KR100191705B1 - New pyrrolidinyl thio carbapenem derivatives and a process for preparing thereof - Google Patents

Abstract

The present invention relates to novel pyrrolidyl thiocarbapenem derivatives represented by the following general formula (I) and pharmaceutically acceptable salts thereof.

In which R ¹ is hydrogen or lower alkyl; R ² , R ³ , R ⁴ are hydrogen, lower alkyl, substituted lower alkyl or aminoprotecting groups, where R ³ , R ⁴ may be saturated or unsaturated cyclic groups formed with the nitrogen atoms bonded to one another, and The cyclic group may comprise at least one hetero element of oxygen, sulfur or nitrogen; X ¹ is hydrogen or a hydroxy protecting group; X ² is hydrogen, a carboxy protecting group, an alkali or alkaline earth metal, a nonmetal salt; Y ² represents hydrogen or an aminoprotecting group; In (R ⁵ ) n R ⁵ is -CH ₂ -and n is an integer of 0-5.

Description

[Name of invention]

Novel pyrrolidinyl thiocarbapenem derivatives and preparation method thereof

Detailed description of the invention

The present invention relates to novel pyrrolidinyl thio carbapenem derivatives having a broad antimicrobial range and a method for preparing the same.

Carbapenem antibiotics, which are beta-lactam antibiotics, are already well known, such as imipenem, meropenem, and (1R, 5S, 6S) -6-[(R) -1-hydroxyethyl] -1-methyl-2. -[(2S, 4S) -2-sulfamoylmethylpyrrolidin-4-yl thio] -1-carbafen-2-m-3-carboxylic acid (JP-A-578360) and (1R , 5S, 6S) -6 [(R) -1-hydroxyethyl] -1-methyl-2-[(2S, 4S) -2-sulfamidomethylpyrrolidin-4-yl thio] -1-carba Phen-2-m-3-carboxylic acid (European publication 528,698 A ₁ ) and (4R, 5S, 6S) -3 [(2R, 4S) -2-N'-amidinomethylpyrrolidine-4 -Yl] thio-6-[(1R) -1-hydroxyethyl] -4-methyl-7-oxo-1-azabicyclo [3, 2, 0] hept-4-ene-2-carboxylic acid (JP-A-7-157483).

All of the carbapenem derivatives have high activity against gram (+) and gram (-) strains, but due to the emergence of various resistant strains, there is a continuous need for antibiotics with stronger antibacterial activity. have.

An object of the present invention is to provide a novel pyrrolidyl thio carbapenem derivative represented by the following general formula (I), a pharmaceutically acceptable salt thereof, and a preparation method thereof.

In which R ¹ is hydrogen or lower alkyl; R ² , R ³ , R ⁴ are hydrogen, lower alkyl, substituted lower alkyl or aminoprotecting groups, where R ³ , R ⁴ may be saturated or unsaturated cyclic groups formed with the nitrogen atoms bonded to one another, and The cyclic group may comprise at least one hetero element of oxygen, sulfur or nitrogen; X ¹ is hydrogen or a hydroxy protecting group; X ² is hydrogen, a carboxy protecting group, an alkali or alkaline earth metal, a nonmetal salt; Y ² represents hydrogen or an aminoprotecting group; In (R ⁵ ) n R ⁵ is -CH ₂ -and n is an integer of 0-5.

Preferred compounds are those wherein R ¹ is a methyl group, X ¹ is hydrogen, X ² is hydrogen or an alkali metal, and Y ² is hydrogen. More preferably, R ^{2 is} also hydrogen, R ³ and R ⁴ are each hydrogen, R ³ is methyl, R ⁴ is hydrogen, R ³ , R ⁴ are each methyl. In addition, it is preferable that n is an integer of 1 or 2.

In the present invention, particularly preferred compounds are compounds of the following chemical names or pharmaceutically acceptable salts thereof.

Carbapenem derivatives of the general formula (I) may be prepared by the following method.

