7-Acylamino-3-Substituted Cephalosporanic Acid Derivatives

To a solution of N-tert-butoxycarbonyl-2-(3-methanesulfonamidophenyl)-D-glycine (3.3 g) and triethylamine (1.34 ml) in tetrahydrofuran (50 ml) was added dropwise a solution of ethyl chloroformate (0.91 ml) in tetrahydrofuran (10 ml) at -10.degree. to -7.degree. C. with stirring, and the stirring was continued at the same temperature for 40 minutes to give a solution of the activated acid. This solution was added dropwise to a solution of benzhydryl 7-amino-3-methylthiomethyl-3-cephem-4-carboxylate (3.0 g) in methylene chloride (100 ml) at -30.degree. C. over a period of 5 minutes with stirring, and the stirring was continued at the same temperature for an hour. After addition of water, the reaction mixture was stirred for half an hour, followed by extracting with methylene chloride (100 ml). The extract was washed twice with 5% aqueous solution of sodium bicarbonate (50 ml) and an aqueous solution of sodium chloride, and then dried over anhydrous magnesium sulfate, followed by evaporation under reduced pressure to give benzhydryl 7-[N-tert-butoxycarbonyl-2-(3-methanesulfonamidophenyl)-D-glycinamido]-3-m ethylthiomethyl-3-cephem-4-carboxylate (6.5 g).

I.R. (Nujol): 1780, 1710, 1680, 1152 cm.sup.-1

NMR .delta.ppm (DMSO-d.sub.6): 1.36 (9H, s), 1.76 (3H, s), 2.98 (3H, s), 3.3-3.8 (4H, m), 5.12 (1H, d, J=5Hz), 5.73 (1H, dd, J=5Hz, 8Hz), 6.93 (1H, s), 7.1-7.4 (14H, m), 9.93 (1H, d, J=8Hz)

Benzhydryl 7-[N-tert-butoxycarbonyl-2-(3-methanesulfonamidophenyl)-D-glycinamido]-3-e thylthiomethyl-3-cephem-4-carboxylate (5.4 g) was obtained by reacting benzhydryl 7-amino-3-ethylthiomethyl-3-cephem-4-carboxylate (3.08 g) with N-tert-butoxycarbonyl-2-(3-methanesulfonamidophenyl)-D-glycine (3.3 g) in substantially the same manner as that of Example 1.

I.R. (Nujol): 3250, 1780, 1700, 1530, 1490, 1455 cm.sup.-1

NMR .delta.ppm (DMSO-d.sub.6): 0.98 (3H, t, J=7Hz), 1.38 (9H, s), 2.28 (2H, q, J=7Hz), 2.97 (3H, s), 3.4-3.8 (4H, m), 5.12 (1H, d, J=5Hz), 5.24 (1H, m), 5.73 (1H, dd, J=5Hz, 7Hz), 6.93 (1H, s), 7.0-7.7 (14H, m), 9.89 (1H, d, J=8Hz), 10.43 (1H, s)

(1) To a suspension of tert-butyl 7-amino-3-methoxymethyl-3-cephem-4-carboxylate tosylate (13.1 g) in methylene chloride (200 ml) was added water (100 ml), followed by adjusting to pH about 6 with an aqueous solution of sodium bicarbonate. After separating out the methylene chloride layer, the remaining aqueous solution was extracted with methylene chloride (50 ml). The combined methylene chloride solution was washed with an aqueous solution of sodium chloride, dried over anhydrous magnesium sulfate and then treated with an activated charcoal, followed by evaporation under reduced pressure to give tert-butyl 7-amino-3-methoxymethyl-3-cephem-4-carboxylate (5.7 g).

I.R. (Nujol): 3370, 1760, 1740, 1710 cm.sup.-1

NMR .delta.ppm (DMSO-d.sub.6): 1.5 (9H, s), 2.3 (2H, broad s), 3.24 (3H, s), 3.51 (2H, s), 4.15 (2H, s), 4.8 (1H, d, J=5Hz), 5.04 (1H, d, J=5Hz)

(2) On the other hand, to a solution of N-tert-butoxycarbonyl-2-(3-methanesulfonamidophenyl)-D-glycine (8.89 g) and triethylamine (2.61 g) in dry tetrahydrofuran (95 ml) was added dropwise a solution of ethyl chloroformate (2.8 g) in dry tetrahydrofuran (25 ml) at -10.degree. to -7.degree. C. over a period of 10 minutes with stirring, and stirring was continued at the same temperature for 40 minutes to prepare a solution of the activated acid.

(3) To a solution of the compound (5.65 g) obtained according to Example 3-(1) in dry methylene chloride (190 ml) was added dropwise the solution of the activated acid prepared above at -30.degree. C. over a period of 10 minutes with stirring, and the stirring was continued at the same temperature for an hour. After addition of water (100 ml), the reaction mixture was stirred for half an hour. The methylene chloride layer was separated out therefrom and the remaining aqueous solution was extracted with methylene chloride. The combined methylene chloride solution was washed twice with 5% aqueous solution of sodium bicarbonate (100 ml) and an aqueous solution of sodium chloride, dried over anhydrous magnesium sulfate, and then treated with an activated charcoal, followed by evaporation under reduced pressure to give tert-butyl 7-[N-tert-butoxycarbonyl-2-(3-methanesulfonamidophenyl)-D-glycinamido]-3-m ethoxymethyl-3-cephem-4-carboxylate (17.8 g).

