CEPHEM COMPOUNDS WITH ANTIMICROBIAL ACTIVITY

Technical Field

The present invention relates to new cephem compounds and pharmaceutically acceptable salts thereof. More particularly, it relates to new cephem compounds and pharmaceutically acceptable salts thereof, which have antimicrobial activities, to processes for preparation thereof, to pharmaceutical composition comprising the same, and to a method for preventing and/or treating infectious diseases in human beings and animals. Accordingly, one object of the present invention is to provide the cephem compounds and pharmaceutically acceptable salts thereof, which are highly active against a number of pathogenic microorganisms.

Another object of the present invention is to provide processes for the preparation of the cephem compounds and salts thereof.

A further object of the present invention is to provide pharmaceutical composition comprising, as an active ingredient, said cephem compounds or their pharmaceutically acceptable salts.

Still further object of the present invention is to provide a method for preventing and/or treating infectious diseases caused by pathogenic microorganisms, which comprises administering said cephem compounds to infected human beings or animals.

Background Art

Cephem compounds having high antimicrobial activity against wide variety of pathogenic microorganisms

including Gram-positive and Gram-negative microorganisms have been known in, for example, EP-A-306863, but they are still insufficient as antimicrobial agents against methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa.

Disclosure of the Invention

The object cephem compounds of the present invention are novel and can be represented by the following general formula [I] :

R

wherein R ¹ is amino or protected amino,

R ² is ethyl, propyl, cyclo(lower)alkyl,

1-methylpropenyl or 2-methylpropenyl, R3 is hydroxy(lower)alkyl or protected hydroxy(lower)alkyl, and R4 is amino or protected amino.

As to the object compound [I], the following points are to be noted.

That is, the partial structure of the formula :

(wherein R ¹ and R ² are each as defined above)

shows that the object compound [I] and the starting compound [III] mentioned below are syn isomers.

Another point to be noted is that the pyrazolio moiety of the compound [I] can also exist in the tautomeric form, and such tautomeric equilibrium can be represented by the following schemes.

R- R-

(A) (B)

(wherein R^ and R^ are each as defined above).

Both of the above tautomeric isomers are included within the scope of the present invention, and in the present specification and claim, however, the object compound [I] is represented for the convenient sake by one expression of the pyrazolio group of the formula (A) .

The cephem compound [I] or the present invention can be prepared by a process as illustrated in the following.

Process 1

R-

[II] [III] or its reactive or its reactive derivative at the derivative at the amino group carboxy group or a salt thereof or a salt thereof

R-

[I] or its salt

wherein R-^-, R ² , R^ and R ⁴ are each as defined above.

In the above and subsequent descriptions of this specification, suitable examples of the various definitions are explained in detail as follows : The term "lower" is intended to mean 1 to 6 carbon atom(s), unless otherwise indicated.

Suitable "amino protective group" in the "protected amino" may be an acyl group as mentioned below, substituted or unsubstituted ar(lower)alkylidene [e.g. benzylidene, hydroxybenzylidene, etc.], ar(lower)alkyl

such as mono or di or triphenyl(lower)alkyl [e.g. benzyl, phenethyl, benzhydryl, trityl, etc.], or the like.

Suitable "acyl" may be lower alkanoyl [e.g. formyl, acetyl, propionyl, hexanoyl, pivaloyl, etc.], mono(or di or tri)halo(lower)alkanoyl [e.g. chloroacetyl, tri- fluoroacetyl, etc.], lower alkoxycarbonyl [e.g. methoxy- carbonyl, ethoxycarbonyl, tert-butoxycarbonyl, tert- pentyloxycarbonyl, hexyloxycarbonyl, etc. ] , carbamoyl, carboxy, aroyl [e.g. benzoyl, toluoyl, naphthoyl, etc.], ar(lower)alkanoyl [e.g. phenylacetyl, phenylpropionyl, etc.], aryloxycarbonyl [e.g. phenoxycarbonyl, naphthyloxycarbonyl, etc.], aryloxy(lower)alkanoyl [e.g. phenoxyacetyl, phenoxypropionyl, etc.], arylglyoxyloyl [e.g. phenylglyoxyloyl, naphthylglyoxyloyl, etc. ] , ar(lower)alkoxycarbonyl which may have suitable substituent(s) [e.g. benzyloxycarbonyl, phenethyloxy- carbonyl, p-nitrobenzyloxycarbonyl, etc.], or the like.