A pyrrolidine derivative represented by the following general formula (II);

In formula (II), R ² , R ³ , R ⁴ may be a cyclic group including hydrogen, lower alkyl, substituted lower alkyl, an amino protecting group or a nitrogen atom to which R ³ , R ⁴ is bonded. , Each cyclic group may comprise at least one or more groups from elements such as oxygen sulfur and nitrogen, and each cyclic group may be substituted; (R ⁵⁾ n in R ⁵ is -CH ₂ -, and; n is an integer of 0-5; Y ¹ is hydrogen or a mercapto protecting group; Y ² is hydrogen or an amino protecting group.

By reacting the carbapenem derivative represented by the following general formula (III), pyrrolidyl thio carbapenem derivative represented by the general formula (I) is prepared.

In the formula (III), R ¹ is hydrogen or lower alkyl; X ¹ is hydrogen or a hydroxy protecting group; X ² is hydrogen, a carboxy protecting group, a nonmetal salt, an alkali metal or an alkaline earth metal; OX ³ is a leaving group (ie, alkyl sulfonyl, aryl sulfonyl, diphenyl phosphoryl, etc. as the active ester of the hydroxy group).

The scope of each group so far is as follows.

The number of carbon atoms of the lower alkyl group means 1 to 5, and examples of such alkyl groups include methyl, ethyl, normal-propyl, iso-propyl, normal-butyl, tertiary-butyl, and pentyl. As for carbon number of a lower alkyl group, 1-3 are comparatively preferable, and methyl and ethyl group are preferable among these.

Examples of the amino protecting group and the hydroxy protecting group are lower alkoxycarbonyl, lower alkenyloxycarbonyl, halogeno alkoxycarbonyl, aralkyloxycarbonyl, trialkylsilyl group, and the like. Lower alkoxycarbonyl is t-butyloxycarbonyl. Lower alkenyl oxycarbonyl is allyloxycarbonyl, halogenoalkoxy carbonyl is 2-iodoethyloxycarbonyl and 2,2,2-trichloroethyloxycarbonyl; Examples of aralkyloxycarbonyl include benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, diphenylmethoxycarbonyl and the like; Examples of the trialkylsilyl include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl and the like.

R ³ and R ⁴ may be a saturated or unsaturated cyclic group formed together with the nitrogen atom to which R ³ and R ⁴ are bonded, and a cyclic group of 3 to 8 atoms saturated or unsaturated may be one or more nitrogen, It may have a sulfur or oxygen atom, and if necessary it is preferred to include heteroatoms as monocyclic groups consisting of 5 or 6 atoms.

Examples include the following: pyrrolidin-1-yl, pyrrole-1-yl, imidazolidin-1-yl, imidazol-1-yl, pyrazolidin-1-yl, pyrazol- 1-yl, piperidino, piperazino, piperazin-1-yl with a substituent at the 4 position, morpholino, thiomorpholino and the like.

These groups may be substituted with one or more of the following groups.

Amino, protected amino, carbamoyl, lower alkyl, hydroxy, protected hydroxy, lower alkoxy, oxo, lower alkylsulfonyl, hydroxy lower alkyl, carbamoyl lower alkyl, lower alkoxycarbonyl and cyano groups. And, if the cyclic group is imidazolidin-1-yl, pyrazolidin-1-yl or piperazin-1-yl, the imino moiety can be protected as a commonly used imino protecting group.

Carboxy protecting groups typically use those used in the art; Lower alkanoyloxy (lower) alkyl ester groups such as acetoxymethyl ester, butyloxymethyl ester, pivaloyloxymethyl ester group and the like; Lower alkoxycarbonyloxy (lower) alkyl groups such as methoxycarbonyloxymethyl ester group, ethoxycarbonyloxymethyl ester group, t-butoxycarbonyloxymethyl group and the like; Lower alkenyl groups such as vinyl ester groups, allyl ester groups, isopropenyl ester groups and the like; Ar (lower) alkyl groups having at least one substituent such as benzyl ester group, 4-methoxybenzyl ester group, 4-nitrobenzyl ester group, trityl ester group, benzhydryl ester group and the like; Arylester groups having at least one substituent such as phenylester group, 4-chlorophenylester group, tolylester group and t-butylphenylester group and the like can be used.

Mercapto protecting groups are commonly used and include, for example, acyl and aryl substituted lower alkyls such as benzyl, p-methoxybenzyl, phenethyl, trityl and benzohydryl.