I.R. (Nujol): 3250, 1780, 1710, 1680 cm.sup.-1

NMR .delta.ppm (DMSO-d.sub.6): 1.36 (9H, s), 1.46 (9H, s), 2.97 (3H, s), 3.18 (3H, s), 3.4 (2H, broad s), 4.07 (2H, broad s), 5.03 (1H, d, J=5Hz), 5.24 (1H, d, J=8Hz), 5.73 (1H, dd, J=5Hz, 8Hz), 6.93-7.43 (4H, m), 7.5 (1H, d, J=8Hz), 9.16 (1H, d, J=8Hz), 9.73 (1H, broad s),

The compounds described in the following Examples 4 to 7 were obtained by reacting the corresponding 7-aminocephalosporanic acid derivative with 2-(3-methanesulfonamidophenyl)-D-glycine in substantially the same manner as that of Example 3.

7-[2-(3-Methanesulfonamidophenyl)-D-glycinamido]-3-methylthiomethyl-3-cephe m-4-carboxylic acid.

I.R. (Nujol): 1758, 1687 (shoulder), 1666, 1144, 974 cm.sup.-1

7-[2-(3-Methanesulfonamidophenyl)-D-glycinamido]-3-ethylthiomethyl-3-cephem -4-carboxylic acid.

I.R. (Nujol): 3500, 3150, 1780, 1685, 1460 cm.sup.-1

7-[2-(3-Methanesulfonamidophenyl)-D-glycinamido]-3-methoxymethyl-3-cephem-4 -carboxylic acid.

I.R. (Nujol): 3500, 3150, 1760, 1685 cm.sup.-1

7-[2-(3-Methanesulfonamidophenyl)-D-glycinamido]-3-allylthiomethyl-3-cephem -4-carboxylic acid trifluoroacetate.

I.R. (Nujol): 1760, 1680, 1600, 1140 cm.sup.-1

A mixture of benzhydryl 7-[N-tert-butoxycarbonyl-2-(3-methanesulfonamidophenyl)-D-glycinamido]-3-m ethylthiomethyl-3-cephem-4-carboxylate (6.5 g), anisole (5.0 ml) in trifluoroacetic acid (20 ml) was stirred at 25.degree. C. for 15 minutes. After the reaction, the reaction mixture was added dropwise to diisopropyl ether (300 ml), and the precipitated solid was collected by filtration, washed with diisopropyl ether and then dissolved in a mixture of water (50 ml) and ethyl acetate (50 ml). After the aqueous layer was separated out, it was washed with ethyl acetate, followed by removal of ethyl acetate from the aqueous solution completely under reduced pressure. The resultant aqueous solution was adjusted to pH 3.8 with an aqueous solution of sodium bicarbonate and subjected to column chromatography using "Diaion HP-20" (90 ml). After washing with water (180 ml), elution was carried out with 30% isopropyl alcohol, and the fractions containing the desired compound were collected and lyophilized to give 7-[2-(3-methanesulfonamidophenyl)-D-glycinamido]-3-methylthiomethyl-3-ceph em-4-carboxylic acid (4.8 g).

I.R. (Nujol): 1758, 1687 (shoulder), 1666, 1144, 974 cm.sup.-1

NMR .delta.ppm (D.sub.2 O+DCl): 1.98 (3H, s), 3.15 (3H, s), 3.45 (2H, broad s), 3.56 (2H, broad s), 5.12 (1H, d, J=5Hz), 5.30 (1H, s), 5.70 (1H, d, J=5Hz), 7.45 (4H, s)

7-[2-(3-Methanesulfonamidophenyl)-D-glycinamido]-3-ethylthiomethyl-3-cephem -4-carboxylic acid (1.6 g) was obtained by reacting benzhydryl 7-[N-tert-butoxycarbonyl-2-(3-methanesulfonamidophenyl)-D-glycinamido]-3-e thylthiomethyl-3-cephem-4-carboxylate (3.7 g) with trifluoroacetic acid (7.4 ml) in the presence of anisole (7.4 ml) in substantially the same manner as that of Example 8, mp 188.degree. C. (dec.).

I.R. (Nujol): 3500, 3150, 1780, 1685, 1460 cm.sup.-1

NMR .delta.ppm (D.sub.2 O+DCl): 1.13 (3H, t, J=7Hz), 2.48 (2H, q, J=7Hz), 3.11 (3H, s), 3.3-3.8 (4H, m), 5.30 (1H, s), 5.65 (1H, d, J=5Hz), 7.43 (4H, s)

To a solution of tert-butyl 7-[N-tert-butoxycarbonyl-2-(3-methanesulfonamidophenyl)-D-glycinamido]-3-m ethoxymethyl-3-cephem-4-carboxylate (15 g) in anisole (11.25 ml) was added dropwise trifluoroacetic acid (33.75 ml) below 15.degree. C. over a period of 10 minutes with stirring, and the stirring was continued at 15.degree. to 20.degree. C. for half an hour. The reaction mixture was poured into diisopropyl ether (750 ml), followed by stirring at ambient temperature for 20 minutes. After the precipitated solid was collected by filtration and washed with diisopropyl ether, it was poured into a mixture of ethyl acetate (100 ml) and water (100 ml) and stirred for a while. The aqueous solution was separated out therefrom, and the remaining organic layer was extracted with water. The combined aqueous solution was concentrated under reduced pressure, and the concentrate was adjusted to pH about 3.8 with an aqueous solution of sodium bicarbonate, followed by subjecting to column chromatography using non-ionic absorption resin "Diaion HP-20" (225 ml), which was washed with water (450 ml) and then eluted with 30% isopropyl alcohol. The fractions containing the desired compound were collected and evaporated under reduced pressure, and the residue was lyophilized to give 7-[2-(3-methanesulfonamidophenyl)-D-glycinamido]-3-methoxymethyl-3-cephem- 4-carboxylic acid (4.45 g).

