CA1041087A - SUBSTITUTED .alpha.-CARBOXY ACETAMIDOCEPHALOSPORINS - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Novel cephalosporins with the .alpha.-carboxy-.alpha.-thienylacetamido group at position 7 and 1-methyl-tetrazol-5 ylthiomethyl group at position 3 are prepared. These compounds have antibacterial activity.

Description

:` iO~087 This invention relates to cephalosporin compounds with a-carboxy~a-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 3-- thienyl 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 a-carboxy acetamido groups have been used at position 7 of the cephalosporin nucleus in the search for improved antibiotics. For example, a-carboxy-a-thienylacetamido cephalosporanic acid has been disclosed in ~elgian patent N 794,844 and in , 15 South African patent n 68/1208, respectively, but cephalosporins with a-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 :
. ....................................................................... .

` ~ CH-CONH I ~ ~ N
COOH O ~ ~ CH2S ~

', , The side chain at position 7 presents an asymmetrical carbon atom and the corresponding cephalo-sporins may thus exist in the form of DL, D and L isomers each of which being part of the invention.

~ , ...... .
. .:. . :. :: . ~. . ..

1041~7 Also within the scope of the invention are the non-toxic pharmaceutically acceptable salts o~ the acidic compounds de~ined 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 and the like.
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-heterocyclic-thiomethyl 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-(1-methyltetrazol-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-(or benzyI)-oxycarbonyl~ 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.

.' .

104~0~
. ~
:.`. SCHEMA I : ~-: ~ H2 COOH : :
~,~ S

~CH2--COCl S
esterification ~ ~3}CH2-CooR

carboxylation ¦ BuLi/Co2 .
. ~ CH-COOH
S COO,R
. activation¦ HO-N

.; 15 0 . ~ CH-COO-N ~ -S COOR ~--.:: .
,'.'.' 1 S~ ~ i . ~'V
.~ - acylationH2N I ~ ~ N-N
H2S ~ N
COOH CH3 ~

j~ ~ CH-CONH ~ ~ N - ~ :
r': S COOR O ~ N ~ H2S ~ N~
. . COOH CH
hydrolysis 3 . ~

CH-CONH I ~ ~ N - liN
COOH ~ ~ 2S~N~ `
COOH CH3 :
wherein R is a labile organic radical such as benzyl or tert-b ~l.
'': ~`.
: 4 - ::

. .
: ~ - .~: ~ ,, , :
, . . . . .

~ ~0410~37 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. ~47-53 (1949) and in C.R. Acad. Sci. 7 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-thioT~hene acetic acid can ., also be prepared according to the procedure described in Belgian patent N 777,570.
:.
Also a coupling reagent such as dicyclohexyl-,.~ . , carbodiimide or N,N'-carbonyldiimidazole may be used to acylate the 7-amino-3-(1-methyltetrazol-5-ylthiomethyl)-3-, . ................................... .
cephem-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 ~-chymotrypsin, respectively.
The compounds of this invention have broad-spectrum antibacterial activity with minimum inhibitory concentrations (MIC) ranging from 0.2 to >200 Jlg/ml. when -~ determined by standard agar inclusion methods.
Table 1 shows MIC's for the compounds of the ; 25 invention, i.e. DL-7-~-carboxy-a-(2-thienyl)-acetamido~-