Examples of pharmaceutically acceptable salts are salts with bases, salts with acids, salts with basic or acidic amino acids and intermolecular or intramolecular quaternary salts.

Examples of salts with bases include alkali metal salts such as sodium and potassium salts; Alkaline earth metal salts such as calcium and magnesium salts; Ammonium salts; And organic amine salts such as triethylamine salt, pyridine salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt and dibenzylamine salt; Examples of salts with acids include inorganic acid addition salts such as hydrochloride, hydrobromide, sulfates and phosphates.

Examples of the organic acid addition salts include formic acid salts, acetate salts, maleic acid salts, tartaric acid salts, and methanesulfonic acid salts; Examples of salts with amino acids include salts with arginine, aspartic acid or glutamic acid.

If necessary from the compound of the general formula (I) obtained by the reaction as described above, a reaction for the removal of protecting groups such as a hydroxyl group, a carboxy group, an amino group and an imino group is carried out to obtain a compound of the general formula (I) a. You can get it.

In Formula (I) a, the meanings of R ¹ , R ² , R ³ , R ⁴ , R ⁵ and X ¹ , X ² , Y ² , n are as defined above.

The method of removing the protecting group varies depending on the type of protecting group, and uses contact hydrogen and reaction using a palladium catalyst which is commonly used in the synthesis of carbapenem antibiotics.

Pyrrolidinylthiocarbapenem derivative (I) may be prepared using pyrrolidine derivative (II) obtained by using 4-hydroxypyrrolidine-2-carboxylic acid as a starting material, wherein general formula (II) The preparation method of pyrrolidine derivatives is as follows.

(Method 1)

The meaning of the abbreviation shown by said formula is as follows.

Ac: Acetyl

Me: Methyl

Ms: methanesulfonyl

PNB: p-nitrobenzyl

PMB: p-methoxybenzyl

PNZ: p-nitrobenzyloxycarbonyl

TFA: trifluoroacetic acid

In the synthesis of Method 1, Y ² , R ² , R ³ , and R ⁴ are the same as those mentioned in the general formulas (I) and (II).

This synthesis process starts with trans-4-hydroxy-L-proline (IV), introduces an amino protecting group to give compound (V), then esterifies the carboxy group to synthesize compound (VI), Compound (VII) was synthesized by introducing a methanesulfonin group to a hydroxy group at the 4-position, and compound (VIII) was synthesized by a reduction reaction, and a methanesulfonyl group was introduced to a hydroxy group of a hydroxymethyl group to give a compound (IX). ) Is synthesized.

Then, a cyano group is introduced into compound (IX) to obtain compound (X), and then a thioacetate group is introduced at position 4 to synthesize compound (XI). Subsequently, the 4-position thioacetate group of compound (XI) is hydrolyzed to obtain a thiol body, that is, compound (XII), and p-methoxybenzyl group is introduced thereto to obtain compound (XIII). Conversion to methylimidate yields compound (XIV), which is reacted with sulfamide derivatives to give compound (XV) and the removal of thiol protecting groups gives compound (II-a).

If the starting material represented by formula (III) is R ¹ is a hydrogen atom methods, such as only Salzburg (Salzmann) [J. Am. Chem. Soc. Vol. 102, 6161 (1980); If R ₁ is a methyl group, such as Shih et al. [Heterocycles, Vol. 21, 29 (1984)] and the like.

The invention is intended to be described in more detail in the following Preparation Examples and Examples, which do not limit the scope of the invention.

In the preparation examples and examples, the thin layer chromatography used silica gel 60F ₂₅₄ (Merck) as a plate, UV detector or ninhydrin (KnM), KM _n O ₄ color as the detection method. Silica gel (Silicagel 60 (Merck)) was used as the silica gel for the column and Maxima System (Waters) was used as the high performance liquid chromatography (HPLC). NMR spectra were measured in heavy dimethyl sulfoxide (DMSO-d ₆ ) or heavy chloroform (CDCl ₃ ) solution using tetramethylsilane (TMS) as internal reference and in heavy aqueous solution (D ₂ O). In this case, 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) was used and measured using an ARX300 (300 MHz: Bruker) model, and all δ values were expressed in ppm. The meaning of the abbreviation used in the NMR measurement is as follows.