I.R. (Nujol): 3500, 3150, 1760, 1685 cm.sup.-1

NMR .delta.ppm (D.sub.2 O+DCl): 3.13 (3H, s), 3.26 (3H, s), 3.42 (2H, q, J=18Hz), 4.25 (2H, s), 5.06 (1H, d, J=5Hz), 5.27 (1H, s), 5.73 (1H, d, J=5Hz), 7.42 (4H, s)

Benzhydryl 7-amino-3-allylthiomethyl-3-cephem-4-carboxylate (3.3 g) and N-tert-butoxycarbonyl-2-(3-methanesulfonamidophenyl)-D-glycine (3.44 g) were treated in substantially the same manner at that of Example 1 to give an oily product (6.5 g). A mixture of this oil, trifluoroacetic acid (15 ml) and anisole (15 ml) was stirred at 20.degree. C. for half an hour, followed by adding dropwise to diisopropyl ether. The presipitated solid was collected by filtration, washed with diisopropyl ether and then dissolved in a mixture of water (50 ml) and ethyl acetate (50 ml). The aqueous layer was separated out and washed with diethyl ether, followed by removal of the organic solvent therefrom completely. The resultant aqueous solution was lyophilized to give 7-[2-(3-methanesulfonamidophenyl)-D-glycinamido]-3-allylthiomethyl-3-cephe m-4-carboxylic acid trifluoroacetate (2 g).

I.R. (Nujol): 1760, 1680, 1600, 1140 cm.sup.-1

NMR .delta.ppm (D.sub.2 O+DCl): 3.20 (2H, m), 3.23 (3H, s), 3.6 (7H, m), 5.2 (1H, d, J=5Hz), 5.45 (1H, s), 5.80 (1H, d, J=5Hz), 7.55 (3H, m)

To a solution of benzhydryl 7-[4-bromoacetoacetamido)-3-methylthiomethyl-3-cephem-4-carboxylate (6.0 g) in tetrahydrofuran (30 ml) was added dropwise a solution of thiourea (0.85 g) and sodium bicarbonate (0.94 g) in tetrahydrofuran (30 ml) and water (24 ml) at 25.degree. C. with stirring, and the stirring was continued at 28.degree. to 30.degree. C. for an hour. The reaction mixture was poured into a mixture of ethyl acetate (100 ml) and water (100 ml), followed by separating out the organic layer, which was washed twice with an aqueous solution of sodium chloride, dried over anhydrous magnesium sulfate and then evaporated to give benzhydryl 7-[2-(2-aminothiazol-4-yl)-acetamido]-3-methylthiomethyl-3-cephem-4-carbox ylate (4.0 g).

IR (Nujol): 1773, 1715, 1653 cm.sup.-1

NMR .delta.ppm (DMSO-d.sub.6): 1.85 (3H, s), 3.3-3.8 (6H, m), 5.25 (1H, d, J=5Hz), 5.80 (1H, dd, J=5Hz, 8Hz), 6.32 (1H, s), 7.00 (1H, s), 7.43 (10H, s), 8.95 (1H, d, J=8Hz)

tert-Butyl 7-amino-3-methoxymethyl-3-cephem-4-carboxylate tosylate (2.0 g) was dissolved in a mixture of acetone (40 ml) and a saturated aqueous solution of sodium bicarbonate (15 ml), and thereto was added dropwise a solution of the activated acid, which was prepared from 2-(2-formamidothiazol-4-yl)-2-methoxyiminoacetic acid (syn isomer) (1.06 g), phosphorus oxychloride (0.85 g) and N,N-dimethylformamide (0.41 g) in tetrahydrofuran (10 ml), at 0.degree. to 5.degree. C. over a period of 10 minutes. During the addition, the pH value of the reaction mixture was maintained at pH 7.0 to 7.5 with a saturated aqueous solution of sodium carbonate. After stirring for an hour, the reaction mixture was diluted with water (50 ml), followed by extracting twice with ethyl acetate. The combined extract was washed with 5% aqueous solution of sodium bicarbonate and water, dried and then evaporated to dryness under reduced pressure to give a residue, which was triturated with diethyl ether to obtain tert-butyl 7-[2-(2-formamidothiazol-4-yl)-2-methoxyiminoacetamido]-3-methoxymethyl-3- cephem-4-carboxylate (syn isomer) (1.14 g).

IR (Nujol): 3250, 3100, 1790, 1710, 1660 cm.sup.-1

NMR .delta.ppm (DMSO-d.sub.6): 1.49 (9H, s), 3.21 (3H, s), 3.28 (2H, broad s), 3.89 (3H, s), 4.1 (2H, s), 5.16 (1H, d, J=5Hz), 5.80 (1H, dd, J=5Hz, 8Hz), 7.36 (1H, s), 8.48 (1H, s), 9.6 (1H, d, J=8Hz), 12.66 (1H, broad s)

tert-Butyl 7-[2-(2-formamidothiazol-4-yl)-2-methoxyiminoacetamido]-3-methylthiomethyl -3-cephem-4-carboxylate (syn isomer) (0.91 g) was obtained by reacting tert-butyl 7-amino-3-methylthiomethyl-3-cephem-4-carboxylate (0.90 g) with 2-(2-formamidothiazol-4-yl)-2-methoxyiminoacetic acid (syn isomer) (0.85 g) according to the similar manner to that of Example 13.

IR (Nujol): 3250, 3050, 1780, 1690 cm.sup.-1

NMR .delta.ppm (DMSO-d.sub.6): 1.47 (9H, s), 1.97 (3H, s), 3.29 (2H, broad s), 3.55 (2H, ABq, J=13Hz), 3.87 (3H, s), 5.21 (1H, d, J=5Hz), 5.76 (1H, dd, J=5Hz, 8Hz), 7.38 (1H, s), 8.49 (1H, s), 9.66 (1H, d, J=8Hz), 12.56 (1H, broad s)

The compounds described in the following Examples 15 to 17 were obtained by reacting the 7-aminocephalosporanic acid derivatives with the corresponding acid according to the similar manner to that of Example 13.