Suitable lower alkyl moieties in the "hydroxy(lower)alkyl" and "protected hydroxy(lower)alkyl" may be a straight or branched one such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl or the like.

Suitable "hydroxy(lower)alk l" may be mono- or dihydroxy(lower)alkyl such as hydroxymethyl, hydroxyethyl, hydroxypropyl, dihydroxyeth l, dihydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl or the like, preferably mono- or dihydroxy(Cι-C4)alkyl, more preferably hydroxy(C -C2)alkyl and the most preferably hydroxyethyl. Suitable "cyclo(lower)alkyl" may be one having 3 to 6 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl .

Suitable "protected hydroxy"^ in the "protected hydroxy(lower)alkyl" may be acyloxy group or the like. Suitable "acyl moiety" in the "acyloxy" may be lower alkanoyl [e.g. formyl, acetyl, propionyl, hexanoyl,

o -

pivaloyl, etc.], mono(or di or tri)halo(lower)alkanoyl [e.g. chloroacetyl, trifluoroacetyl, etc.], lower alkoxycarbonyl [e.g. ethoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, tert-pentyloxycarbonyl, exyloxycarbonyl, etc.], carbamoyl or the like.

Suitable pharmaceutically acceptable salts of the object compound [I] are conventional non-toxic salts and include a metal salt such as an alkali metal salt [e.g. sodium salt, potassium salt, etc.] and an alkaline earth metal salt [e.g. calcium salt, magnesium salt, etc.], an ammonium salt, an organic base salt [e.g. trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, etc.], an acid addition salt such as an organic acid addition salt [e.g. formate, acetate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc.], an inorganic acid addition salt [e.g. hydrochloride, hydrobromide, sulfate, phosphate, etc.], a salt with an amino acid [e.g. arginine salt, aspartic acid salt, -glutamic acid salt, etc.], and the like.

Preferred embodiments of the object compound [I] are as follows.

Preferred embodiment of ! is amino,

R ² is ethyl, propyl, cyclo(lower)alkyl [more preferably cyclo(C4-C5)alkyl] _; 1-methylpropenyl or 2- methylpropenyl (more preferably l-methyl-2-propenyl or 2-methyl-2-propenyl), in which the most preferable one is ethyl, R ³ is hydroxy(lower)alkyl [more preferably mono- or di- hydroxy(C _] _-C )alkyl, most preferably hydroxyethyl], R ⁴ is amino.

The process for preparing the object compound of the present invention is explained in detail in the following.

Process 1 The compound [I] or its salt can be prepared by reacting the compound [II] or its reactive derivative at the amino group or a salt thereof with the compound [III] or its reactive derivative at the carboxy group or a salt thereof. Suitable reactive derivative at the amino group of the compound [II] may include Schiff's base type i ino or its tautomeric enamine type isomer formed by the reaction of the compound [II] with a carbonyl compound such as aldehyde, ketone or the like; a silyl derivative formed by the reaction of the compound [II] with a silyl compound such as bis(trimethylsilyDacetamide, mono(trimethylsilyDacetamide [e.g. N-(trimethylsilyD¬ acetamide], bis(trimethylsilyl)urea or the like; a derivative formed by reaction of the compound [II] with phosphorus trichloride or phosgene, and the like.

Suitable salts of the compound [II] and its reactive derivative can be referred to the ones as exemplified for the compound [I] .

Suitable reactive derivative at the carboxy group of the compound [III] may include an acid halide, an acid anhydride, an activated amide, an activated ester, and the like. Suitable examples of the reactive derivatives may be an acid chloride; an acid azide; a mixed acid anhydride with an acid such as substituted phosphoric acid [e.g. dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoric acid, dibenzylphosphoric acid, halogenated phosphoric acid, etc.], dialkylphosphorous acid, sulfurous acid, thiosulfuric acid, sulfuric acid, sulfonic acid [e.g. methanesulfonic acid, etc.], aliphatic carboxylic acid [e.g. acetic acid, propionic acid, butyric

