FI63943B - VID FRAMSTAELLNING AV 7-ACYLAMIDO-3- (1-CARBOXY-LAEGRE-ALKYL-TETRAZOLE-5-YLTHOMETHYL) -3-CEFEM-4-CARBOXYLSYROR OR DERAS SALTER SAOSOM MELLANPRODUKTER ANVAENDBARA FOERENAR - Google Patents

Description

63943 Intermediate compounds used in the preparation of 7-acylamido-3- (1-carboxy-lower alkyl-tetrazol-5-ylthiomethyl) -3-cefine-4-carboxylic acids and their salts 5

This invention relates to intermediates for the preparation of 7-acylamido-3- (1-carboxy-lower alkyl-tetrazol-5-yl-10-thiomethyl) -3-cephem-4-carboxylic acids of the formula R1-NH_ | f \ - L | il i, 5 <S?

COOH (^ CH.) -COOH

Wherein n1 is acyl and n is an integer from 1 to 3, and the 7-amino-3- (1-carboxy-alerapi-20-alkyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acids of formula is S \

H-N - f N-N

I I! J B

25 -HY ^ CV- <b ^

COOH (CH_) -COOH

λ n where n means the same as above, and their salts.

The preparation of the final product is described in FI application 752434 (pat. No. 62 841); the intermediate of the invention (formula B) is reacted with an acylating agent of the formula α-ch2-cox 35 in which A represents a group 63943 _s CH2NHR '^ x ^ .CH2NHR' _✓CH2NHR 'R - \ ^ y— or II or 5 in which formulas R is hydrogen, hydroxy or methoxy and R 'is hydrogen or methyl, and X represents a halide or a functional equivalent thereof.

A new intermediate is prepared by removing the 7-side chain of formula 1 ° .ς R -NH-C-CH CHO N - N.

Il i2 J

0 / N \ ^ - C ~ CH2

, c COOH (CH_) COOH

Of a compound of formula 2 wherein n is as defined above and R is an acyl group by converting said reagent, preferably in silyl form, to an iminochloride (e.g. phosphorus pentoxide) and then to an iminoether (e.g. methanol) and then to the desired product by hydrolysis (e.g. water).

The details of carrying out such reactions are as follows: are known in the art and are described, for example, in U.S. Patents 3,575,970, 3,573,295 and 3,573,296.

According to another method, a new intermediate is prepared by reacting 7-aminocephalosporanic acid or a salt or easily hydrolyzable ester Schiff's base of formula

N N

30 J I, hs — il ja

OF

(CH_) COOH 2 with a compound of formula n wherein n is the same as above.

63943

Suitable salts include carboxylic acid salts including metal salts, e.g. sodium, potassium, calcium, aluminum, ammonium and substituted ammonium salts, e.g. salts formed by such amines, such as trialkyl-5-amines, including triethylamine, procaine, dibenzyl. , N-benzyl-yk -phenethylamine, 1-efenamine, N, N'-dibenzylethylenediamine, dehydroabietylamine, Ν, Ν'-bid-dehydroabiethylethylenediamine, N-lower alkylpiperidine, e.g. N-ethylpiperidine and other amines used to form salts with benzylpensillin and their non-toxic acid addition salts (i.e. amine salts) including mineral acid addition salts, e.g. , tartrate, fu-malate, malate, mandelate and ascorbate.

The amino group of a compound of the invention may be protected by a substituent, e.g. 2-iodoethoxycarbonyl (GB Patent 1,349,673), tert-butoxycarbonyl, carbobenzyl solutions, formyl, o-nitrophenylsulphenyl, trichloroethoxycarbonyl, 2-oxo-2-oxo-2-oxo-2 or 1-carbomethoxy-1-propenyl-2. In particular, such protecting groups include ketones (especially acetone) and aldehydes (especially formaldehyde and acetaldehyde) disclosed in, e.g., U.S. Patents 3,198,804 and 3,347,851, and β-ketoesters and β-diketones which are disclosed in U.S. Patent 3,325,479 and ketoamides disclosed in JP Patent 71/24714 (Farmdoc 47,321S).