7-[2-(2-Aminothiazol-4-yl)acetamido]-3-methylthiomethyl-3-cephem-4-carboxyl ic acid

IR (Nujol): 1763, 1654 cm.sup.-1

7-[2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-methoxymethyl-3-cephe m-4-carboxylic acid hydrochloride (syn isomer)

IR (Nujol): 3300, 1780, 1720, 1660, 1640 cm.sup.-1

7-[2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-methylthiomethyl-3-ce phem-4-carboxylic acid (syn isomer)

IR (Nujol): 3350, 1770, 1670 cm.sup.-1

The mixture of benzhydryl 7-[2-(2-aminothiazol-4-yl)acetamido]-3-methylthiomethyl-3-cephem-4-carboxy late (3.3 g), trifluoroacetic acid (10 ml) and anisole (10 ml) in methylene chloride (10 ml) was stirred at 10.degree. C. for an hour. After benzene (50 ml) was added to the reaction mixture, the trifluoroacetic acid therein was azeotropically removed under reduced pressure. The remained aqueous solution was poured into a mixture of ethyl acetate (100 ml) and water (100 ml), followed by adjusting to pH 7.5 with sodium bicarbonate. After separating out the aqueous solution, the organic solvent was removed therefrom by evaporation completely under reduced pressure, followed by adjusting to pH 3.0 with 10% hydrochloric acid. The precipitated solid was collected by filtration and then dried to give 7-[2-(2-aminothiazol-4-yl)acetamido]-3-methylthiomethyl-3-cephem-4-carboxy lic acid (0.95 g).

IR (Nujol): 1763, 1654 cm.sup.-1

NMR .delta.ppm (DMSO-d.sub.6): 2.01 (3H, s), 3.48 (2H, s), 3.65 (4H, broad s), 5.17 (1H, d, J=5Hz), 5.67 (1H, dd, J=5Hz, 8Hz), 6.38 (1H, s), 9.00 (1H, d, J=8Hz)

To a cold suspension of tert-butyl 7-[2-(2-formamidothiazol-4-yl)-2-methoxyiminoacetamido]-3-methoxymethyl-3- cephem-4-carboxylate(syn isomer)(1.0 g) in methylene chloride (20 ml) were added trifluoroacetic acid (4.4 g) and anisole (0.2 ml), and the mixture was gradually warmed at 40.degree. C., followed by stirring at 10.degree. C. for 3 hours. After evaporation of the reaction mixture, to the residue was added ethyl acetate (20 ml), followed by extracting with an aqueous solution of sodium bicarbonate. The resultant aqueous solution was adjusted to pH 2.0 with diluted hydrochloric acid and then extracted with ethyl acetate. The extract was washed with a saturated aqueous solution of sodium bicarbonate, water and an aqueous solution of sodium chloride in turn, dried and then evaporated to dryness under reduced pressure. The residue was stirred for 30 minutes in diethyl ether, and the remained substance was collected by filtration to give 7-[2-(2-formamidothiazol-4-yl)-2-methoxyiminoacetamido]-3-methoxymethyl-3- cephem-4-carboxylic acid (syn isomer) (0.56 g).

IR (Nujol): 3250, 1780, 1660 cm.sup.-1

NMR .delta.ppm (DMSO-d.sub.6): 3.22 (3H, s), 3.55 (2H, broad s), 3.90 (3H, s), 4.19 (2H, s), 5.17 (1H, d, J=5Hz), 5.81 (1H, dd, J=5Hz, 8Hz), 7.43 (1H, s), 8.51 (1H, s), 9.67 (1H, d, J=8Hz), 12.58 (1H, broad s)

7-[2-(2-Formamidothiazol-4-yl)-2-methoxyiminoacetamido]-3-methylthiomethyl- 3-cephem-4-carboxylic acid (syn isomer) (0.64 g) was obtained by reacting tert-butyl 7-[2-(2-formamidothiazol-4-yl)-2-methoxyiminoacetamido]-3-methylthiomethyl -3-cephem-4-carboxylate (syn isomer) (0.9 g) with trifluoroacetic acid (5.8 g) in the presence of anisole (0.9 ml) according to the similar manner to that of Example 19.

IR (Nujol): 3250, 1780, 1660 cm.sup.-1

NMR .delta.ppm (DMSO-d.sub.6): 1.99 (3H, s), 3.38-4.1 (4H, m), 3.93 (3H, s), 5.25 (1H, d, J=5Hz), 5.79 (1H, dd, J=5Hz, 8Hz), 7.45 (1H, s), 8.43 (1H, s), 9.69 (1H, d, J=8Hz), 12.68 (1H, broad s)

The compounds described in the following Examples 21 and 22 were obtained by reacting tert-butyl ester of the corresponding cephalosporanic acid derivatives with trifluoroacetic acid in the presence of anisole according to the similar manner to that of Example 19.

7-[2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-methoxymethyl-3-cephe m-4-carboxylic acid hydrochloride (syn isomer)

IR (Nujol): 3300, 1780, 1720, 1660, 1640 cm.sup.-1

7-[2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-methylthiomethyl-3-ce phem-4-carboxylic acid (syn isomer)

IR (Nujol): 3350, 1770, 1670 cm.sup.-1

To a suspension of 7-[2-(2-formamidothiazol-4-yl)-2-methoxyiminoacetamido]-3-methoxymethyl-3- cephem-4-carboxylic acid (syn isomer) (0.52 g) in a mixture of methanol (3 ml) and tetrahydrofuran (2 ml) was added conc. hydrochloric acid (0.18 g), and the mixture was stirred at 30.degree. C. for 4 hours. After the reaction mixture was cooled and diluted with diisopropyl ether, the precipitated crystals were collected by filtration, washed with diisopropyl ether and then dried to give 7-[2-(2-aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-methoxymethyl-3-ceph em-4-carboxylic acid hydrochloride (syn isomer) (0.45 g).