3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid disodium salt or (RIT 3733) and DL-7-~a-carboxy-a-(3--- thienyl)-acetamido~-3~1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid disodium salt (RIT 3838) against representative Gram-positive and Gram-negative bacteria , `:; ; : t ;:
10410~7 by comparison oÇ MIC~s for 7-~ -(2-thienyl)-acetamido~-3-- (1-methyltetrazol-5-yl-thiomethyl)-3-cephem-4-carboxylic -~
. acid (RIT 3734)~ for DL-7-~ -(3-thienyl)-acetamido~-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid . ., ~ 5 (RIT 3839), for 7-~-carboxy-~(2-thienyl)-acetamido~-i, ~
~;i cephalosporanic acid (RIT 3759), for a cephalosporin compound known for its particularly high activity (cePazolin) : j and Por 6-(a-carboxy-phenylacetamido) penicillanic acid i''''?i'' (carbenicillin). The figures demontrate the specially high ?~;l 10 activity of the productsof the invention against Serratia .,. . : , ;,;~ marc. and Proteus morganii.
, TABLE 1 :~ RIT RIT RIT RIT RIT Cefa- Carbe- ^ i Compound 3838 3733 3839 3734 3759 zolin nliicnl- ~ -~; 15 staph. Aureus ATCC 6538P4 <0.25 <0.25 ~0.25 <0.25 (0.25 1 staph. Aureus HH 127 41.6 <0.25 ND ND ~0.25 1 staph. Aureus SK 23390 16 0.4 <0.25 NDND <0.25 8 E. coli ATCC 10536 0.5o.5 1 <0.25 2 1 >256 .-` Kleb. pneumo. SK 4200 2 2 2 <0.25 4 0.5 4 Kleb. pneumo. SK 1200<0.250.5 ~0.25 ND ND <0.25 256 -' Salmonella RIT 23 22 <0.25 0.5 4 <0.25 ND
~ ! 20 Shigella RIT 26 <0.25 <0.25 <0.25 <0.25 1 <0.25 1 !.,C' .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 ND256 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
;~ 25 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 12fl 32 8256<0.25 Proteus mirabilis RIT 11 <0.25 0.5 <0.25 1 4 ~0.25 <0.25 ; , :.~ .
? ND = not determined.
, -, .
' ' ... .
--q~Ji - !
~ 1041087 . ` , ~.
~ Upon in vivo subcutaneous administration the .
compound also shows significant activity.

These results are summarized in Table 2.

ED50 (mg/kg) in vivo Comvound Enterobacter Serratia marc Proteu5 Proteus mor-cloacae RIT272 RIT 270 mRoIr an~09 ganii RIT446 RIT 3838 44 35.7 40 41 RIT 3733 77.9 36.6 54 34.08 Ce~azolin ~120 >120 >120 >120 Carbenicillin ~ _ _ ~120 80 >120 ,., : The compounds o~ this invention are formulated and administered by lnjection in the same manner as other cephalosporins in dosages o~ Çrom 250 mg to 1 g. The dail-y dosage, which may be divided, may range ~rom 1 to 5 g and is dependent on the age and weight of the subject and on the in~ection 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 ~ollowing examples illustrate the invention but are not to be construed as limiting the scope thereo~.

In the examples, the indicated R~ values have been -.
determined by thin layer chromatography on SELECTA TLC Plastic Sheets F 1500 LS 254 Silica gel (a product o~ ~.- SCHLEICHER

& SCHULL, W. Germany) and the H nmr spectra have been recorded on a Perkin Elmer R-12 (60 Mc), the chemical shi~t being expressed in ppm, using tetra~ethylsilane or sodium-2,2-dimethyl-2-~ilapentane-5-sul~onate as internal reference (~=O.o~n E~AMPLE 1 A solution o~ 18.3 g o~ 2-thiophene acetyl chloride (prepared according to the procedure described by P. Cagniant , , : ~ ~r . ~ .,:.

1~J4~.0~
in Bull. Soc. Chim. ~rance 847-53, 1949), in 9.25 g of tert- ~
i ,.
- butyl alcohol and 50 ml. o~ diethyl ether is heated to reflux and 15.2 ml. o~ dimethylaniline dissolved in 25 ml. of diethyl -~ ether is added dropwise. The mixture is refluxed for 5 hours ,; .
and ~iltered. The filtrate is washed 3 times with 20 ml.
;~ N H2S04, once with water, twice with 20 ml. N NaHC03 solution, once with water, and then dried over anhydrous Na2S04 and . ~ .. i ~; distilled under reduced pressure (87C/1.2mm) to yield tert-butyl 2-thienylacetate.
..
, 10 Rf = 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 (2H) (J 3.5 c./sec.), 3.67 s (2H), 1.45 s (9H).
, - .