acid, isobutyric acid, pivalic acid, pentanoic acid, isopentanoic acid, 2-ethylbutyric acid, trichloroacetic acid, etc.] or aromatic carboxylic acid [e.g. benzoic acid, etc.]; a symmetrical acid anhydride; an activated amide with imidazole, 4-substituted imidazole, di ethylpyrazole, triazole or tetrazole; or an activated ester [e.g. cyanomethyl ester, methoxymethyl ester, dimethyliminomethyl [ {CH3) ] ester, vinyl ester, propargyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, trichlorophenyl ester, pentachlorophenyl ester, mesylphenyl ester, phenylazophenyl ester, phenyl thioester, p-nitrophenyl thioeter, p-cresyl thioester, carboxymethyl thioester, pyranyl ester, pyridyl ester, piperidyl ester, 8-quinolyl thioester, etc.], or an ester with a N-hydroxy compound [e.g. N,N-dimethylhydroxylamine, l-hydroxy-2-(1H)-pyridone, N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxy-lH-benzotriazole, etc.], and the like. These reactive derivatives can optionally be selected from them according to the kind of the compound [III] to be used.

Suitable salts of the compound [III] and its reactive derivative can be referred to the ones as exemplified for the compound [I] .

The reaction is usually carried out in a conventional solvent such as water, alcohol [e.g. ethanol, ethanol, etc.], acetone, dioxane, acetonitrile, chloroform, ethylene chloride, ethylene chloride, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, pyridine or any other organic solvent which does not adversely influence the reaction. These conventional solvent may also be used in a mixture with water.

In this reaction, when the compound [III] is used in a free acid form or its salt form, the reaction is preferably carried out in the presence of a conventional condensing agent such as N,N'-dicyclohexylcarbodiimide;

N-cyclohexyl- ' -morpholinoethylcarbodiimide; N-cyclohexyl-N' -(4-diethylaminocyclohexyl)carbodiimide; N,N'-diethylcarbodii ide, N,N'-diisopropylcarbodiimide; N-ethyl-N'-(3-dimethylaminopropyl)carbodii ide; N,N' -carbonylbis-(2-methylimidazole) ; pentamethyleneketene-N-cyclohexylimine; diphenylketene-N- cyclohexylimine; ethoxyacetylene; 1-alkoxy-l- chloroethylene; trialkyl phosphite; ethyl polyphosphate; isopropyl polyphosphate; phosphorus oxychloride (phosphoryl chloride); phosphorus pentachloride; phosphorus trichloride; thionyl chloride; oxalyl chloride; lower alkyl haloformate [e.g. ethyl chloroformate, isopropyl chloroformate, etc.]; triphenylphosphine; 2-ethyl-7-hydroxybenzisoxazolium salt; 2-ethyl-5-( - sulfophenyDisoxazolium hydroxide intramolecular salt; l-(p-chlorobenzenesulfonyloxy)-6-chloro-lH-benzotriazole; so-called Vilsmeier reagent prepared by the reaction of N,N-dimethylformamide with thionyl chloride, phosgene, trichloromethyl chloroformate, phosphorus oxychloride, etc.; or the like.

The reaction may also be carried out in the presence of an inorganic or organic base such as an alkali metal bicarbonate, tri(lower)alkylamine, pyridine, N-(lower)alkylmorpholine, N,N-di(lower)alkylbenzylamine, or the like.

The reaction temperature is not critical, and the reaction is usually carried out under cooling to warming.

The object compound [I] and its pharmaceutically acceptable salt are novel and exhibit high antimicrobial activity, inhibiting the growth of a wide variety of pathogenic microorganisms including Gram-positive and Gram-negative microorganisms, especially, MRSA and Pseudomonas aeruginosa, and are useful as antimicrobial agents.

Now in order to show the utility of the object compound [I], the test data on MIC (minimal inhibitory concentration) of a representative compound of this invention are shown in the following.

Test method :

In vitro antibacterial activity was determined by the two-fold agar-plate dilution method as described below.

One loopful of an overnight culture of each test strain in Trypticase-soy broth (10 ⁶ viable cells per ml) was streaked on heart infusion agar (Hl-agar) containing graded concentrations of representative test compound, and the minimal inhibitory concentration (MIC) was expressed in terms of μg/ml after incubation at 37°C for 20 hours.