IR (Nujol): 3300, 1780, 1720, 1660, 1640 cm.sup.-1

NMR .delta.ppm (DMSO-d.sub.6): 3.26 (3H, s), 3.58 (2H, broad s), 4.0 (3H, s), 4.24 (2H, s), 5.24 (1H, d, J=5Hz), 5.82 (1H, dd, J=5Hz, 8Hz), 7.01 (1H, s), 9.87 (1H, d, J=8Hz)

7-[2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-methylthiomethyl-3-ce phem-4-carboxylic acid (syn isomer) (0.23 g) was obtained by reacting 7-[2-(2-formamidothiazol-4-yl)-2-methoxyiminoacetamido]-3-methylthiomethyl -3-cephem-4-carboxylic acid (syn isomer) (0.6 g) with conc. hydrochloric acid (0.4 g) in a mixture of methanol (3 ml) and tetrahydrofuran (1 ml) according to the similar manner to that of Example 23.

IR (Nujol): 3350, 1770, 1670 cm.sup.-1

NMR .delta.ppm (DMSO-d.sub.6): 2.01 (3H, s), 3.2-4.1 (4H, m), 3.88 (3H, s), 5.25 (1H, d, J=5Hz), 5.78 (1H, dd, J=5Hz, 8Hz), 6.82 (1H, s), 7.24 (2H, broad s), 9.64 (1H, d, J=8Hz)

To a solution of benzhydryl 7-amino-3-methylthiomethyl-3-cephem-4-carboxylate (10 g) and trimethylsilylacetamide (18.4 g) in methylene chloride (100 ml) was added at -15.degree. C. an activated acid, which was prepared from (2-formamidothiazol-4-yl)glyoxylic acid (6.56 g), N,N-dimethylformamide (2.92 ml) and phosphorus oxychloride (3.46 ml) in a conventional manner, and the mixture was stirred at -20 .degree. to -15.degree. C. for 15 minutes. After the reaction mixture was poured into water, it was extracted with ethyl acetate. The extract was washed with an aqueous solution of sodium bicarbonate and an aqueous solution of sodium chloride, followed by drying over anhydrous magnesium sulfate. Removal of the solvent gave a residue (13.4 g), which was chromatographed on silica gel (100 ml) using a mixture of benzene and ethyl acetate (5:1 by volume) as an eluent. The fractions containing the desired compound were collected and then evaporated to dryness to obtain benzhydryl 7-[(2-formamidothiazol-4-yl)glyoxylamido]-3 -methylthiomethyl-3-cephem-4-carboxylate (7.1 g).

I.R. (Nujol): 3300, 1780, 1700, 1656 cm.sup.-1

N.M.R. .delta.ppm (DMSO-d.sub.6): 1.83 (3H, s), 3.58 (2H, broad s), 3.67 (2H, broad s), 5.33 (1H, d, J=5Hz), 5.87 (1H, dd, J=5Hz, 8Hz), 6.90 (1H, s), 7.40 (10H, s), 8.47 (1H, s), 8.58 (1H, s), 9.88 (1H, d, J=8Hz), 12.68 (1H, broad s)

The following compounds were obtained by reacting 7-amino-3-substituted cephalosporanic acid derivatives with the corresponding acids according to the similar manner to that of Example 25.

7-[(2-Aminothiazol-4-yl)glyoxylamido]-3-methylthiomethyl-3-cephem-4-carboxy lic acid.

I.R. (Nujol): 3300, 1762, 1522 cm.sup.-1

7-[2-(2-Aminothiazol-4-yl)-DL-glycinamido]-3-methylthiomethyl-3-cephem-4-ca rboxylic acid.

I.R. (Nujol): 3300, 1755, 1686, 1600 cm.sup.-1

To a mixture of benzhydryl 7-amino-3-methylthiomethyl-3-cephem-4-carboxylate (4.92 g) and N-tert-butoxycarbonyl-2-(2-formamidothiazol-4-yl)-DL-glycine (4.0 g) in methylene chloride (100 ml) and tetrahydrofuran (80 ml) was added N,N'-dicyclohexylcarbodiimide (2.39 g), and the mixture was stirred at ambient temperature for an hour. After the insoluble substance was removed by filtration, the filtrate was evaporated to dryness to give a residue, which was dissolved in ethyl acetate. This solution was washed with an aqueous solution of sodium bicarbonate and an aqueous solution of sodium chloride, and then dried over anhydrous magnesium sulfate. Removal of the solvent gave benzhydryl

7-[N-tert-butoxycarbonyl-2-(2-formamidothiazol-4-yl)-DL-glycinamido]-3-meth ylthiomethyl-3-cephem-4-carboxylate (9.4 g).

I.R. (Nujol): 3300, 1770, 1710, 1680, 1615 cm.sup.-1

N.M.R. .delta.ppm (DMSO-d.sub.6): 1.36 (9H, s), 1.80 (3H, s), 3.58 (4H, m),

5.38 (1H, d, J=8Hz), 5.6-5.9 (1H, m), 6.90 (1H, s), 7.13 (1H, s), 7.33 (10H, broad s), 8.47 (1H, s), 9.08 (1H, d, J=8Hz)

A mixture of 7-[(2-formamidothiazol-4-yl)-glyoxylamido]-3-methylthiomethyl-3-cephem-4-c arboxylic acid (3.0 g) and conc. hydrochloric acid (3 ml) in methanol (50 ml) was stirred at ambient temperature for 2 hours. The reaction mixture was adjusted to pH 5-6 with an aqueous solution of sodium bicarbonate and then concentrated under reduced pressure. The precipitated crystals in the concentrate were collected by filtration to give 7-[(2-aminothiazol-4-yl)glyoxylamido]-3-methylthiomethyl-3-cephem-4-carbox ylic acid (1.1 g).