To a solution of 11.8 ml. o~ diisopropylamine in 55 ml. o~ tetrahydro~uran maintained under dry nitrogen atmos-phere there is added 36 ml. oÇ 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 o~ tert-butyl 2-thienylacetate as pre-; pared in example 1, the mixture being stirred ~or 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 Por 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. o~ ether. The combined ethereal solutions are washed with water, dried over anhydrous Na2S04 and evaporated , :

" ~, ,, , . ~

. .

~ 10410~7 ~l under reduced pressure to yield crystallized mono tert-butyl 2-thienylmalonate.
Rf = 0.30 (+ 0.10) in the system benzene/acetone : (8/2), detection with U.V~ or bromocresol green.
nmr (CCl4) : 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).

To a cooled (-5 C) solution of 3.65 g of tert-butyl .,.~,j .
2-thienylmalonate (as prepared in example 2) dissolved in 30 ml.
of tetrahydroPuran thére is added 1.84 g of N-hydroxysuccinimide and 3.75 g of dicyclohexylcarbodiimide. The mixture is stirred ~i for 4 hours ~rom -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 ~- 15 and the filtrate is evaporated lmder reduced pressure. The residue is crystallized from diisopropyl ether to yield N-~a-tert-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 (CDCl3) : 7.46-6.9 m (3H),-5.16 s (1H), 2.80 s (4H), 1.53 s (9H); nmr (acetone-d6) 7.64-6.94 m (3~), 5.46 s (1H), 2.88 s (4H), 1.52 s (9H).
:

To 13.02 g of 1-methyl-5-mercapto-1,2,3,4-tetrazole prepared according to the procedure described by Stollé R. I
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 . - ~ -, - , .................... , ,.i. : .: . - . . . .-. , . . - . ..................... , ,, , . . : :

-: .:: .. . .: .. , ~. . - :

; 10410~7 -; addition of21.5 ml. of triethylamine, heated at 70 C ~or 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. oP diethyl ether and dried under reduced pressure to yield a crude product , ~ which is dissolved in 300 ml. o~ 2N HCl, treated with DARC0 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. oP concentrated ammonium hydroxide.
The crystallization medium is stirred for 30 minutes at 25 C and filtered under reduced pressure.
-- 15 The precipitate is washed with 200 ml. oP ~ater 200ml. o~ methanol and 200 ml. of diethylether and -. ~
dried at 40 C over P205 under reduced pressure to yield i 7-amino-3~ methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.
-~ 20 R~ = 0.44 (+ 0.10) in the system acetonitrile-water (80/20), detection with U.V. or bromocresol green.
.; nmr (D20 - Na2C03) 5.48 d (jH) (J 4.7 c./sec.), 5.08 d (1H) (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.).

- ~o a cooled mixture of 1.00 g o~ 7-amino-3-;~ (1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid (as prepared in example 4) and 0.84 gr. oP triethyl-amine in 30 ml. of acetonitrile there is added 1.02 g of -~ 30 N-~a-tert-butoxycarbonyl-a-(2-thienyl)-acetoxy~succinimide ,',''' .
'. . .

. .
" . . - ~ .
, ,. .. ~ - :

, .. , - .. .

~ 10410~7 ..... .
(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. o~ water/ethyl acetate (1/1) is added thereto. The - 5 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 DARC0 G60 (a product manufactured and sold by Atlas Chemical Industries, New Murphy Road, Wilmington, DEL 19899) and evaporated under reduced pressure. The residue is taken up in ethyl acetate (5 ml.) and precipitated in 25 ml. of diisopropyl ether to yield ., DL-7-~a-tert-butoxycarbonyl-a-(2-thienyl)-acetamidoJ-3-(1-methyl-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.
Rf =i0.62 (+ 0.10) in the system methylisobutyl-ketone/methanol/formic acid(80/18/2), detection with U.V. or : bromocresol green.
' nmr (DMS0-d6) : 7.57 m (1H), 7.11 m (2H), 5.75 m -. . .
(1H), 5.18 m (1H + lH), 4.37 s (2H), 4.01 s (3H), 3.73 m (2H), ` 1.44 s (9H); nmr (acetone-d6) 7.38 d (lH) (J 5.0 c./sec.), 7.04 m (2H), 5.77 q (lH) (J 4.5 c./sec.), 5.13 m (lH + 1H),

- 4.28 s (2H), 3.96 s (3H), 3.76 m (2H), 1.45 s (9H).