Test compound :

7β-[2-(5-Amino-l,2,4-thiadiazol-3-yl)-2-ethoxyimino- acetamido]-3-[3-amino-2-(2-hydroxyethyl)-1- pyrazolio]methyl-3-cephem-4-carboxylate hydrochloride (syn iso er)

Test results :

Test Strain MIC (μg/ml)

S. aureus 2538 3.13

E. coli 31 ≤0.025

P. aeruginosa 2 0.39

For therapeutic administration, the object compound [I] and its pharmaceutically acceptable salt of the

present invention are used in the form of conventional pharmaceutical preparation which contains said compound as an active ingredient, in admixture with pharmaceutically acceptable carriers such as an organic or inorganic solid or liquid excipient which is suitable for oral, parenteral such as intravenous, intramuscular, subcutaneous or intraarticular, and external (topical)administration. The pharmaceutical preparations may be in solid form such as tablet, granule, powder, capsule, suppository, or liquid form such as solution, suspension, syrup, emulsion, lemonade and the like.

If needed, there may be included in the above preparations auxiliary substances, stabilizing agents, wetting agents and other commonly used additives such as lactose, citric acid, tartaric acid, stearic acid, magnesium stearate, terra alba, sucrose, corn starch, talc, gelatin, agar, pectin, peanut oil, olive oil, cacao butter, ethylene glycol, arginine, sodium bicarbonate, sodium carbonate, and the like. While the dosage of the compound [I] may vary from and also depend upon the age, conditions of the patient, a kind of diseases, a kind of the compound [I] to be applied, etc. In general amounts between 1 g and about 4,000 mg or even more per day may be administered to a patient. An average single dose of about 50 mg, 100 mg, 250 mg, 500 mg, 1000 mg, 2000 mg of the object compounds [I] of the present invention may be used in preventing and/or treating diseases infected by pathogenic microorganisms.

Examples

The following Preparations and Examples are given for the purpose of illustrating the present invention in more detail.

Preparation 1

To a suspension of ethyl 2-cyano-3-ethoxyacrylate (5.7 g) in ethanol (100 ml) and water (10 ml) was added 2,3-0-benzylidene-2,3-dihydroxypropylhydrazine (10 g) , and the reaction mixture was refluxed for 2 hours. After removal of ethanol under reduced pressure, the residue was subjected to column chromatography on silica gel (150 g) and eluted with a mixture of dichloro ethane and ethyl acetate (1:2, V/V) . The fractions containing the object compound were combined and evaporated in vacuo to give 4- ethoxycarbonyl-1-(2,3-dihydroxypropyl)-5-aminopyrazole (830 mg) .

NMR (DMSO-d ₆ , δ) : 1.24 (3H, t, J=7Hz) , 3.25-3.40

(2H, m), 3.70-4.10 (3H, m) , 4.16 (2H, q, J=7Hz), 4.78 (1H, t, J=6Hz), 5.09 (1H, d, J=4Hz), 6.05

(2H, br s) , 7.46 (1H, s)

Preparation 2

A suspension of 4-ethoxycarbonyl-l-(2,3- dihydroxypropyl)-5-aminopyrazole (4.2 g) in lN-sodium hydroxide solution (37 ml) was stirred at 70°C for 30 minutes. The reaction mixture was poured into ethyl acetate (80 ml) and water (40 ml), and then the mixture was adjusted to pH 3.0 with 6N hydrochloric acid. The separated organic phase was washed with brine (40 ml) and dried over magnesium sulfate and evaporated in vacuo to give 4-carboxy-l-(2,3-dihydroxypropyl)-5-aminopyrazole (2.95 g).

NMR (DMSO-d ₆ , δ) : 3.34 (2H, d, J=4Hz), 3.40-4.10 (3H, m), 4.77 (1H, br s), 5.08 (1H, br s), 6.00

(2H, br s), 7.43 (1H, s), 11.08 (1H, br s)

Preparation 3

A solution of 4-carboxy-l-(2,3-dihydroxypropyl)-5- aminopyrazole (400 mg) in cyclohexanol (4 ml) wa-s stirred

at 135°C for 30 minutes. The reaction mixture was subjected to column chromatography on silica gel (20 g) and eluted with a mixture of ethyl acetate and methanol (10:1, V/V) . The fractions containing the object compound were combined and evaporated in vacuo to give l-(2,3- dihydroxypropyl)-5-aminopyrazole (140 mg) .