Production Example

[Preparation of Pyrrolidine Derivatives]

Preparation Example 1

[Step 1]

[Preparation of (2S, 4R) -4-hydroxy-1- (P-nitrobenzyloxycarbonyl) proline]

After dissolving 50 g of trans-4-hydroxy-L-proline in 380 ml of 2N-sodium hydroxide solution and keeping the temperature at 0 ° C., 95.36 g of 4-nitrobenzyl chloroformate was dissolved in dichloromethane and slowly added dropwise thereto for 2 hours. Stir while. 64 ml of 2N-sodium hydroxide solution is added, washed twice with dichloromethane, an excess of ethyl acetate is added to the aqueous layer, the temperature is maintained at 0 to 5 ° C., and c-sulfuric acid is added to maintain pH 2. The ethyl acetate layer was washed with water, dehydrated with anhydrous magnesium sulfate, concentrated to about two thirds of the reaction solution under reduced pressure, and recrystallized with nucleic acid to obtain 96.63 g of the target compound.

Production Example 2

[Step 2]

[Preparation of (2S, 4R) -4-hydroxy-2-methoxycarbonyl-1- (p-nitrobenzyloxycarbonyl) pyrrolidine]

96.63 g of the product of Preparation Example 1 was dissolved in 800 ml of methanol, and then 1.7 ml of c-sulfuric acid was added to reflux for 3 hours. After the reaction solvent was removed under reduced pressure, 1000 ml of ethyl acetate was added, followed by washing with saturated sodium bicarbonate and brine. After dehydration filtration with anhydrous magnesium sulfate, the filtrate was concentrated under reduced pressure and recrystallized with nucleic acid to obtain the title compound 95g.

Preparation Example 3

[Step 3]

[Preparation of (2S, 4R) -2-methoxycarbonyl-4-methanesulfonyloxy-1- (p-nitrobenzyloxycarbonyl) pyrrolidine]

After dissolving 95 g of the product of Preparation Example 2 in 1000 ml of dichloromethane, the temperature was cooled to -30 ° C. 61.42 ml of triethylamine are added and 34.1 ml of methanesulfonylchloride are added and stirred for 30 minutes. The reaction solution was washed with 500 ml of 1N hydrochloric acid solution and 500 ml of water, and the filtrate was concentrated under reduced pressure by dehydration filtration with anhydrous magnesium sulfate. The concentrated solution was dissolved in a small amount of ethyl acetate and recrystallized with nucleic acid to obtain 106 g of the target compound.

Production Example 4

[Step 4 Preparation of (2S, 4R) -2-hydroxymethyl-4-methanesulfonyloxy-1- (p-nitrobenzyloxycarbonyl) pyrrolidine]

After dissolving 106 g of the product of Preparation Example 3 in 500 ml of ethanol and 400 ml of tetrahydrofuran, the temperature was maintained at 0 ° C. and 15 g of sodium borohydride was added. The reaction temperature is stirred at 0 ° C. for 1 hour 30 minutes and at room temperature for 10 hours. After acidifying to pH 4 with 2N hydrochloric acid, the reaction solution was concentrated under reduced pressure. 1000 ml of ethyl acetate was added to the concentrate, washed with saturated brine and water, followed by dehydration filtration with anhydrous magnesium sulfate and concentrated under reduced pressure. The concentrate was separated by column chromatography on silica gel using ethyl acetate-nucleic acid (2: 1) as eluent to obtain 68.9 g of the target compound.

Production Example 5

[Step 5]

[Preparation of (2S, 4R) -4-methanesulfonyloxy-2-methanesulfonyloxymethyl-1- (p-nitrobenzyloxycarbonyl) pyrrolidine]

21.7 g of the product of Preparation Example 4 are dissolved in 400 ml of dichloromethane, the temperature is maintained at 0 ° C., and 12.13 ml of triethylamine are added. 6.73 ml of methanesulfonyl chloride are added and stirred for 30 minutes, followed by washing with 200 ml of saturated brine. The organic layer was dehydrated with anhydrous magnesium sulfate, filtered and the filtrate was concentrated under reduced pressure, dissolved in a small amount of ethyl acetate, and recrystallized with nucleic acid to obtain 23.6 g of the target compound.