I.R. (Nujol): 3300, 1762, 1522 cm.sup.-1

N.M.R. .delta.ppm (DMSO-d.sub.6): 2.00 (3H, s), 3.65 (4H, broad s), 5.22 (1H, d, J=5Hz), 5.68 (1H, dd, J=5Hz, 8Hz), 7.37 (2H, broad s), 7.83 (1H, s), 9.73 (1H, d, J=8Hz)

A mixture of benzhydryl 7-[N-tert-butoxycarbonyl-2-(2-formamidothiazol-4-yl)-DL-glycinamido]-3-met hylthiomethyl-3-cephem-4-carboxylate (9.4 g) and conc. hydrochloric acid (4.16 ml) in methanol (100 ml) and tetrahydrofuran (25 ml) was stirred at 30.degree. to 35.degree. C. for 5 hours. The reaction mixture was adjusted to pH 4.5 with sodium bicarbonate and then evaporated to dryness to give a residue, which was dissolved in ethyl acetate. This solution was washed with an aqueous solution of sodium chloride, dried over anhydrous magnesium sulfate and then evaporated to obtain benzhydryl 7-[N-tert-butoxycarbonyl-2-(2-aminothiazol-4-yl)-DL-glycinamido]-3-methylt hiomethyl-3-cephem-4-carboxylate (7.4 g).

I.R. (Nujol): 3300, 1772, 1716, 1685, 1623, 1244, 1170, 1160 cm.sup.-1

N.M.R. .delta.ppm (DMSO-d.sub.6): 1.40 (9H, s), 1.80 (3H, s), 3.6 (4H, m), 5.0-5.4 (2H, m), 5.6-5.9 (2H, m), 6.93 (1H, s), 7.30 (1H, s), 7.43 (10H, broad s), 8.93 (1H, d, J=8Hz)

The following compounds were obtained by reacting 7-acylamino-3-substituted cephalosporanic acid derivatives having a formamido group with hydrochloric acid according to the similar manner to that of Example 30.

7-[2-(2-Aminothiazol-4-yl)-DL-glycinamido]-3-methylthiomethyl-3-cephem-4-ca rboxylic acid.

I.R. (Nujol): 3300, 1755, 1686, 1600 cm.sup.-1

7-[2-(2-Aminothiazol-4-yl)-DL-glycolamido]-3-methylthiomethyl-3-cephem-4-ca rboxylic acid.

I.R. (Nujol): 3300, 1752, 1675, 1600 cm.sup.-1

Benzhydryl 7-[(2-formamidothiazol-4-yl)glyoxylamido]-3-methylthiomethyl-3-cephem-4-ca rboxylate (7.0 g) was dissolved in a solution of methylene chloride (70 ml), anisole (7 ml) and trifluoroacetic acid (14 ml), and the mixture was stirred at ambient temperature for an hour. After the solvent was removed by distillation under reduced pressure, the residue was dissolved in water, adjusted to pH 2.0 with conc. hydrochloric acid and then extracted with ethyl acetate. The extract was washed with an aqueous sodium chloride and dried over magnesium sulfate, followed by evaporation. The residue was pulverized with diisopropyl ether to obtain 7-[(2-formamidothiazol-4-yl)glyoxylamido]-3-methylthiomethyl-3-cephem-4-ca rboxylic acid (3.3 g).

I.R. (Nujol): 3120, 1762, 1731, 1677 cm.sup.-1

N.M.R. .delta.ppm (DMSO-d.sub.6): 2.00 (3H, s), 3.63 (4H, broad s), 5.24 (1H, d, J=5Hz), 5.73 (1H, dd, J=5Hz, 8Hz), 8.43 (1H, s), 8.57 (1H, s), 9.90 (1H, d, J=8Hz), 12.80 (1H, broad s)

The following compounds were obtained by reacting 7-acylamino-3-substituted cephalosporanic acid derivatives having benzhydryl ester with 2,2,2-trifluoroacetic acid in the presence of anisole according to the similar manner to that of Example 33.

7-[(2-Aminothiazol-4-yl)glyoxylamido]-3-methylthiomethyl-3-cephem-4-carboxy lic acid.

I.R. (Nujol): 3300, 1762, 1522 cm.sup.-1

7-[2-(2-Aminothiazol-4-yl)-DL-glycolamido]-3-methylthiomethyl-3-cephem-4-ca rboxylic acid.

I.R. (Nujol): 3300, 1752, 1675, 1600 cm.sup.-1

A mixture of benzhydryl 7-[N-tert-butoxycarbonyl-2-(2-aminothiazol-4-yl)-DL-glycinamido]-3-methylt hiomethyl-3-cephem-4-carboxylate (7.0 g), anisole (7 ml) and 2,2,2-trifluoroacetic acid (21 ml) was stirred at 5.degree. C. for half an hour. To the reaction mixture was added dropwise diisopropyl ether (300 ml), and the precipitated substance was collected by filtration and then washed with diisopropyl ether, followed by dissolving in a mixture of ethyl acetate (50 ml) and water (100 ml). After the aqueous layer was separated out, the ethyl acetate in the aqueous layer was completely removed, and then the remained aqueous solution was adjusted to pH 4.2 with 5% aqueous solution of sodium bicarbonate. The resultant aqueous solution was chromatographed on nonionic adsorption resin "Diaion HP-20" (Trade Mark, made by Mitsubishi Chemical Industries Ltd.) (100 ml). After washing with water (250 ml), elution was carried out with 30% aqueous isopropyl alcohol. The fractions containing the desired compound were collected and then evaporated, followed by lyophilization to obtain 7-[2-(2-aminothiazol-4-yl)-DL-glycinamido]-3-methylthiomethyl-3-cephem-4-c arboxylic acid (2.9 g).