4,63943

Example 1 7-Amino-3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid 5

.11 H2N. S

N 2 + I-0 10 NaS ^ - ^ «2C ° 2Na CH 2 -O-i-CH 3

2N I h I

J-N c-i - N -CH-CO-H

20 0 ^ CH2 "S 2 2 co2h 1. To a three-necked flask equipped with a stirrer, thermostat, thermometer, and nitrogen inlet tube, add 18 g (0.066 moles) of 7-aminocephalosporanic acid and 300 mL of 0.1 M pH 6.4. phosphate buffer (20.7 g of monobasic sodium phosphate. Η2 <0 and 8.5 g of anhydrous dibasic sodium phosphate in 2 liters).

2. Mix the mixture described in step 1 and add 1.5 g of sodium bisulfite and 16 g (0.078 mol) of disodium 1-carboxymethyl-5-mercaptotetrazole.

3. Stirring is continued and nitrogen is bubbled through the mixture for 10 minutes.

4. Stirring and bubbling of nitrogen are continued and the sus-35 suspension is heated for 20 minutes at 56 ° C. During this time, 6.5 g of sodium bicarbonate are added in small portions.

5. Stirring and bubbling of nitrogen are continued and the temperature of the solution is maintained at 56 ° C for four hours. In this case, the pH must be between 6.2 and 6.6.

6. Cool the reaction mixture in an ice bath to 5 ° C.

7. Add 50 ml of a 1: 1 aqueous phosphoric acid solution or concentrated hydrochloric acid to a pH of 2.0 to 3.0.

8. The product is isolated by filtration. The filter cake is washed with 20 ml of cold water and 200 ml of cold methanol.

9. The solid is air dried to constant weight. In a typical run, 14.5 g of product were obtained. The color 10 of this product may vary from yellow to dark brown.

10. The product is sieved through a 200 mesh stainless steel sieve.

11. Suspend 10 g of 200 mesh powder with vigorous stirring in 200 ml of n-propanol.

15. Add 2.0 ml of concentrated hydrochloric acid and stir vigorously for half an hour at room temperature.

13. Filter the suspension. The brown solid is washed with 20 ml of n-propanol and the washings are added to the filtrate (the filter cake is recovered to further isolate the product).

14. To the n-propanol filtrate of step 13 add 1.5 g of charcoal (Darco G-60). Suspend for half an hour. The carbon is removed by filtration. The carbon is washed with 20 ml of n-propanol and the washings are added to the filtrate.

15. Stir vigorously and add triethylamine to the apparent pH of 3.0 to the n-propanol filtrate. Crystals form. Suspend for 10 minutes.

16. The white crystals are isolated by filtration, washed with 30 ml of n-propanol and 50 ml of methanol and dried under vacuum at 40 ° C for 24 hours. Yield 4-8 g of 7-amino-3- (1-30 carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid, m.p. 157 ° C (decomposes).

17. An alternative method for purifying 7-amino-3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid is as follows: a) suspend 10 g of the product of step 10 200 mesh in 75 ml of 1- n hydrochloric acid for 10 to 15 minutes at room temperature. Filter to remove dark brown solids.

__. I

6 sex. «94 3 b) Add 2.5 g of charcoal (Darco G-60) and suspend for half an hour.

c) The carbon is removed by filtration. The charcoal is washed with 15 ml of water and the washing liquid is added to the filtrate.

d) Stir vigorously and add concentrated ammonium hydroxide to the filtrate to pH 2.5 to 3.0. Crystals are formed.

e) The crystal mass is suspended for 25 minutes. The crystals are isolated by filtration. The crystals are washed with 30 ml of water and 50 ml of methanol and dried in vacuo at room temperature. Yield 4 to 7 g of off-white crystals; mp. 157 ° C. Preparation of starting materials 1-carboxymethyl-5-mercaptotetrazole 15

N-N

P II

HS - c N

20 |

CH 2 COOH

a) Recrystallization of 1-methyl-5-mercaptotetrazole Method: 1. Suspend 110 g of 1-methyl-5-mercaptotetrazole in 350 ml of boiling chloroform. Dissolution is almost complete.

2. The hot solution (50-60 ° C) is rapidly filtered through a vacuum heated Buchner funnel (11 cm, paper SS No. 604, 6.4-8.5 mm concentrated filter aid (Supercel)).