- A mixture of 0.675 g of DL-7-~a-tert-butoxycarbon a-(2-thienyl)-acetamido~-3-(1-methyltetrazol-5-ylthiomethyl)-: 30 3-cephem-4-carboxylic acid (as prepared according in example 5), ,'~ ' ' '~' ;,' ::

~ i~
104~087 i~, 7 ml. of trifluoroacetic acid and 0.7 ml. o~ thiophenol is `~
stirred ~or 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 DARC0 G60 (a product manufactured and sold by Atlas Chemical Industries, New Murphy Road, Wilmington, DEL 19899), The mixture is filtered and pouredi~ diisopropyl ether to yield a preci-pitate of DL-7-~a-carboxy-~-(2-thienyl)-acetamido~-3-(1-methyl-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.
Rf = 0.55 (+ 0.10) in the system methylisobutylketone/
~ methanol/formic acid(80/18/2)or 0.29 (+0.10) in the system aceto-; nitrile/water (8/2), detection with U.V. or bromocresol green.
nmr (acetone-d6) 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 (DMS0-d6) : 9.41 d (1H) (J
8.5 c./sec.), 7.54 d (1H) (J 5.0 c./sec.3, 7.~0 m (2H) (J 5~0 c./sec.), 5.76 dd (1H) (J 8.5 & 4.7 c./sec.), 5.16 d+s (lH + 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 N NaHC03 solution is added dropwise up to pH 4.4 and the aqueous solution is freeze-dried.
., .

A mixture o~ 22.5g of 2-thiophene acetyl chloride (pre-pared according to the procedure described by P. Cagniant in Bull.
Soc. Chim. France 847-53,1949), 50 ml. o~ 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 ~ther. The ethereal solution is washed once !

' .

'' :.: ` . . ~ ` . : . . ~ : ' . .
.

i 10410 917 ~

with 50 ml. of a 8.5% NaHC03 solution, twice with 25 ml. of water, dried over anhydrous Na2S04, evaporated under reduced pressure and the residue is distilled to yield benzyl 2-thienylacetate.
Rf = 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),

5.12 s (2H), 3.70 s (2H).

To 7.7 ml. of cooled (-10~ 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, ; 15 11.6 g of benzyl 2-thienylacetate (as prepared in example 7) 7 and the obtained mlxture 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 eth~real 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 ~' . 11 with 2 portions of 50 ml. of diethyl ether and the combined ethereal solutions are washed with water, dried over anhydrous ; Na2S04 and evaporated under reduced pressure.
, 25 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 .', . ' i' , - . . - . . . . . . . . .

.,, . ~ . . . . ; . :.

1 04~087 `,;d To 2.5g of cooled (- 5 C) monobenzyl 2-thienyl malonate(as prepared in example 8) and dissolved in 100 ml.
- of tetrahydroÇuran there are added 1.15 g of N-hydroxy-succinimide and 2.06 g of dicyclohexylcarbodiimide. The mixture is stirred for 15 minutes between 0 and -5 C, thereaÇter at 20 C for 135 minutes and then filtered. The filtrate is evaporated under reduced pressure. The obtained crude N-~a-benzyloxycarbonyl-a-(2-thienyl)-acetoxy~-succinimide is dissolved in 50 ml. o~ acetonitrile and this solu-tion is added, at once, to a mixture of 3.3g o~ DL-7-amino-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic ~cid (as pre-- pared 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 resldue 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 Çiltered.
,,., 11 After concentration under reduced pressure,the solution is poured into diisopropylether to precipitate Dl-7-~ -benzyloxycarbonyl-~-(2-thienyl)acetamido~-3-(1-methyl-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.
Rf = 0.79 (+ 0.10~ in the system acetonitrile/
~ methanol/water(6/3/1), detection with U.V. or bromocresol ; -green.
nmr (DMS0-d6) : 9.44 d (1H) (J 7.2 c./sec.), 7.60-

7.45 m (1H), 7.34 s (5H), 7.06 m (2H), 5.67 q (1H) (J 7.2 and -- 1 4 _ . ' ' ~ . - ~-- ~ .
: :.
-. .