I.R. (Nujol): 3300, 1755, 1686, 1600 cm.sup.-1

N.M.R. .delta.ppm (D.sub.2 O+DCl): 2.07 (3H, s), 3.5-4.0 (4H, m), 5.27 (1H, d, J=5Hz), 5.50 (1H, s),

7.27 (1H, s)

To a solution of 7-[(2-aminothiazol-4-yl)glyoxylamido]-3-methylthiomethyl-3-cephem-4-carbox ylic acid (1.1 g) in methanol (80 ml) was added sodium borohydride (150 mg) at 5.degree. to 10.degree. C., and the mixture was stirred at the same temperature for half an hour. After the reaction mixture was adjusted to pH 5.0 with 10% hydrochloric acid, the solvent was removed by distillation. To the residue was added water (50 ml), followed by adjusting to pH 5.0 with 10% hydrochloric acid. The resultant aqueous solution was chromatographed on nonionic adsorption resin "Diaion HP-20" (50 ml). After washing with water (100 ml), elution was carried out with 30% aqueous isopropyl alcohol. The fractions containing the desired compound were collected and then evaporated to dryness, followed by lyophilization to obtain 7-[2-(2-aminothiazol-4-yl)-DL-glycolamido]-3-methylthiomethyl-3-cephem-4-c arboxylic acid (0.75 g).

I.R. (Nujol): 3300, 1752, 1675, 1600 cm.sup.-1

N.M.R. .delta.ppm (DMSO-d.sub.6): 2.00 (3H, s), 3.48 (2H, broad s), 3.67 (2H, broad s), 4.93 (1H, s), 5.07 (1H, d, J=5Hz), 5.57 (1H, m), 6.50 (1H, s), 7.03 (2H, broad s),

Vilsmeier reagent prepared from phosphorus oxychloride (1.23 ml) and N,N-dimethylformamide (1.1 ml) was suspended in dry tetrahydrofuran (40 ml). To the suspension was added 2-(2-formamidothiazol-4-yl)-2-tert-butoxycarbonylmethoxyiminoacetic acid (syn isomer) (4.0 g) under ice-cooling with stirring, and the mixture was stirred at the same temperature for 50 minutes to prepare the activated acid solution. On the other hand, benzhydryl 7-amino-3-methylthiomethyl-3-cephem-4-carboxylate (4.66 g) and trimethylsilylacetamide (8.6 g) were dissolved in methylene chloride (50 ml). To the solution was added the activated acid solution at -20.degree. C. at a time, and the mixture was stirred at the same temperature for an hour. Water (100 ml) and ethyl acetate (200 ml) were added to the resultant solution, and the organic layer was separated, washed with 5% aqueous sodium bicarbonate and an aqueous sodium chloride, and then dried over magnesium sulfate, followed by evaporation under reduced pressure to give benzhydryl 7-[2-(2-formamidothiazol-4-yl)-2-tert-butoxycarbonylmethoxyiminoacetamido] - 3-methylthiomethyl-3-cephem-4-carboxylate (syn isomer) (8.0 g), mp 132.degree.-138.degree. C.

IR (Nujol): 3260, 1783, 1725, 1687 cm.sup.-1

NMR .delta.ppm (DMSO-d6.delta.): 1.47 (9H, s), 1.83 (3H, s), 3.60 (2H, broad s), 3.66 (2H, broad s), 4.98 (2H, broad s), 5.32 (1H, d, J=5Hz), 5.92 (1H, dd, J=5Hz, 8Hz), 6.95 (1H, s), 7.4 (10H, m), 7.48 (1H, s), 8.54 (1H, s), 9.63 (1H, d, J=8Hz), 12.65 (1H, broad s)

Benzhydryl 7-[2-(2-formamidothiazol-4-yl)-2-benzhydryloxycarbonylmethoxyiminoacetamid o]-3-methylthiomethyl-3-cephem-4-carboxylate (syn isomer) (7.8 g) was obtained by reacting benzhydryl 7-amino-3-methylthiomethyl-3-cephem-4-carboxylate (4.0 g) with the activated acid solution prepared from 2-(2-formamidothiazol-4-yl)-2-benzhydryloxycarbonylmethoxyiminoacetic acid (syn isomer) (6.18 g), phosphorus oxychloride (1.42 ml) and N,N-dimethylformamide (1.21 ml), according to a similar manner to that of Example 38, mp 135.degree.-142.degree. C.

IR (Nujol): 3250, 1780, 1722, 1685 cm.sup.-1

NMR .delta.ppm (DMSO-d6): 1.83 (3H, s), 3.63 (4H, m), 5.0 (2H, broad s), 5.35 (1H, d, J=5Hz), 5.98 (1H, dd, J=5Hz, 8Hz), 6.95 (1H, s), 6.98 (1H, s), 7.37 (20H, m), 7.50 (1H, s), 8.57 (1H, s), 9.82 (1H, d, J=8Hz), 12.73 (1H, broad s)

Benzhydryl 7-[2-benzhydryloxycarbonylmethoximino-2-{2-(2,2,2-trifluoroacetamido)thiaz ol-4-yl}acetamido]-3-methylthiomethyl-3-cephem-4-carboxylate (syn isomer) (5.6 g) was obtained by reacting benzhydryl 7-amino-3-methylthiomethyl-3-cephem-4-carboxylate (3.0 g) with the activated acid solution prepared from 2-benzhydryloxycarbonylmethoximino-2-[2-(2,2,2-trifluoroacetamido)thiazol- 4-yl]acetic acid (syn isomer) (3.68 g), phosphorus oxychloride (0.89 ml) and N,N-dimethylformamide (0.75 ml), according to a similar manner to that of Example 38, mp 165.degree.-169.degree. C.