The filter cake is washed with 50 ml of 50-60 ° C hot chloroform, which is added to the filtrate.

3. Cool the filtrate to about 0 to 6 ° C and keep at this temperature for two hours. The formed crystals are isolated by filtration at 0 to 6 ° C and washed with 60 ml of 0 to 6 ° C chloroform, which is added to the filtrate. The crystals (part A) are dried for 18 hours at 37-45 ° C.

4. Concentrate the filtrate on a rotary evaporator 7 63943 (bath temperature 60 ° C) to about half volume. This suspension is cooled to 0-6 ° C and kept at this temperature for two hours. The crystals are isolated by filtration at 0 to 6 ° C, washed with 40 ml of chloroform at 0 to 6 ° C, which is added to the filtrate. The crystals (Part B) are dried in air for 18 hours at 37-45 ° C. The crystalline parts A and B are combined to give a yield of about 65% by weight.

5. The filtrate from Part 4, Step B can be re-treated twice as described in Step 4 to give a yield of 15%.

b) Preparation of the disodium salt of 1-carboxymethyl-5-mercaptotetrazole 1. Cool 500 ml of almost dry and pure tetrahydrofuran in a 2-liter three-necked flask equipped with a stirrer in a salt-acetone-ice bath to about -10 ° C. Dry nitrogen gas is introduced to the surface of the liquid.

2. To tetrahydrofuran, add 500 mL of 15.06% (1,6-n) butyllithium in hexane (Foote Mineral Co.) over 10 minutes under dry nitrogen and, with stirring, cool the almost complete solution to -5 to -10 ° C.

3. Dissolve 46.4 g of 1-methyl-5-mercaptotetrazole (recrystallized as above) in 200 ml of almost pure and dry tetrahydrofuran. If the solution is cloudy, filter and cool to 5-10 ° C.

4. The cooled solution of step 3 is added to the butyllithium solution over 10 minutes with stirring under dry nitrogen. The temperature must be -5 ... + 10 ° C. A precipitate may form.

5. Stir the mixture for half an hour in a dry nitrogen blanket at 0 ... + 10 ° C.

6. Dry carbon dioxide is bubbled into the mixture at high speed and with vigorous stirring for 15-30 minutes from about ambient temperature (0-10 ° C) to a maximum of + 20 ° C.

7. The white precipitate formed is preferably isolated by filtration at a low humidity location. The precipitate is washed with about 75 ml of tetrahydrofuran.

8 63943 8. Dissolve the precipitate in 250 ml of water (pH 8.5 to 9.5). A second layer of tetrahydrofuran may be present. This can be removed in a rotary evaporator (bath temperature 50 ° C).

9. The pH of the aqueous solution is adjusted to 1.6 to 2.0 with concentrated hydrochloric acid.

10. The acidic aqueous solution is extracted twice with 250 ml portions of ethyl acetate. Each 250 ml of ethyl acetate extract is extracted with 10 ml portions of water. The aqueous extracts are discarded. The ethyl acetate extracts (without the aqueous layer) are filtered and combined.

11. The combined ethyl acetate extracts are evaporated to dryness on a rotary evaporator (bath temperature 60 ° C).

12. Boil the crystals in the flask and 300 ml of chloroform for about 2 minutes. The hot suspension (50-60 ° C) is filtered through a vacuum heated Buchner funnel (paper 11 cm SS 604). The crystals are washed with about 75 ml of warm chloroform at 50 ° C. The crystals are dried for about 3 hours at room temperature and ground to 100-200 mesh.

13. Crystals of 100 to 200 mesh are treated with boiling 20 chloroform exactly as in 12 (hot chloroform removes most of the unreacted 1-methyl-5-mercapto-tetrazole). Yield about 45-50 g of crystalline 1-carboxymethyl-5-mercaptotetrazole. These crystals may contain 0.02 to 0.05 moles of 1-methyl-5-mercaptotetrazole.

14. The crystals of step 13 are suspended for 3-5 minutes in 250 ml of ethyl ether at room temperature. The mixture is filtered. The insoluble matter (0.5 to 5%) may consist of a symmetrical symmetrical mercaptotetrazole ketone with an experimental formula of 30 j — t ϊ ”1 N N - CH2-C-CH2-N f.» f f

35 SH SH

Note. This compound explodes at a temperature of about 205-210 ° C.

r 63943 15. The ether filtrate of step 14 is evaporated to dryness on a rotary evaporator (bath temperature 50 ° C). N.