114.7 c./sec.), 5.40-5.00 m~s (2 x 1H + 2H), 4.31 m (2H), 3.94 s (3H), 3.64 m (2H).

100 mg o~ DL-7-~a-benzyloxycarbOnyl-a-(2-thienyl)-acetamido~-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid (as prepared in example 9) and 20 mg of a-chymotrypsin dissolved in 20 ml. o~ a bu~fer phosphate solution (0.2 M and pH 7.0 are heated at 37 C for 75 minutes.
:. ,.
The mixture is acidi~ied to pH 2.0 by addition o~
10N HCl and extracted with ethyl acetate; the organic layer is dried over anhydrous Na2S04, most o~ the solvent is eva-porated and the residue is poured into 100 ml. o~ diisopropyl ether to precipitate DL-7-~a-carbOxy a-(2-thienyl)-acetamido~-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-~-carboxylic acid -~ 15 showing the same characteristics as those indicated ~or the ~ product o~ example ~.
,f ~ .
~ The sodium salt is prepared by dissolving the acid - -....
in ethyl acetate, adding a sodium 2-ethylhexanoate solution, and then slowl~ adding ether until the sodium salt is precipitated.

1~ ' An injectable pharmaceutical composition is prepared by dissolving 100-500 mg o~ sodium DL-7-~a-carb a-(2-thienyl)-acetamido~-3-(1-methyltetrazol-5-ylthiomethyl) 3-cephem-4-carboxylate (as prepared in example 10) in sterile water (1-5 ml.l~.

. . ' .

;

.,:' " . , . ' ,' ' - ' ' ' . ' ' ~ ~ '`

- 104~087 .~ ~
; EXAMPLE 12 A solution of 9.25 g (0.058M) ofl3-thiophene ~; acetyl chloride in 7.5 ml. of tert-butyl alcohol and i:~
;~ 30 ml. of diethyl ether is heated to re~lux and 8.5 ml.
. . .
~ 5 of dimethylaniline dissolved ln 20 ml. of diethyl ether - ~ is added dropwise. The mixture is refluxed Çor 5 hours i and ~iltered. The filtrate is washed twice with 20 ml~ water, ~-~ once with 20 ml. N HCl, once with 20 ml. water, twice with 20 ml. N NaHC03 and twice wlth 20 ml. H20, and then dried over anhydrous ~aC12 and distilled under reduced pressure (107-120C/

8 mm) to yield tert-butyl 3-thienyl acetate.
R~= 0.65 (~ 0.10) in chloroPorm, detection with bromocresol green.
nmr (CDCl ) : 7.15 m (3H)j 3.51 s (2H), 1.44 s (9H)-.," .~ .
;: EXAMPLE 13 : . :
~- To a solution of 7 ml. of diisopropylamine in : 50 ml. of tetrahydrofuran maintained under dry nitrogen i.. .-..
~ atmosphere th~re is quickly added 65 ml. o~ a 5 % solution ; ~ 20 of n-butyllithium in hexane, the reaction medium being - maintained between -10 and -15 C. A~ter 15 minutes standing, 8.5 g of tert-butyl 3-thienylacetate (as prepared in example 12) in 21 ml.\o~ tetrahydrofuran is - 11 .
; added thereto. After 5 minutes standing, the mixture is poured onto solid carbon dioxide and stirred for one hour.
, The solvent is evapor~ated and the residue i5 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 12 N HCl and extra~ted once with 90! ml. ether, once with , , , .