Preparation Example 6

[Step 6 Preparation of (2S, 4R) -4-methanesulfonyloxy-2-cyanomethyl-1- (p-nitrobenzyloxycarbonyl) pyrrolidine]

3.17 g of the product of Preparation Example 5 was dissolved in a mixed solution of 20 ml of N, N-dimethylformamide and 35 ml of ethanol, and 1.4 g of cyano sodium and 0.28 g of sodium iodine were added. The mixture is stirred for 4 hours while maintaining the reaction temperature at 92 ° C. under a nitrogen stream. After adding 50 ml of ethyl acetate and 40 ml of saturated brine, the organic layer was washed with anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and separated by column chromatography on silica gel using ethyl acetate-nucleic acid (2: 1) as an eluent. 2.28 g of compound was obtained.

Production Example 7

[Step 7]

[Preparation of (2S, 4R) -4-acetylthio-2-cyanomethyl-1- (p-nitrobenzyloxycarbonyl) pyrrolidine]

20 g of the product of Preparation Example 6 was dissolved in 500 ml of N, N-dimethylformamide and 20 g of potassium thioacetate was added, followed by stirring for 3 hours while maintaining the reaction temperature at 65 ° C. 1000 ml of ethyl acetate is added, washed with 500 ml × 3 of saturated brine, dehydrated with anhydrous magnesium sulfate and concentrated under reduced pressure. The concentrated solution was separated by column chromatography on silica gel using nucleic acid ethyl acetate (1: 2) as an eluent to obtain 15 g of the target compound.

Preparation Example 8

[Step 8, 9]

[Preparation of (2R, 4S) -2-cyanomethyl-2-methoxybenzylthio-1- (p-nitrobenzyloxycarbonyl) pyrrolidine]

1 g of the product of Preparation Example 7 was dissolved in 20 ml of methanol and 5 ml of dichloromethane, the reaction temperature was cooled to 0 ° C., 0.25 g of sodium hydroxide was dissolved in 2 ml of water, and then stirred for 20 minutes. 0.45 ml of 4-methoxybenzylchloride is added and the reaction temperature is raised to room temperature and stirred for 1 hour. The reaction solution was concentrated, 50 ml of ethyl acetate was added, and the mixture was washed with 40 ml of saturated brine. The organic layer is dehydrated with anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure, and silica gel-ethyl acetate (2: 1) was used as an eluent on silica gel to obtain 1.19 g of the target compound by column chromatography.

Preparation Example 9

[Step 10]

[Preparation of (2R, 4S) -2- (2-imino-2-methoxy) ethyl-4- (p-methoxybenzyl) thio-1- (p-nitrobenzyloxycarbonyl) pyrrolidine]

Hydrochloric acid gas was collected in 50 ml of methanol for 20 minutes at the temperature of -45 degreeC. 4.1 g of the product of Preparation Example 8 was dissolved in 30 ml of chloroform, added to the reaction solution, and the reaction temperature was gradually raised to -5 to 5 ° C and stirred for 72 hours. Neutralize to pH 7 with saturated calcium carbonate solution at 0 ° C, and extract by adding 100 ml of ethyl acetate. The organic layer was dehydrated with anhydrous magnesium sulfate, filtered and the filtrate was concentrated under reduced pressure. On the silica gel, nucleic acid-ethyl acetate (1: 3) was used as the eluent to separate the residue by column chromatography to obtain 3 g of the target compound.

Preparation Example 10

[Step 11]

[Preparation of (2R, 4S) -2- (N'-aminosulfonylamidinomethyl) -4- (p-methoxybenzyl) thio-1- (p-nitrobenzyloxycarbonyl) pyrrolidine]

After dissolving 4 g of the product of Preparation Example 9 in 50 ml of ethanol, 1.78 g of sulfamide was added. The reaction temperature was stirred at 70 ° C for 12 hours. The reaction solvent is concentrated under reduced pressure, 50 ml of ethyl acetate is added, and the mixture is washed twice with 50 ml of saturated brine. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. On silica gel, nucleic acid-ethyl acetate (1: 2) was used as an eluent to separate column chromatography to obtain 3.1 g of the target compound.