42 to 48 g of crystalline 1-carboxymethyl-5-mercaptotetrazole containing about 0.01 to 0.05 moles of 1-methyl-5-mercaptotetrazole.

16. The crystals are dissolved in 420 with any absolute ethanol (about 100 mg / ml). The solution is heated to 50-60 ° C.

17. 310 ml of a 41% solution of 2-ethylhexanoate (SEH) in isopropanol are added to the hot solution of step 16 over 10 minutes with very vigorous stirring. A crystalline precipitate forms. The mixture is suspended for 20 minutes at 50-60 ° C.

18. Filter the mixture through a heated (50-60 ° C) heated Biichner funnel (11 cm paper, SS No. 604). The crystals are washed with 75 ml of 50 ° C ethanol.

19. The ethanol wet crystals of step 18 are suspended in 200 to 300 ml of ethanol. The suspension is screened through a 200 mesh screen. The suspension is heated for five minutes at 50-60 ° C with vigorous stirring (unreacted sodium 1-methyl-5-mercaptotetrazole is very soluble in hot ethanol.

20. The crystals are isolated by heating at 50-60 ° C

Using a Biichner funnel, paper 11 cm, SS No. 604. The crystals are washed with 75-100 ml of ethanol and dried under vacuum at 50-60 ° C for 24-48 hours. Yield 40-48 g of disodium 1-carboxymethyl-5-mercaptotetrazole (according to NMR it does not contain 1-25 methyl-5-mercaptotetrazole).

Preparation of 1-carboxyethyltetrazole-5-thiol

JP

30 HS-N

r (ch2) 2-co2h A) 2-Carbethoxyethyl isocyanate 35.3 g of β-alanine ethyl ester hydrochloride, 123.5 g of triethylamine and 400 ml of methylene chloride are mixed and cooled to 10 DEG C. 46.5 g of carbon disulphide dissolved in 150 ml of chloroform are added to the solution over two hours and the temperature is maintained at about -10 ° C. After the addition was complete, the solution was allowed to warm to 10 ° C for about 10 minutes. The solution is re-cooled to -10 ° C, 66.8 g of ethyl chloroformate in 60 ml of chloroform are added dropwise with stirring over 40 minutes. The temperature is allowed to rise to room temperature for 30 minutes and re-cooled to 0 ° C. At this temperature, 61.6 g of triethylamine are added and the solution is stirred at room temperature for 3 hours.

The mixture is treated with water and the organic phase is isolated, washed twice with 250 ml portions of 2N hydrochloric acid, then twice with 250 ml portions of sodium bicarbonate and then twice with 250 ml portions of water. The organic phase is dried over sodium sulfate and the solvent is removed in vacuo to give 93.7 g of an oil which was found to be the desired product. The IR and NMR spectra correspond to the structure.

B) 1-Carboxyethyltetrazole-5-thiol

Dissolve 29.7 g of sodium azide in 400 ml of water and heat to 60 ° C under a nitrogen blanket. To the hot sodium azide solution is added 46.9 g of 2-carboxyethyl isocyanate dissolved in 50 ml of Skellysolve B (mainly n-hexane). The solution is stirred for about 150 minutes at about 70-72 ° C, cooled to 30 ° C in an ice bath and 50% sodium hydroxide solution is added so that the pH is 12. The mixture is heated for 40 minutes at 70 ° C and cooled to 15 ° C. ° C in an ice bath. The pH is adjusted to 2 with concentrated hydrochloric acid and extracted four times with 150 ml portions of ethyl acetate. The ethyl acetate extracts are washed with water and dried over sodium sulfate.

The solvent is evaporated in vacuo and the product is isolated as crystals from methylene chloride to give 19.5 g of the title product; mp. 147-150 ° C.