:

'; ' : ' ~04~087 45 ml. ether and once with 25 ml. ether.
The combined ethereal solutions are washed twice with 20 ml. water, dried over anhydrous CaCl2 and evaporated under reduced pressure to yield crystallized mono tert-butyl 3-thienyl malonate.
Rf = 0.60 (+ 0.10) in chloroform, detection with bromocresol green and alc. KMnO4.
nmr (CDCl3): 10.31 s (1H), 7.29 m (3H), 4.71 s (1H), 1.45 s (9H).
, . .

To a cooled (-15 C) solution of 7.9 g (32 mmol) ;~ mcnotert-butyl 3-thienyl malonate (as prepared in example 13) ,. ~
~ dissolved in 100 ml. o~ tetrahydrofuran, there are added ., .
;~ 4.2 g of N-hydroxysuccinimide and 8.3 g of dicyclohexyl-- 15 carbodiimide. The mixture is stirred at -15 C for 3 hours ~- and then Piltered. The filtrate i-s evaporated to dryness ,' and the residue is treated twice with 75 ml. of tetra-~; hydrofuran to eliminate dicyclohexyl urea. The solvent ~ -is evaporatedito dryness and the oily residue is treated . , :
20 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-~-tert- ~-butoxycarbonyl-~-(3-thienyl)-aceto~cy~7-succinimide.
Melting point: 97-98 C.
- 25 Rf = 0.77 (+ 0.10) in the system chloroform/aceto-nitrile/water/formic lacid (35/58/5/~), detection with U.V.
nmr (CDC13): 7.33 m (3H), 4.96 s (1H), 2.80 s (4H), 1.50 s (9H).

30 To 13.02,g of 1-methyl-5-~nercapto-1,2,3,4-. , , - .................................... . ~ ... ,, . . :

- 10~1087 tetrazole prepared according to the procedure described by Stollé R. et al. in J. Prakt. Chem. ~2~!~ 124, 261-79 - (1930) and dissolved in 8,oo ml. of a 0.2 M phosphate bu~er 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 triethyl-amine, heated at 70 C for 80 minutes, at once cooled to ` 20 C, acidified to pH 3.5 by addition of 65 ml. of 2 N HCl and then stirred for 15 minutes.
c 10 The obtained precipitate is filtered, washedwith 200 ml~ oÇ water, 200 ml. of methanol and 200 ml.
of diethylether and dried under reduced pressure to yield :.
-~ a crude product which is dissolved in 300 ml. of 2 N HCl, treated with DARC0 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. jl The crystallization medium is stirred for 30 minutes at 25 C and filtered under reduced pressure. The li precipitate is washed with 200 ml. of water, 200 ml. of methan~l and 200 ml. of diethylet~er and dried at 40 C
over P205 under reduced pressure to yield 7-amino-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.
Rf = 0.44 (~ 0.10) in thelsystem acetonitrile/
water (80/20), detection with U.V. or bromocresol green.
nmr (D20 - Na2C03) 5.48 d (1H) (J 4-7 c-/sec-), 5.08 d (1H) (J 4.7 c./sec.), 4.40 and 4.06 2d (2 x lH) (J 13 c./sec.), 3.~5 s (3H), 3.86 a~d 3.43 2d (2 x 1H)(J18c./sec.) ,`~
~ - 18 -.
; - ~
.