NMR (DMSO-d ₆ , δ) : 3.29 (2H, t, J=4Hz) , 3.40-3.80

(2H, ), 3.80-4.00 (1H, m) , 4.74 (1H, t, J=4Hz), 5.03 (2H, br s), 5.07 (1H, d, J=4Hz), 5.28 (1H, d, J=3Hz), 7.04 (1H, d, J=3Hz)

Preparation 4

A mixture of acetic anhydride (5 ml) and formic acid (2.8 ml) was stirred for 20 minutes at 30°C. l-(2,3- Dihydroxypropyl)-5-aminopyrazole (1.67 g) was added thereto with stirring under ice-cooling, and then the mixture was stirred for 30 minutes at 10 to 20°C. The reaction mixture was poured into ethyl acetate (30 ml) and water (30 ml), and then the mixture was adjusted to pH 6.0 with 5% aqueous sodium hydrogen carbonate solution. The separated organic phase was washed with brine (30 ml), dried with magnesium sulfate and evaporated in vacuo to give 1-[2,3-bis(formyloxy)propyl]-5-formylaminopyrazole (2 g). NMR (DMSO-d ₆ , δ) : 4.10-4.45 (2H + 2H, m) , 5.35-5.55

(1H, m), 6.20 (0.2H, d, J=3Hz) , 6.32 (0.8H, d, J=3Hz), 7.40 (1H, d, J=3Hz) , 8.13 (1H, s), 8.26 (1.8H, s), 8.34 (0.2H, s), 10.43 (1H, br s)

Preparation 5

A suspension of diphenylmethyl 7β-tert- butoxycarbonylamino-3-chloromethyl-3-cephem-4-carboxylate

(2.1 g) , 1-[2,3-bis(formyloxy)propyl]-5- formylaminopyrazole (2.0 g) and sodium iodide (620 mg) in N,N-dimethylformamide (4 ml) was stirred at room

temperature for 36 hours. The resulting mixture was poured into a mixture of ethyl acetate (50 ml) and water (50 ml), the separated organic layer was washed with water (50 ml) twice, dried over magnesium sulfate and concentrated to about 10 ml. The concentrated solution was dropwise added to diisopropyl ether (200 ml) with stirring. The resulting precipitate was collected by filtration, and dried in vacuo to give diphenylmethyl 7β- tert-butoxycarbonylamino-3-[2-[2,3-bis(formyloxy)propyl]- 3-formylamino-l-pyrazolio]methyl-3-cephem-4-carboxylate iodide (3 g) .

NMR (DMSO-d ₆ , δ) : 1.46 (9H, s), 3.20-3.65 (2H, m) , 4.10-4.50 (4H, m) , 4.70-4.85 (1H, m) , 5.07 (1H, d, J=5Hz), 5.13-5.65 (2H, m) , 5.66 (1H, dd, J=5Hz, 8Hz), 6.90 (1H, d, J=3Hz), 7.07 (1H, s),

7.20-7.60 (10H, m) , 8.10 (1H, d, J=8Hz), 8.27 (1H, d, J=3Hz)

Preparation 6 To a solution of diphenylmethyl 7β-tert- butoxycarbonylamino-3-[2-[2,3-bis(formyloxy)propyl]-3- formylamino-1-pyrazolio] ethyl-3-cephem-4-carboxylate iodide (3 g) and anisole (3 ml) in dichloromethane (9 ml) was added trifluoroacetic acid (6 ml) under ice-cooling with stirring and the mixture was stirred for 2 hours at the same temperature. The resulting solution was added to diisopropyl ether (250 ml). The precipitate was collected by filtration, dried under reduced pressure to give 7β- amino-3-[2-[2,3-bis(formyloxy)propyl]-3-formylamino-1- pyrazolio]methyl-3-cephem-4-carboxylate ditrifluoroacetate (1.21 g).

NMR (DMSO-d ₆ , δ) : 3.30-3.80 (2H + 2H, m) , 4.20-

4.60 (1H + 2H, m), 5.10-5.60 (1H + 2H + 1H, m) , 7.11 (1H, d, J=3Hz), 8.29 (1H, d, J=3Hz)

Preparation 7

To a suspension of 7β-amino-3-[2-[2, 3- bis(formyloxy)propyl]-3-formylamino-l-pyrazolio]methyl-3- cephem-4-carboxylate ditrifluoroacetate (1 g) in methanol (7 ml) was added hydrochloric acid (1.2 ml) under stirring at 25°C, and stirring was continued for 3 hours at 30°C. The reaction mixture was poured into ethyl acetate (100 ml). The precipitate was collected by filtration and dried under reduced pressure to give 7β-amino-3-[2-(2,3- dihydroxypropyl)-3-amino-l-pyrazolio]methyl-3-cephem-4- carboxylate trihydrochloride (482 mg) .