35 5 11 63943

Example 2 7- (2-Aminomethyl-phenyl-acetamido) -3- (1-carboxymethyl-tetrazol-5-yl-thiomethyl) -3-cephem-4-carboxylic acid CH2NH2 10 [mu] M | J ”—C 2 0 T l

COOH CH2COOH

1. To a three-necked flask containing 100 ml of deionized water equipped with a stirrer and a thermometer is added 7.6 g (0.02 Moles) of 7-amino-3- (1-carboxymethyl-tetrazol-5-yl-thiomethyl) -3 -cephem-4-carboxylic acid and 3.4 g (0.034 mol) of N-methylmorpholine. Cool to 0 ° C.

While stirring, the solution is kept at 0 ° C with an ice bath.

2. Add 9.6 g (0.03 moles) of sodium 2- (1-methoxycarbonyl-1-propen-2-ylaminomethyl) -phenyl acetate and 184 ml of tetrahydrofuran to a separate stirred flask. With stirring, the suspension is cooled to -30 ° C with a dry ice-acetone bath. While stirring at -30 ° C, 20 drops of dimethylbenzylamine and 4.4 g (0.03 mol) of isobutyl chloroformate are added. The resulting mixture is stirred for 5 minutes.

3. Add the mixture formed in step 2 to the stirred solution of step 1 at a time. The resulting solution is stirred for one hour at 3 ° C.

35 12 6394 3 4. Tetrahydrofuran is evaporated from the reaction mixture at 30 ° C under vacuum (15 mm Hg).

5. The pH of the remaining aqueous solution is adjusted to 4.0 with concentrated hydrochloric acid.

5 6. Add 2.5 g of charcoal (Darco G-60) to the solution and stir for 20 minutes. The carbon is removed by filtration.

7. Pour 120 ml of ethyl acetate into the filtrate and lower the pH to 3.8 with concentrated hydrochloric acid with stirring. A small amount of pale precipitate is formed which is removed by filtration 10 (saved for post-treatment and recovery).

8. Cool the filtrate in an ice bath to 5 ° C and lower the pH to 2.5-2.8 with concentrated hydrochloric acid with stirring. Stirring is continued for one hour at 5 ° C.

9. The product is isolated by filtration. The filter cake is washed with 5 to 15 ml of cold deionized water and 5 ml of cold methanol.

Crystalline 7- (2-aminomethylphenylacetamido) -3- (1-carboxymethyltetrazo-5-ylmethyl) -3-cephem-4-carboxylic acid is air dried to constant weight, 4.1 g.

11. The product from step 10 is screened through a 200 mesh stainless steel screen.

12. Suspend 10 g of this 200 mesh product in 100 ml of chloroform. 5 ml of triethylamine are added and the mixture is heated to 50 ° C with vigorous stirring. The mixture is suspended for 5 minutes at 50 ° C.

13. The mixture is filtered hot (7-amino-3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid, pigments and other impurities are soluble in hot chloroform-triethylamine solution). The filter cake is washed with 25 ml of chloroform and air-dried for 2 hours. Yield 1-8 g of 7- (2-aminomethylphenylacetamido) -30- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid.

14. The product from step 13 is screened through a 200 mesh screen.

15. Suspend 10 g of 200 mesh product in 75 ml of 0.1 N hydrochloric acid for 10-15 minutes. The mixture is filtered, the filter cake 35 is washed with 25 ml of water and 50 ml of methanol and air-dried at room temperature for 2-3 hours. Yield 10 g.

13-394 3 16. Suspend 10 g of 7- (2-aminomethylphenylacetamido) -3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid obtained in step 15 in 65 ml of methanol, and (a) 2 ml of concentrated hydrochloric acid are added; stirring for 5 to 5 minutes, b) 130 ml of water, pH 1.3-1.6, are added rapidly and vigorously with stirring, a precipitate (containing most of the color) immediately forms.

c) the mixture is suspended for one minute and filtered rapidly (saving solids for work-up and recovery); d) the filtrate is inoculated and mixed gently; crystallization begins after about 15-30 minutes, e) the mixture is stirred at ambient temperature for 2 hours after the onset of crystallization, and - 15 f) the crystals are isolated by filtration, washed with 25 ml of a mixture of 65% water and 35% methanol (v / v). t) and dried under vacuum for 1 day at 50 ° C. Yield 9 g of purified white 7- (2-aminomethylphenylacetamido) -3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid.

