl)-acetamido/-3_(-| -methyl>· tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.

Rp = 0.55 (+ 0.10) in the system methylisobutylketone/ methanol/formic acid (flO/18/2) or 0.29 /+0.10) in the system acetonitrile/water (8/2), detection with U.V. or bromocresol green. nmr (acetone-dg) 8.44 d (1H) (j 8.5 c./sec.), 7.47 d (1H) (J 5.0 c./sec.), 7.14 m (2H) (j 5.0 c./sec.), 5.87 dd (1H) (j 8.5 & 4.7 c./sec.), 5.19 d+s (1H + 1H) (j 4.7 c./sec.), 4.44 s (2H), 4.00 s (3H), 3.78 m (2H). nmr (DMSO-dg) : 9.41 d (1H) (J 8.5 c./sec.), 7.54 d (1H) (J 5.0 c./sec.), 7.10 m (2H) (J 5.0 c./sec.), 5.76 dd (1H) (J 8.5 & 4.7 c./sec.), 5.16 d+s (1H + 1H) (J 4.7 c./sec.), 4.35 S (2H), 3.99 s (3H), 3.72 m (2H).

The sodium salt is prepared by dissolving the acid in 100 ml. of ethyl acetate. After the addition of 5 ml. of water,a M NaHCOj solution is added dropwise up to pH 4.4 and the aqueous solution is freeze-dried.

EXAMPLE 7 A mixture of 22.5g of 2-thiophene acetyl chloride (pre25 pared according to the procedure described by P. Cagniant in Bull Soc. Chim. France 847-53,1949), 50 ml. of ether and 16 ml. of benzyl alcohol is gently refluxed for 18 h. The reaction mixture is concentrated under reduced pressure and the residue is taken up with 100 ml. of ether. The ethereal solution is washed once with 50 ml. of a 8.5% NaHCO^ solution, twice with 25 ml. of water, dried over anhydrous NagSO^, evaporated under reduced pressure and the residue is distilled to yield benzyl 2-thienylacetate.

Rp = 0.59 (+ 0.10) in the system benzene/acetone (8/2), U.V. detection. nmr (CC14) 7.32 s (5H), 7.18 m (1H), 6.93 m (2H), .12 s (2H), 3.70 s (2H).

EXAMPLE 8 To 7.7 ml. of cooled (-10°(5 diisopropylamine dissolved in 35 ml. of anhydrous tetrahydrofuran there is added dropwise and under dry nitrogen atmosphere 25.6 ml. of a 15 % solution of n-butyllithium (3.9 g) in hexane, the temperature being maintained under -10° C. At this temperature there is added dropwise and within 30 minutes, 11.6 g of benzyl 2-thienylacetate (as prepared in example 7) and the obtained mixture is poured onto solid carbon dioxide.

The solvent is evaporated under reduced pressure and the residue is treated with 25 ml. of water and 75 ml. of diethyl ether; the ethereal layer containing the unreacted benzyl 2-thienylacetate is separated and evaporated to dryness; the aqueous layer is brought to pH 2 with 12N HCl, extracted 'ί with 2 portions of 50 ml. of diethyl ether and the combined ethereal solutions are washed with water, dried over anhydrous Na^SO^ and evaporated under reduced pressure.

The residue is concentrated under reduced pressure to yield monobenzyl 2-thienylmalonate.

Rf = 0.30 (+ 0.10) in the system benzene/acetone (8/2), detection with U.V. or bromocresol green. nmr (CC14-CDC13 1/1) 7.30 s (5H), 7.20-6.80 m (3H), 6.82 s (1H), 5.19 s;(2H), 4.92 s (1H). - 13 / EXAMPLE 9 To 2.5g of cooled (- 5° C) monobenzyl -2-thienyl malonate (as prepared in example 8) and dissolved in 100 ml. of tetrahydrofuran there are added 1.15 g of N-hydroxysuccinimide and 2.06 g of dicyclohexylcarbodiimide. The mixture is stirred for 15 minutes between 0 and -5° C, thereafter at 20° C for 135 minutes and then filtered. The filtrate is evaporated under reduced pressure. The it obtained crude N-/re-benzyloxycarbonyl-a-(2-thienyl)-acetoxy/succinimide is dissolved in 50 ml. of acetonitrile and this solution is added, at once, to a mixture of 3.3g of DL-7-amino-3-(lmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid (as prepared in example 4) in 50 ml. of acetonitrile and 2.8 ml. of triethylamine. The mixture is stirred for one hour, the solvent is removed and the residue is taken up in water, acidified to pH 1.0 with 12N HCl and extracted with ethyl acetate. The organic layer is dried over anhydrous Na2S04, evaporated to dryness and the residue is taken up in acetone, decolorized with charcoal ^nd filtered on Silica gel. The filtrate is evaporated, taken up again in 100 ml. of ethyl acetate and filtered.

K After concentration under reduced pressure,the solution is poured into diisopropylether to precipitate Dl-7-/abenzyloxycarbonyl-«-(2-thienyl)acetamido/-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxyli c acid.