Production Example 11

[Step 12]

[Preparation of (2R, 4S) -2- (N'-aminosulfonylamidinomethyl) -4-mercapto-1- (p-nitrobenzyloxycarbonyl) pyrrolidine]

After adding 2.17 ml of anisole to 1.1 g of the product of Preparation Example 10, the temperature was maintained at 0 ° C. 11.1 ml of trifluoroacetic acid and 0.35 ml of trifluoromethanesulfonic acid are added and stirred for 1 hour. The reaction solution is concentrated under reduced pressure and concentrated under reduced pressure by adding 30 ml of diethyl ether. 30 ml of ethyl acetate was added to the concentrate, followed by washing with a saturated aqueous sodium hydrogen carbonate solution. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated solution was dissolved in a small amount of ethyl acetate and recrystallized from diethyl ether to obtain 0.5 g of the target compound.

Preparation Example 12

[Preparation of (2R, 4S) -2- (N'-methylaminosulfonylamidinomethyl) -4- (p-methoxybenzyl) thio-1- (p-nitrobenzyloxycarbonyl) pyrrolidine]

Using the product of Preparation Example 9 and N-methylsulfamide using the method of Preparation Example 10 to obtain 1.4g of the target compound.

Preparation Example 13

[Preparation of (2R, 4S) -2- (N'-methylaminosulfonylamidinomethyl) -4-mercapto-1- (p-nitrobenzyloxycarbonyl) pyrrolidine]

The product of Preparation Example 12 was reacted according to the method of Preparation Example 11 to obtain 0.6g of the target compound.

Preparation Example 14

[Preparation of (2R, 4S) -2- (N'-methylaminosulfonyl amidinomethyl) -4- (p-methoxybenzyl) thio-1- (p-nitrobenzyloxycarbonyl) pyrrolidine]

1.5 g of the target compound was synthesized according to the method of Preparation Example 10, using the target compound of Preparation Example 9 and N, N-dimethylsulfamide.

Preparation Example 15

[Preparation of (2R, 4S) -2- (N'-methylaminosulfonyl amidinomethyl) -4-mercapto-1- (p-nitrobenzyloxycarbonyl) pyrrolidine]

The product of Preparation Example 14 was reacted according to the method of Preparation Example 11 to obtain 0.8 g of the target compound.

Example 1

[p-nitrobenzyl (4R, 5S, 6S) -3-[(2R 4S) -2- (N'-aminosulfonyl amidinomethyl-1- (p-nitrobenzyloxycarbonyl) pyrrolidine-4 -Yl] thio-6-[(1R) -1-hydroxyethyl] -4-methyl-7-oxo-1-azabicyclo [3. 2 0] hept-2-ene-2-carboxylate Manufacture of

p-nitrobenzyl (4R, 5S, 6S, 8R) -3 (diphenylphosphoryloxy) -4-methyl-6-[(1R) -1-hydroxyethyl] -1-azabicyclo [3. 2. 0.4 g of 0] hept-2-ene-7-one-2-carboxylate is dissolved in 40 ml of acetonitrile, and the reaction temperature is maintained at -30 ° C. 0.334 ml of N, N-diaisopurofylethylamine was added to the reaction solution, 0.3 g of the product of Preparation Example 11 was added thereto, stirred for 3 hours, and the reaction solution was concentrated under reduced pressure. 40 ml of ethyl acetate is added to the concentrate, and 30 ml of saturated saline is added to the solution. The organic layer was dehydrated with anhydrous magnesium sulfate, filtered and the filtrate was concentrated under reduced pressure. Ethyl acetate-methanol (20: 1) was used as an eluent on silica gel to obtain 0.185 g of the target compound by column chromatography.