2g 17. The following are two alternative methods A) and B) for crystallizing 7- (2-aminomethylphenylacetamido) -3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-4-carcoxylic acid; A) 1. Suspend 10 g of the product of step 15 in 100 ml of methanol.

2. Add 2 ml of concentrated hydrochloric acid.

3. Add 1.5 g of charcoal (Darco G-60) and suspend the mixture for half an hour.

4. The carbon is removed by filtration and washed with 20 ml of methanol. The washing liquid is added to the filtrate.

5. Add 120 ml of water to the filtrate (the small amount of precipitate formed is removed by filtration and saved for reprocessing and recovery).

6. Stir the solution of step 5 vigorously and adjust the pH to 2.5-3.0 with 10% sodium hydroxide solution. A precipitate forms.

7. Mix mixture for 1 lot for half an hour. The precipitate is isolated by filtration, washed with 20 ml of 50% methanol-water (v / v), 30 ml of methanol and dried in vacuo for 1 day at 50 ° C. 9 g are obtained

_ I

14 9 3 9 4 3 Pure 7- (2-aminomethylphenylacetamido) -3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid.

B) 1. Suspend 10 g of the product of step 15 in 75 ml of water.

5. Add 10% sodium hydroxide solution until the pH is 6.8 to 7.2.

3. Add 1.5 g of charcoal (Darco G-60) and stir the mixture for half an hour at pH 6.8 to 7.2 (continuously add 0.1 to 1 N sodium hydroxide solution).

10 4. Carbon is removed by filtration; the precipitate is washed with 20 ml of water, which is added to the filtrate.

5. The solution of step 4, pH 6.8 to 7.2, can be crystallized at pH 1.2 to 1.5 (by adding hydrochloric acid) and as described in steps 16, d, e, and f. In both cases, 15 9 g of crystalline 7- (2-aminomethylphenylacetamido) -3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid.

Example 3 7- (2-Aminomethyl-1,4-cyclohexadienyl-acetamido) -3- (1-carboxymethyl-tetrazol-5-yl-thiomethyl) -3-cephem-4-carboxylic acid

ch2conh-j — f "" '• j | OF

cooh 1: h2cooh

A solution of 0.80 g (0.003 mol) of 2-tert-butoxy-30-carbonylaminomethyl-1,4-cyclohexadienylacetic acid and 0.303 g (0.003 mol) of triethylamine in 19.2 ml of tetrahydrofuran is stirred at 0 ° C. and 0.41 g (0.003 mol) of isobutyl chloroformate is added. The mixture is stirred for 30 minutes at 0 ° C and added to a solution of 0.003 moles of 7-amino-3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid and 0.61 g (0.006 mol) of triethylamine in 9.2 ml of 50% tetrahydrofuran. The resulting solution is stirred for 1.5 hours at 25 ° C. Tetrahydrofuran is evaporated at 30 ° C / 15 mm 15 63 94 3

At Hg, the residue is washed twice with 30 ml portions of ether and half diluted with water. The solution is acidified to pH 3.5 with dilute hydrochloric acid, the product is isolated and dried in vacuo for 18 hours with phosphorus pentoxide at 25 ° C to give 1.55 g (54.0%) of a white powder. A total of 3.4 ml of trifluoroacetic acid is added to the above 7- (2-tert-butoxycarbonylaminomethyl-1,4-cyclohexadienylacetamido) -3- (1-carboxy-> 2-methyltetrazol-5-ylthiomethyl) -3-cephem-4 carboxylic acid and stirred for one hour at 0 ° C. The solution is diluted with 150 ml of ether and the precipitate is isolated by filtration. The trifluoroacetate salt is suspended in 3.4 ml of water and the pH is adjusted to 4.5 with dilute ammonium hydroxide solution. The gummy residue is triturated in water, isolated and washed with water and acetone. The product is dried under vacuum for 18 hours with phosphorus pentoxide at 25 ° C to give 53 mg (15.72%) of 7- (2-aminomethyl-1,4-cyclohexadienylacetamido) -3- (1-carboxymethyltetrazol-5- ylthiomethyl) -3-cephem-4-carboxylic acid, m.p. above 160 ° C (decomposes slowly).