NMR (DMSO-dg, δ) : 3.20-3.60 (2H, m) , 3.70-4.30 (2H + 2H, ), 4.30-4.50 (1H, ) , 5.10-5.50 (1H + 1H + 2H, m), 5.91 (1H, d, J=3Hz), 7.50 (2H, br s), 8.12 (1H, d, J=3Hz)

Example 1

To a suspension of phosphorus pentachloride (0.89 g) in methylene chloride (18 ml) was added (Z)-2-(5-amino- 1,2,4-thiadiazol-3-yl)-2-cyclopentyloxyiminoacetic acid (1 g) at -15°C. After being stirred for 30 minutes at -10°C ~ -15°C, the mixture was evaporated in vacuo to remove methylene chloride. The residue was suspended in tetrahydrofuran (16 ml), and then added to a solution of 7β-amino-3-[3-amino-2-(2-hydroxyethyl)-1-pyrazolio]methyl- 3-cephem-4-carboxylate hydrochloride dihydrate (1.6 g) and N-(trimethylsilyl)acetamide (5.1 g) in a mixture of N,N-dimethylformamide (16 ml) and tetrahydrofuran (16 ml) at -10°C. After being stirred under ice-cooling for 1 hour, the mixture was added dropwise to ethyl acetate. The resulting precipitate was collected by filtration, dissolved in water and adjusted to pH 4 with an aqueous solution of sodium bicarbonate. The aqueous solution was subjected to column chromatography on Diaion HP-20 (Trademark : Mitsubishi Kasei Corporation), and eluted

with 10% isopropyl alcohol. The fractions containing the object compound were collected, and evaporated in vacuo to remove isopropyl alcohol . The aqueous solution was lyophilized to give 7β-[2-(5-amino- 1, 2,4-thiadiazol-3-yl)-2-cyclopentyloxyiminoacetamido]-3- [3-amino-2-(2-hydroxyethyl)-l-pyrazolio]methyl-3-cephem-4- carboxylate (syn isomer) (0.16 g) .

IR (Nujol) : 3300, 1765, 1660 cm ^"1

NMR (DMSO-d,, δ) : 1.27-2.02 (8H, ) , 2.94 and 3.23 (2H, ABq, J=18Hz), 3.67-3.82 (2H, ) , 4.20-4.52

(2H, m), 4.57-4.83 (1H, ) , 5.01 (1H, d, J=5Hz), 5.02 and 5.28 (2H, ABq, J=15Hz), 5.61 (1H, dd, J=5Hz, 8Hz), 5.81 (1H, d, J=3Hz) , 7.31 (2H, br s), 8.06 (1H, d, J=3Hz), 8.07 (2H, br s), 9.38 (1H, d, J=8Hz)

Example 2

7β-[2-(5-Amino-l,2,4-thiadiazol-3-yl)-2-cyclobutoxy- iminoacetamido]-3-[3-amino-2-(2-hydroxyethyl)-1-pyrazolio]- methyl-3-cephem-4-carboxylate (syn isomer) was obtained according to a similar manner to that of Example 1. IR (Nujol) : 3250, 1765, 1660 cm ^"1

NMR (DMSO-dg, δ) : 1.45-1.70 (2H, m) , 2.10-2.30 (4H, m), 2.99 and 3.20 (2H, ABq, J=18Hz), 4.20-4.60 (4H, ), 4.71 (1H, m) , 4.90-5.40 (2H, m) , 5.04

(1H, d, J=5Hz), 5.65 (1H, dd, J=8Hz, 5Hz) , 5.82 (1H, d, J=3Hz), 7.38 (2H, br s), 8.10 (1H, d, J=3Hz), 8.19 (2H, br s), 9.53 (1H, d, J=8Hz)

Example 3

A solution of 7β-amino-3-[3-amino-2-(2-hydroxyethyl)- l-pyrazolio]methyl-3-cephem-4-carboxylate hydrochloride (50 g) in a mixture of acetonitrile (500 ml) and water (500 ml) was adjusted to pH 6.0 with aqueous sodium bicarbonate solution. To the resulting solution was added