Example 2

[(4R, 5S, 6S) -3-[(2R 4S) -2- (N'-aminosulfonylamidinomethyl) pyrrolidin-4-yl] thio-6-[(1R) -hydroxyethyl ] -4-methyl-7-oxo-1-azabicyclo [3. 2. 0] Preparation of Hept-2-ene-2-carboxylic Acid]

185 mg of the product of Example 1 was dissolved in 10 ml of tetrahydrofuran and 10 ml of 0.1 N-MOPS buffer, stirred at room temperature for 2 hours in the presence of hydrogen gas (30 psi), and then the catalyst was filtered using Celite and the tetrahydrofluoric acid under reduced pressure. The aqueous solution obtained by removing hydrofuran was washed with 10 ml of dichloromethane, 10 ml of ethyl acetate, and 10 ml of ethyl ether, and then the volatiles remaining in the aqueous solution were removed under reduced pressure and then lyophilized. The freeze-dried residue was dissolved in a small amount of water, purified using reverse phase chromatography (using C ₁₈ silica gel) using water containing 5% methanol as an eluent, and lyophilized to obtain 50 mg of the target compound as a white powder.

Example 3

[p-nitrobenzyl (4R, 5S, 6S) -3-[(2R 4S) -2- (N'-methylaminosulfonyl amidinomethyl) -1- (p-nitrobenzyloxycarbonyl) pyrrolidine 4-one] thio-6-[(1R) -1-hydroxyethyl] -4-methyl-7-oxo-1-azabicyclo [3. 2. 0] Preparation of Hept-2-ene-2-carboxylate]

The product of Preparation Example 13 was reacted according to the method of Example 1 to obtain 210 mg of the target compound.

Example 4

[(4R, 5S, 6S) -3-[(2R, 4S) -2- (N'-methylaminosulfonylamidinomethyl) pyrrolidin-4-one] thio-6-[(1R) -1 -Hydroxyethyl] -4-methyl-7-oxo-1-azabicyclo [3. 2. 0] Preparation of Hept-2-ene-2-carboxylic Acid]

The product of Example 3 was reacted according to the method of Example 2 to obtain 41 mg of the target compound.

Example 5

[p-nitrobenzyl (4R-5S, 6S) -3-[(2R, 4S) -2- (N'-dimethylaminosulfonylamidinomethyl) -1- (p-nitrobenzyloxycarbonyl) pyrroli Din-4-yl] thio-6-[(1R) -1-hydroxyethyl] -4-methyl-7-oxo-1-azabicyclo [3. 2. 0] Preparation of Hept-2-ene-2-carboxylate]

The product of Preparation Example 15 was reacted according to the method of Example 1, obtaining 250 mg of the target compound.

Example 6

[(4R, 5S, 6S) -3-[(2R, 4S) -2- (N'-dimethylaminosulfonylamidinomethyl) pyrrolidin-4-yl] thio-6-[(1R) -1 -Hydroxyethyl] -4-methyl-7-oxo-1-azabicyclo [3. 2. 0] Preparation of Hept-2-ene-2-carboxylic Acid]

The product of Example 5 was reacted according to the method of Example 2 to obtain 62 mg of the target compound.

Example 7

[Antibacterial Test]

Antimicrobial activity was measured by the Japanese Chemotherapeutic Society standard method [Chemotheraphy, Vol. 29, 76-79 (1981)], the agar plate dilution method was used as follows. Imipenem was used as an internal standard. Each test strain was preincubated at 37 ° C. for 18 hours on sterile Mueller Hinton agar plates. Take an appropriate amount of each compound to dissolve to 1mg / ml in sterile distilled water, and then diluted sequentially two times in a sterile test tube to prepare an antibiotic solution. After dispensing Mueller hinton agar into a tube of 14 ml and sterilizing, the mixture was cooled to 50 ° C. in an aqueous phase, mixed with 1 ml of antibiotic dilution, and then mixed in a Petri dish to prepare antibiotic media for each concentration. Precultured test bacteria were taken and diluted to sterile distilled water to Mcfaland tube scale 0.5.

Test bacteria were inoculated into the antibiotic medium for each concentration using a replicator, and cultured at 37 ° C. for 18 hours to make the minimum concentration of antibiotics suppressed by the growth of bacteria as the minimum inhibitory concentration (MIC). The results are shown in Table 1 below.