Analysis for C20H23N7O6S2.1 / 2 H., 0

Calculated: C 45.18 H 4.55 N 18.44 20 Found: C 45.46 H 4.68 N 17.09

Example 4 7- (2-Aminomethyl-phenyl-acetamido) -3- (1-carboxymethyl-tetrazol-5-yl-thiomethyl) -3-cephem-4-carboxylic acid monosodium salt trihydrate 1. Suspend 10 g of crystalline 7- (2-aminomethyl-phenyl-acetamido) -3- (1-Carboxymethyl-tetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid (described and prepared in Example 1) in 50 ml of ion exchange water.

2. Sodium hydroxide solution is slowly added with vigorous stirring to maintain a constant pH of 7.4 to 7.7.

3. Add 2 g of activated carbon (Darco G-60) and stir the mixture for half an hour. The pH is maintained at 7.4 to 7.7 with 1 N sodium hydroxide solution.

4. Remove the carbon by filtration and wash with 10 ml of 35 water. Wash water is added to the filtrate.

5. The solution of step 4, pH 7.4 to 7.7, is sterilized and pyrogen-free by aseptic filtration and methods.

16 6394 3

The total time of steps 3, 4 and 5 must not exceed 5 hours at room temperature.

6. Using the sterile technique, 65 ml of 5 (approximately equal volume) of sterile, pyrogen-free acetone is added over 5 minutes to the vigorously stirred, sterile solution of step 5.

7. Add 2 volumes (120 mL) of sterile, pyrogen-free acetone with vigorous stirring over 15-20 minutes to form crystals.

8. Suspend the mixture for 10 minutes.

9. Add 3 volumes (180 ml) of sterile acetone over 15 minutes. The mixture is stirred for half an hour.

10. The precipitate is isolated by filtration, washed with 75 ml of sterile acetone and vacuum dried at 45-50 ° C or air dried at 50-56 ° C for 24 hours. Yield about 8.8 g.

Properties of 7- (2-aminomethylphenylacetamido) -3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid monosodium salt trihydrate: water = 9.2% (theoretical = 9.05% ) sodium (flame photometric) = 4,0% (theoretical 3,86%) 20 solubility in water = more than 500 mg / ml stability in water = stable for at least 24 hours at room temperature at a concentration of 250 mg / ml.

Example 5 7- (3-Aminomethyl-2-thienyl-acetamido) -3- (1-carboxymethyl-tetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid A) 7- (3-tert-butoxycarbonylaminomethyl-2- thienylacetamido) -3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid

A mixture of 542 mg (2 mmol) of 3-tert-butoxycarbonyl-30-aminomethyl-2-thienylacetic acid and 412 mg (2 mmol) of dicyclohexylcarbodiimide in tetrahydrofuran is stirred for 1.5 hours at room temperature. The precipitated urea is removed and the filtrate is evaporated in vacuo. The 1.0 g of active ester formed is dissolved in 10 ml of tetrahydrofuran and added at 0 ° C with stirring to a mixture of 656 mg (2 mmol) of 7-amino-3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3 -cephem-4-carboxylic acid and 816 mg (8 mmol) of triethylamine and 5 ml of water.

17 ¢ 394 3

Stirring is continued for 3.5 hours at room temperature. The reaction mixture is added to 20 ml of water and washed four times with 20 ml portions of ether. Ethyl acetate is poured into the aqueous solution and the pH is adjusted to 2 at 5 ° C with concentrated hydrochloric acid. The organic layer is separated and the aqueous layer is extracted twice with 50 ml portions of ethyl acetate. The ethyl acetate extracts are combined, washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate and evaporated in vacuo. The residual oil is chromatographed on 10 g of silica gel. The column is extracted successively with 150 ml of chloroform and 100 ml of 3% methanol-chloroform. 50 mg of 2,4-dinitrophenol are recovered from the chloroform eluate and the title cephalosporin is isolated by evaporation of the methanol-chloroform eluate. Yield 29%, m.p. 185-188 ° C (decomposes).