IR (Nujol): 3300, 1786, 1733, 1675, 1610 cm.sup.-1

NMR .delta.ppm (DMSO-d6): 2.00 (3H, s), 3.56 (4H, m), 4.84 (2H, s), 5.26 (1H, d, J=5Hz), 5.86 (1H, dd, J=5Hz, 8Hz), 6.84 (1H, s), 6.88 (1H, s), 7.3 (20H, m), 9.62 (1H), d, J=8Hz)

To a suspension of benzhydryl 7-[2-(2-formamidothiazol-4-yl)-2-tert-butoxycarbonylmethoxyiminoacetamido] -3-methylthiomethyl-3-cephem-4-carboxylate (syn isomer) (8.0 g) in methanol (160 ml) was added conc. hydrochloric acid (5.6 ml), and the mixture was stirred at 35.degree. C. for an hour. The resultant solution was adjusted to pH 5.0 with a saturated aqueous sodium bicarbonate. After distilling methanol under reduced pressure, the residue was dissolved in water (100 ml) and ethyl acetate (200 ml). The ethyl acetate layer was washed with a saturated aqueous sodium chloride and dried over magnesium sulfate. The solvent was removed by filtration to give benzhydryl 7-[2-(2-aminothiazol-4-yl)-2-tert-butoxycarbonylmethoxyiminoacetamido]-3-m ethylthiomethyl-3-cephem-4-carboxylate (syn isomer) (7.0 g), mp 140.degree.-145.degree. C.

IR (Nujol): 3250, 1780, 1723, 1680 cm.sup.-1

NMR .delta.ppm (DMSO-d6): 1.43 (9H, s), 1.83 (3H, s), 3.63 (4H, m), 4.6 (2H, broad s), 5.29 (1H, d, J=5Hz), 5.87 (1H, dd, J=5Hz, 8Hz), 6.83 (1H, s), 6.95 (1H, s), 7.4 (10H, m), 9.52 (1H, d, J=8Hz)

Benzhydryl 7-[2-(2-aminothiazol-4-yl)-2-benzhydryloxycarbonylmethoxyiminoacetamido]-3 -methylthiomethyl-3-cephem-4-carboxylate (syn isomer) (7.0 g), mp 148.degree.-155.degree. C., was obtained by reacting benzhydryl 7-[2-(2-formamidothiazol-4-yl)-2-benzhydryloxycarbonylmethoxyiminoacetamid o]-3-methylthiomethyl-3-cephem-4-carboxylate (syn isomer) (7.5 g) with conc. hydrochloric acid (3.9 ml) according to a similar manner to that of Example 41.

NMR .delta.ppm (DMSO-d6): 1.83 (3H, s), 3.60 (4H, m), 4.90 (2H, broad s), 5.30 (1H, d, J=5Hz), 5.87 (1H, dd, J=5Hz, 8Hz), 6.86 (1H, s), 6.93 (1H, s), 6.97 (1H, s), 7.4 (20H, m), 9.67 (1H, d, J=8Hz).

A solution of 7-[2-carboxymethoxyimino-2-{2-(2, 2,2-trifluoroacetamido)thiazol-4-yl}acetamido]-3-methylthiomethyl-3-cephem -4-carboxylic acid (syn isomer) (4.1 g) and sodium acetate (9.56 g) in water (41 ml) was stirred for 19.5 hours at ambient temperature. The resultant solution was adjusted to pH 2.0 with conc. hydrochloric acid. The precipitates were collected by filtration and dried over phosphorus pentoxide to give 7-[2-carboxymethoxyimino-2-(2-aminothiazol-4-yl)acetamido]-3-methylthiomet hyl-3-cephem-4-carboxylic acid (syn isomer) (1.8 g), mp 173.degree.-176.degree. C. (dec.).

IR (Nujol): 3370, 1772, 1670 (broad) cm.sup.-1

NMR .delta.ppm (DMSO-d6): 2.00 (3H, s), 3.57 (4H, m), 4.60 (2H, broad s), 5.17 (1H, d, J=5Hz), 5.73 (1H, dd, J=5Hz, 8Hz), 6.77 (1H, s), 9.45 (1H, d, J=8Hz)

To a solution of benzhydryl 7-[2-benzhydryloxycarbonylmethoxyimino-2-{2-(2,2,2-trifluoroacetamido)-thi azol-4-yl}acetamido]-3-methylthiomethyl-3-cephem-4-carboxylate (syn isomer) (5.6 g) and anisole (5.6 ml) in methylene chloride (11.2 ml) was added trifluoroacetic acid (11.2 ml) at 10.degree. C. The mixture was stirred for 1.5 hours at ambient temperature and then poured into a mixture of diisopropyl ether (400 ml) and petroleum ether (100 ml). The precipitates were collected by filtration and washed with petroleum ether to give 7-[2-carboxymethoxyimino-2-{2-(2,2,2-trifluoroacetamido)-thiazol-4-yl}acet amido]-3-methylthiomethyl-3-cephem-4-carboxylic acid (syn isomer) (4.2 g), mp 183.degree.-186.degree. C. (dec.).

IR (Nujol): 3300, 1778, 1723, 1660 cm.sup.-1

NMR .delta.ppm (DMSO-d6): 2.00 (3H, s), 3.62 (4H, m), 4.70 (2H, s), 5.24 (1H, d, J=5Hz), 5.80 (1H, dd, J=5Hz, 8Hz), 7.60 (1H, s), 9.68 (1H, d, J=8Hz)