~i 104~0~7 ; EXAMPLE 16 - Tc a mixture of 9.5 g o~` 7-amino~3-(1-methyl-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid (as prepared in example 15) and 8.1 g o~ triethylamine in 120 ml. o~ acetonitrile there is ~dded at once 11.4 g o~
N-~-tert-butoxycarbonyl-~-(3-thienyl)-acetoxy~-succini-mide (as prepared in example 14). The mixture i5 stirred ~or 30 minutes at 30 C and ~iltered. The solvent i5 then ~'~ removed under reduced pressure and the residue is taken up in ~0 ml. of distilled water and ethyl acetate (~0 ml.) is added ; thereto. The aqueous layer is acidi~ied with 0.1N HCl (pH 2) and ' extracted three times with 40 ml. o~ ethyl acetate and the combi-ned organic layers are dried over anhydrous sodium sul~ate, deco-lorized with charcoal and evaporated under reduced pressure. The residue is ~aken up in ethyl acetate (20 ml.) and 250 ml.
o~ di-isop~opyl ether is added thereto. The obtained crude product is dissolved in 300 ml. o~ ethyl acetate and, a~ter 30 minutes stirring, the solution is ~iltered.
- The ~iltrate ~s decolorized with Darco G 60 (5 g), ~ilteredon Dicalite S.H. (a product sold by DICALITE EUROPE NORD, Brussels, Belgium) and most o~ the solvent is evaporated.
- Di-isopropyl ether (400 ml.) is added thereto to precipitate DL-7-~-tert-butoxycarbonyl--(3~~jhienyl)-acetamido~-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.
R~ = 0.695 (+0.10) in the system chloro~orm/
acetonitrile/water/~qrmic acid (35/58/5/2), detection with ,.
U.V. or bromocresol green.
nmr (DMSO) : 9.20 dd (iH), 7.34 m (3H), 5.6~ m (1H), 5.10 dd (lH), 4.~9 s (1H), 4.31 m (2H), 3.95 s (3H), 3.6~ m (2H), 1.41 s (9H)., , ' 9 ~

... -,~. . ...

'~ ' ' " .' .. ' " ' " ' . ": ' ., :
- . . ' ., . ~,, ' ~ ' , ' . . '. .'' A mixture o~ 4 g (7.25 mmol) o~ DL-7-~a-tert-,', butoxycarbonvl-a-(3-thienyl)-acetamido~-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid (as prepared ; ; 5 in example 1~;), 25 ml. o~ tri~luoroacetic acid and 2.5 ml.
~; o~ thiophenol is stirred for 70 minutes at 0 C. Most o~
the solvent ~s removed under reduced pressure and 300 ml.
of di-isopropyl ether is added thereto. The obtained crude product is dissolved in 350 ml. o~ ethyl acetate and the solution is ~iltered. Most o~ the filtrate is removed under reduced pressure and 300 ml. o~ di-isopropyl ether is poured onto the residue. A~ter ~ilt?~ation , and drying, ;~he product is dissolved in 300 ml. o~
~; ethyl acetate~and then extracted into water (50 ml.) with 0.1N NaHC03 up to pH 5.7.
The aqueous solution is lyophilized to yield DL-7-~a-carboxy-c~-(3-thienyl)-acetamido7-3-(1 methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid, disodiui~ salt.
,20 ~ = 0.52 (+ 0.10) in the system chloro~orm/
acetonitrile/water/~ormic acid (35/58j5/2), detection - with U.V.
nmr (D20): 7.35 m (3H)~j 5.69 d (1H), 5.10 dd -(1H), 4.21 m (2H), 4.05 s (3H), 3.59 m (2H).

An injecta~le pharmaceutilcal composition is prepared by dissolving in sterile water (1-5 ml.) 100-150 mg o~ DL-7-~a-carboxy-a-(3-thienyl)-acetamido7-3-(1-methyltetrazol-5-~- ylthiomethyl)-3-cephem-4-carboxylic acid, disodium salt as prepared in exemple 17.
.,'.; , .

,' -:

Claims (4)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing a compound of the formula:

which comprises acylating of the 7-amino-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid with an activated derivative selected from the group consisting of monotert-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.

2. The process according to claim 1 wherein the activated derivative is N-[?-tert-butoxycarbonyl-(2-thienyl) -acetoxy]-succinimide.

3. The process according to claim 1 wherein the activated derivative is N-[?-benzyloxycarbonyl-?-(2-thienyl)-acetoxy]-succinimide.

4. A compound of the formula or a non toxic pharmaceutically acceptable salt thereof whenever prepared or produced by the process of claim 1 or by any chemical equivalent thereof.