Rp = 0.79 (+0.10) in the system acetonitrile/ methanol/water (6/3/1), detection with U.V. or bromocresol green. nmr (DMSO-dg) : 9.44 d (1H) (j 7.2 c./sec.), 7.607.45 m (IH), 7.34 S;(5H), 7.06 m (2H), 5.67 q (1H) (J 7-2 and 4.7 c./soc.), 5.40-5-00 m+s (2 x 1IJ + 2H), 4.31 m (2IJ), 3.94 £i (3H), 3.64 tn (2H).

EXAMPLE 10 100 mg of DL-7-/«-benzyloxycarbonyl-a-(2-thienyl)5 acetamido/-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4— carboxylic acid (as prepared in example 9) and 20 mg of α-chymotrypsin dissolved in 20 ml. of a buffer phosphate solution (0.2 M and pH 7.0 are heated at 37° C for 75 minutes.

The mixture is acidified to pH 2.0 by addition of 10 10N HCl and extracted with ethyl acetate; the organic layer is dried over anhydrous Na^SO^, most of the solvent is evaporated and the residue is poured into 100 ml. of diisopropyl ether to precipitate DL-7-/re-carboxy a-(2-thienyl)-acetamido/~ 3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid showing the same characteristics as those indicated for the product of example 6.

The sodium salt is prepared by dissolving the acid in ethyl acetate, adding a sodium 2-ethylhexanoate solution, and then slowly adding ether until the sodium salt is precipitated.

EXAMPLE 11 I An injectable pharmaceutical composition is prepared by dissolving 100-500 mg of sodium DL-7-/a-carboxyn-(2-thienyl)-acetamido/-3-(l-niethyltetrazol-5-ylthiomethyl)25 3-cephem-4-carboxylate (as prepared in example 10) in sterile water (1-5 ml.). - 15 41622 EXAMPLE 12 A solution of 9.25 g (0.058M) of ,3-thiophene acetyl chloride in 7-5 ml. of tert-butyl alcohol and 30 ml. of diethyl ether is heated to reflux and 8.5 ml. of dimethylaniline dissolved in 20 ml. of diethyl ether is added dropwise. The mixture is refluxed for 5 hours and filtered. The filtrate is washed twice with 20 ml. water, once with 20 ml. N HCl, once with 20 ml. water, twice with It ml. N NaHCOg and twice with 20 ml. HgO, and then dried over anhydrous OaClp and distilled under reduced pressure (107-120^0/ 8 mm) to yield'tert-butyl 3-thienyl acetate.

R^= 0.65 (+ 0.10) in chloroform, detection with bromocresol green. ' nmr (CDClg) : 7.15 m (3H), 3.51 s (2H), 1.44 s (9H).

EXAMPLE 13 To a solution of 7 ml. of diisopropylamine in ml. of tetrahydrofuran maintained under dry nitrogen atmosphere thtpre is quickly added 65 ml. of a 5 % solution of n-butyllithium in hexane, the reaction medium being maintained between -10 and -15° C. After 15 minutes >l standing, 8,5 g of tert-butyl 3-thienylacetate (as prepared in example 12) in 21 ml. ,of tetrahydrofuran is I1 added thereto. After 5 minutes standing, the mixture is poured onto solid carbon dioxide and stirred for one hour.

The solvent is evaporated and the residue is taken up in 100 ml. of water and extracted twice with 100 ml. ether.

The ethereal layer is washed twice with 20 ml. water and the combined aqueous solutions are brought to pH 2 with_ N HCl and extracted once with 90 ml. ether, once with - 16 41622 ml. ether and once with 25 ml. ether.

The combined ethereal solutions are washed twice with 20 ml. water, 2 and evaporated under reduced pressure to yield crystallized mono tert-butyl 3-thienyl malonate.

Rp = 0.60 (+ 0.10) in chloroform, detection with bromocresol green and ale. KMnO^. nmr (CDC13)_ : 10.31 s (1H), 7.29 m (3H), 4.71 s (1H), 1.45 s (9H).

EXAMPLE 14 To a cooled (-15° c) solution of 7.9 g (32 mmol) nmotert-butyl 3-thienyl malonate (as prepared,,in example 13) dissolved in 100 ml. of tetrahydrofuran, there are added 4.2 g of N-hydroxysuccinimide and 8.3 g of dicyclohexylcarbodiimide. The mixture is stirred at -15° 0 for 3 hours and then filtered. The filtrate is evaporated to dryness and the residue is treated twice with 75 ml. of tetrahydrofuran to eliminate dicyclohexyl urea. The solvent is evaporatedιto dryness and the oily residue is treated with 70 ml. of di-isopropyl ether and solidifies. The solid is filtered, washed twice with 15ml. of di-isopropyl ether and dried under reduced pressure to yield N-/a-tertbutoxycarbony l-a-( 3-thienyl)—aceto; xy/’-succinimide.

Melting point : 97-98° C.