(Z)-2-(5-amino-l,2,4-thiadiazol-3-yl)-2-ethoxyimino- acetyl chloride hydrochloride (40 g) under stirring at 5°C and the mixture was stirred at the same temperature for 1.5 hours, keeping pH 5.0-6.5 with aqueous sodium bicarbonate solution. The reaction mixture was evaporated to remove the containing organic solvent. The aqueous solution was subjected to column chromatography on Sepabeads SP-205 (Trademark : Mitsubishi Kasei Corporation) (1.3 £ ). The column was washed with water (5 H ) and the object compound was eluted with 15% isopropyl alcohol, and the eluate was lyophilized to give 7β-[2-(5- amino-1,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido]-3- [3-amino-2-(2-hydroxyethyl)-1-pyrazolio] ethyl-3-cephem-4- carboxylate (syn isomer) (51.2 g) . IR (Nujol) : 3300, 1750, 1660, 1600 cm ^"1

NMR (DMSO-d ₆ , δ) : 1.23 (3H, t, J=7Hz), 2.93 and 3.18 (2H, ABq, J=18Hz), 3.58 (2H, br s), 4.15 (2H, q, J=7Hz), 4.13-4.25 (2H, m) , 5.01 (1H, d, J=5Hz), 5.03 and 5.23 (2H, ABq, J=16Hz), 5.63 (1H, dd, J=5Hz, 8Hz) , 5.81 (1H, d, J=3Hz) , 7.27

(2H, br s), 8.10 (1H, d, J=3Hz), 8.15 (2H, br s), 9.50 (1H, d, J=8Hz)

Example 4 The following compounds were obtained according to a similar manner to that of Example 3.

(1) 7β-[2-(5-Amino-l,2,4-thiadiazol-3-yl)-2-(n-propoxy- imino)acetamido]-3-[3-amino-2-( 2-hydroxyethyl)-l- pyrazolio]methyl-3-cephem-4-carboxylate (syn isomer)

IR (Nujol) : 3350, 3130, 1765, 1610 cm ^"1 NMR (DMSO-dg, δ) : 0.84-0.93 (3H, m) , 1.53-1.73 (2H, m), 3.00 and 3.22 (2H, ABq, J=18Hz), 3.60 (2H, br s), 4.00-4.10 (2H, m) , 4.15-4.30 (2H, m) , 5.05 (1H, d, J=5Hz) , 5.06 and 5.25 (2H, ABq,

J=16Hz), 5.69 (1H, dd, J=5Hz, 8Hz ) , 5.84 (1H, d, J=3Hz), 7.40 (2H, br s), 8.08 (1H, d, J=3Hz),

8.17 (2H, br s), 9.52 (1H, d, J=8Hz)

(2) 7β-[2-(5-Amino-l,2,4-thiadiazol-3-yl)-2-(l-methyl-2- propenyloxyimino)acetamido]-3-[3-amino-2-(2- hydroxyethyl)-l-pyrazolio]methyl-3-cephem-4- carboxylate (syn isomer) IR (Nujol) : 1755 cm ^"1

(3) 7β-[2-(5-Amino-l,2,4-thiadiazol-3-yl)-2-(2-methyl-2- propenyloxyimino)acetamido]-3-[3-amino-2-(2- hydroxyethyl)-l-pyrazolio]methyl-3-cephem-4- carboxylate (syn isomer) IR (Nujol) : 1760 cm ^"1

(4 ) 7β-[2-(5-Amino-l,2,4-thiadiazol-3-yl)-2-ethoxyimino- acetamido]-3-[2-(2,3-dihydroxypropyl)-3-amino-l- pyrazolio]methyl-3-cephem-4-carboxylate (syn isomer) IR (Nujol) : 1755, 1640, 1610, 1520 cm ^-1

NMR (DMSO-d ₆ , δ) : 1.23 (3H, t, J=7Hz), 2.95 and

3.18 (2H, ABq, J=18Hz), 3.20, 4.00 (2H + 2H, m) , 4.15 (2H, q, J=7Hz), 4.10-4.30 (1H, m) , 5.02 (1H, d, J=5Hz), 4.90-5.35 (2H, m) , 5.66 (1H, dd, J=5Hz, 8Hz), 5.83 (1H, d, J=3Hz), 7.19 (2H, br s), 8.11 (1H, d, J=3Hz), 8.15 (2H, br s), 9.52 (1H, d, J=8Hz)