Analysis 15 Calculated: C 42.91 H 4.54 N 15.23 S 14.94

Found: C 43.02 H 4.17 N 15.07 S 15.04 B) 7- (3-Aminomethyl-2-thienylacetamido) -3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem- 4-Carboxylic acid At 0 ° C, 0.6 ml of trifluoroacetic acid is added to 610 g (1.05 mmol) of the protected cephalosporan of step A and the mixture is stirred for 15 minutes at room temperature. To the reaction mixture is added 15 ml of anhydrous ether to form a precipitate, which is separated by filtration, washed twice with 10 ml portions of anhydrous ether and dissolved in 10 ml of acetonitrile. To the solution is added 25 2 drops of concentrated ammonium hydroxide. The precipitate is filtered off, washed twice with 10 ml portions of acetonitrile and dried for 7 hours at 75 ° C / 1 mm Hg to give the title product. This is treated with one equivalent of ammonium hydroxide to give the monoammonium salt. Yield 83%, m.p. 174-178 ° C (decomposes).

30 Analysis C. ^ gH ^ NgOgSg. ^ O

Calculated: C 38.56 H 4.32 N 19.98 S 17.16 Found: C 38.89 H 4.06 N 18.83 S 15.24

Example 6 7- (2-Aminomethyl-phenyl-acetamido) -3- (1-carboxy-ethyl-tetrazol-2-yl-thiomethyl) -3-cephem-4-carboxylic acid.

A) Potassium o- (1-carbomethoxypropen-2-ylaminomethyl) phenylacetate (enamine) 1. 1000 g of o-aminomethylphenylacetic acid, 340 g of potassium hydroxide, 1412 g of methyl acetoacetate and _-I are added to the tank.

is 63943 32 400 ml of anhydrous methanol.

2. The mixture is heated under reflux and stirring for 4 hours.

3. The reaction solution is concentrated in vacuo and at a temperature below 50 ° C to about one-fifth of the initial volume.

4. Add 10,000 ml of methyl isobutyl ketone to the concentrate and continue concentrating in vacuo until the methanol is removed.

5. Add 10,000 ml of methyl isobutyl ketone to obtain a workable suspension of concentrate.

6. The mixture is stirred and cooled to 5-10 ° C for 30 minutes. 7. The suspension is filtered and the filter cake is washed with 5000 ml of methyl isobutyl ketone and 5000 ml of acetone.

8. Dry the product in a convection oven at about 40 ° C.

9. The yield is 1605-1680 g or 88-92% of white crystalline products, m.p. 140-142 ° C.

B) 7- (2-Aminomethyl-phenyl-acetamido) -3- (1-carboxy-ethyl-tetrazol-2-yl-thiomethyl) -3-cephem-carboxylic acid ή A) 4.09 g of the enamine of step A and 90 ml of tetrahydrofuran are mixed in a stirred and drying tube in a three-necked-20 flask cooled in a dry ice-acetone bath. 8 drops of N, N-dimethylbenzylamine are added and the mixture is cooled to -38 ° C. 1.95 g of isobutyl chloroformate are added and the mixture is stirred for 15 minutes. B) Dissolve 3.5 g of 7-amino-3- (1-carboxyethyltetrazol-2-ylthiomethyl) -3-cephem-4-carboxylic acid in 50 ml of water and 2 .29 to 25 g of N-methylmorpholine and cooled to 2 ° C. The anhydride of step A1 is added to the solution with stirring. The mixture is stirred for 1.5 hours at 2 [deg.] C., the tetrahydrofuran is removed in vacuo, ethyl acetate is poured onto the residue and the pH is adjusted to 3. Stir in an ice bath and the product is separated; yield 250 mg, m.p. 140 ° C 30 (decomposes).

Based on IR and NMR spectra, 20% of impurities are present, mainly starting material.

35

Claims (2)

19 6 3 94 3 Intermediates 7-Acylamido-3- (1-carboxy-lower alkyl-tetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic-5-acids of formula 1 / SR -NH--f N N- N i L i * JN> CHS- ^ ^ IN 10 | COOH (CHO) -COOH 2 n wherein R is acyl and n is an integer from 1 to 3, and the 7-amine-3- (1-carboxy-lower alkyltetrazol-5-ylthiomethyl) -3-cephem 4-Carboxyl-15-silicic acids of the formula S H0N —- f ^ NN 2 1 1 | I J — B 20 COOH (CH 2 -COOH 2 n where n is as defined above, and salts thereof. Compound according to Claim 1, characterized in that n is 1.