Rp = 0.77 (+ 0.10) in the system chloroform/acetonitrile/water/formic (acid (35/58/5/^), detection with U.V. nmr (CDClj) : 7.33 m (3H), 4.96 s (1H), 2.80 s (4H), 1.50 s (9H). - 17 I EXAMPLE T5 Tc a mixture of 9.5 g of 7-amino-j:3-(1-methy 1tetrazol-5-ylthiomethyl)-3-cephem-4-carboixylic acid (as prepared in example 4) and 8.1 g of triethylamine in 120 ml. of acetonitrile there is added at once 11.4 g of N-/a-tert-butoxycarbonyl-re-(3-thienyl)-acetoxy/-suceinimide (as prepared in example 14). The mixture is stirred for 30 minutes at 30° C and filtered. The solvent is then removed under reduced pressure and the residue is taken up in 80 ml. of distilled water and ethyl acetate (80 ml.) is added thereto. The aqueous layer is acidified with 0.1N HCl (pH 2) and extracted three times with 40 ml. of ethyl acetate and the combined organic layers are dried over anhydrous sodium sulfate, decolorized with charcoal and evaporated under reduced pressure. The residue is taken up in ethyl acetate (20 ml.) and 250 ml. of di-isop^opyl ether is added thereto. The obtained crude product is dissolved in 300 ml. of ethyl acetate and, after 30 minutes stirring, the solution is filtered.

The filtrate ts decolorized with Darco G 60 (5 g), filtered on*bicalite S.H. (a product sold by DICALXTE EUROPE NORD, Brussels, Belgium) and most of the solvent is evaporated. Di-isopropyl ether (400 ml.) is added thereto to precipitate DL-7-/a-tert-butoxycarbonyl-a-(3-tjhienyl)-acetamidq/-3(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.

Rp = 0.695 (+0.10) in the system chloroform/ acetonitrile/water/fofrmic acid (35/bfi/5/2), detection with U.V. or bromocresol green. nmr (DMSO) : 9.20 dd (1H), 7-34 m (3ll), 5.68 m (ill), 5.10 fltl (in), 4.89 s (1Π), 4.31 m (2H), 3.95 s (3H), 3.68 m (2H), 1.41 s (9H).: *Dicalite is a trade mark EXAMPLE 16 A mixture of 4 g (7-25 mmol) of E>L-7-/ix-tertbutoxycarbor.yl-«-(3-thienyl)-acetamidq/-3-(l-methyltetrazol5-ylthiomethyl)-3-cephem-4-carboxylic acid (as prepared in example 15), 25 ml. of trifluoroacetic acid and 2.5 ml· of thiophenol is stirred for 70 minutes at 0° C. Most of the solvent is removed under reduced pressure and 300 ml. of di-isopropyl ether is added thereto. The obtained crude product is dissolved in 350 ml. of ethyl acetate and the solution is filtered. Most of the filtrate is removed under reduced pressure and 300 ml. of di-isopropyl ether is poured onto the residue. After filtration and drying, the product is dissolved in 300 ml. of ethyl acetate and then extracted into water (50 ml.) with 0.1N NaHCOg up to pH 5.7.

The aqueous solution is lyophilized to yield DL-7-/ii- Rp = 0.52 (+ 0.10) in the system chloroform/ acetonitrile/water/formic acid (35/58/5/2), detection with U.V. nmr (Do0) : 7.35 m (3H)ψ 5.69 d (1H), 5.10 dd (111), 4.21 m (2H), 4.05 S (3H), 3.59 m (2H).

EXAMPLE 1 7 ι An injectable pharmaceutical composition is prepared by dissolving in sterile water (1-5 ml.) 100-150 mg of DL-7/a-carboxy-a-(3-thienj.1)-acetamidq/-3-(1 -methyltetrazol-5ylthiomethyl)-3-cephem-4-carboxylic acid, disodium salt as prepared in exempl® 16.

Claims (7)

1. A compound of the formula wherein Th is a 2-thienyl or a 3-thienyl ring, or a non toxic 5 pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 wherein Th is a 3. -thienyl ring.

3. A compound according to claim 1 wherein Th is a 2-thienyl ring. io

4. A pharmaceutical composition comprising a compound as claimed in claim 1 or claim 2 and a pharmaceutically acceptable carrier therefor.

5. A pharmaceutical composition comprising a compound as claimed in claim 3 and a pharmaceutically acceptable carrier 15 therefor.

6. A process for preparing a compound according to claim 1 which comprises acylating of the 7. -ami no-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4carboxylic acid with an activated derivative of the 20 monoter.t-butyl-(or benzyl)-2(3)-thienyl malonic acid ester, hydrolyzing of the acylating product and, if desired, transforming the free acid into a non-toxic pharmaceutically acceptable salt.

7. A process according to claim 6, wherein the activated derivative of the monotert-butyl-(or benzyl)-2(3)-thieny1 malonic acid ester is N-(a-tert-but-(or benzyl) oxycarbonyl-ct- 02(3)-thienyl] -acetoxyj) -succinimide. 5 3, A process according to claim 6 as hereinbefore described in Examples 5 and 6, Examples 9 and 10 or Examples 15 and 16.