Example 5 To a solution of 7β-[2-(5-amino-l,2,4-thiadiazol-3- yl)-2-ethoxyiminoacetamido]-3-[3-amino-2-(2-hydroxyethyl)- l-pyrazolio]methyl-3-cephem-4-carboxylate (syn isomer) (30 g) in 4N-hydrochloric acid (22.4 ml) was added acetone (120 ml) under stirring at 20°C. The stirring was continued for 10 hours at the same temperature to give

crystals, which were collected by filtration and dried to give 7β-[2-(5-amino-l,2,4-thiadiazol-3-yl)-2- ethoxyiminoacetamido]-3-[3-amino-2-(2-hydroxyethyl)-1- pyrazolio]methyl-3-cephem-4-carboxylate hydrochloride (syn isomer) (13.46 g) .

IR (Nujol) : 3250, 3150, 3120, 1785, 1705, 1650,

1620, 1575, 1530 cm ^"1 NMR (DMSO-dg, δ) : 1.23 (3H, t, J=7Hz ) , 3.24 and

3.30 (2H, ABq, J=18Hz), 3.60 (2H, br s), 4.03- 4.25 (2H, ), 4.13 (2H, q, J=7Hz) , 5.14 and 5.32

(2H, d, J=16Hz), 5.18 (1H, d, J=5Hz) , 5.86 (1H, dd, J=5Hz, 8Hz), 5.89 (1H, d, J=3Hz), 7.50 (2H, br s), 8.03 (1H, d, J=3Hz) , 8.21 (2H, br s), 8.58 (1H, d, J=8Hz)

Example 6

To a solution of 7β-[2-(5-amino-l,2,4-thiadiazol-3- yl)-2-ethoxyiminoacetamido]-3-[3-amino-2-(2-hydroxyethyl)- l-pyrazolio]methyl-3-cephem-4-carboxylate (syn isomer) (5 g) in water (50 ml) was added 1M sulfuric acid (8 ml). The solution was lyophilized to give 7β-[2-(5-amino- 1,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido]-3-[3- amino-2-(2-hydroxyethyl)-1-pyrazolio]methyl-3-cephem-4- carboxylate sulfate (syn isomer) (5.2 g). IR (Nujol) : 3150, 1760, 1640, 1610 cm ^"1

NMR (DMSO-dg, δ) : 1.23 (3H, t, J=7Hz) , 3.21 and

3.32 (2H, ABq, J=18Hz), 3.62 (2H, br s), 4.03- 4.26 (2H, m), 4.13 (2H, q, J=7Hz), 5.88 (1H, dd, J=5Hz, 8Hz), 5.90 (1H, d, J=3Hz) , 7.50 (2H, br s), 8.04 (1H, d, J=3Hz), 8.21 (2H, br s), 8.59

(1H, d, J=8Hz)

Example 7

Crystals of 7β-[2-(5-amino-l, 2,4-thiadiazol-3-yl)-2- ethoxyiminoacetamido]-3-[3-amino-2-(2-hydroxyethyl)-1-

pyrazolio]methyl-3-cephem-4-carboxylate sulfate (syn isomer) was obtained from 7β-[ 2-(5-amino-l,2,4-thiadiazol- 3-yl)-2-ethoxyiminoacetamido]-3-[3-amino-2-(2- hydroxyethyl)-1-pyrazolio]methyl-3-cephem-4-carboxylate (syn isomer) and 2M sulfuric acid according to a similar manner to that of Example 5.

IR ( Br) : 3257, 3060, 1799, 1697, 1670, 1639, 1579,

1546 cm ^-1 NMR (D ₂ 0, δ) : 1.31 (3H, t, J=7Hz), 3.18 and 3.48 (2H, ABq, J=18Hz), 3.85 (2H, t, J=5Hz) , 4.22-

4.45 (4H, ), 5.20 (2H, s), 5.26 (1H, d, J=5Hz ) , 5.88 (1H, d, J=5Hz), 5.97 (1H, d, J=3Hz ) , 7.89 (1H, d, J=3Hz)