Example 8

[Stability Test for DHP-I Dogs]

500 mg of dog kidney acetone powder obtained from Sigma reagent company was suspended in 50 ml of Tris buffer (50 mM Tris pH 7.0) and stirred vigorously for 2 hours. The mixture was dialyzed with Tris buffer (50 mM Tris pH 7.0) for 48 hours to remove n-butanol, followed by ultracentrifugation at 100,000 xg for 1 hour to obtain the final enzyme supernatant.

Imiphenem was used as a control compound in the stability evaluation of DHP-I, and the maximum degradation rate was measured by measuring the absorbance decreased by the enzymatic reaction according to Campbell's method (Methods in Enzymology 19: 733-729, 1970). The stability for DHP-I is expressed as the relative degradation rate with respect to the imipenem degradation rate. The results are shown in Table 2.

Example 9

Toxicity Test

Acute toxicity test results of the compounds according to the present invention are as follows. Each antibiotic weighed about 300 mg, dissolved in sterile saline solution to 200 mg / ml, and then diluted serially five times to 1.6 mg / ml in a sterile test tube to prepare an antibiotic solution. 0.2 ml each of the antibiotic solution was administered once in the vein of ICR mouse tail of 4-5 weeks old and weighed 20 ± 1g, followed by observation for 1 week, after confirming the growth mice and performing probit analysis. LD was obtained. At this time, 10 males and 10 females were used for each experimental group.

As shown in Table 1, the compound of the present invention exhibits excellent antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, and thus can be used as antibiotics useful for the treatment and prevention of bacterial infections caused by these pathogens. In addition to its wide range of strong antibacterial effects, it is possible to use in addition to pharmaceuticals, animal feed additives, food preservation additives and other industrial applications requiring disinfection. The pharmaceutical formulation of the compound of the present invention may be mixed with a suitable excipient, parenteral administration, injections for oral administration, liquid preparations such as emulsions, etc. solid preparations such as tablets, capsules, and granules; external preparations such as ointments and suppositories. .

The dosage varies depending on the patient's symptoms, weight, age, sex, dosage form, frequency of administration, etc.However, dosages ranging from 5 to 50 mg / kg per day are appropriate for adults, and the number of administrations is 1 or several times. It is preferable to administer separately.

In addition, the compound of this invention can also be administered in combination with DHP-I inhibitors, such as a cilastatin, as needed.

Claims (6)

Novel pyrrolidyl thio carbapenem derivatives represented by the following general formula (I) and pharmaceutically acceptable salts thereof. In which R ¹ is hydrogen or lower alkyl; R ² , R ³ , R ⁴ are hydrogen, lower alkyl or substituted lower alkyl; X ¹ is hydrogen; X ² is hydrogen, an alkali metal or an alkaline earth metal, a nonmetal salt; Y ² represents hydrogen or an aminoprotecting group; In (R ⁵ ) n R ⁵ is -CH ₂ -and n is an integer of 0-5. The novel pyrrolidyl thiocarbapenem represented by the general formula (I) according to claim 1, wherein R ¹ is a methyl group, X ¹ is hydrogen, X ² is hydrogen or an alkali metal, and Y ² is hydrogen. Derivatives and pharmaceutically acceptable salts thereof. The method according to claim 2, wherein R ² is hydrogen, R ³ and R ⁴ are each hydrogen, R ³ is methyl, R ⁴ is hydrogen, R ³ , R ⁴ each methyl, n is an integer of 1 or 2 Novel pyrrolidyl thio carbapenem derivatives represented by formula (I) and pharmaceutically acceptable salts thereof. The novel pyrrolidyl thiocarbapenem derivative and a pharmaceutically acceptable salt thereof according to claim 1, which are a compound represented by the following chemical name and a pharmaceutically acceptable salt thereof. A pyrrolidine derivative represented by the following general formula (II); R ² , R ³ , R ⁴ in formula (II) are hydrogen, lower alkyl or substituted lower alkyl; (R ⁵⁾ n in R ⁵ is -CH ₂ -, and; n is an integer of 0-5; Y ¹ is hydrogen or a mercapto protecting group; Y ² is hydrogen or an amino protecting group. Reacting the carbapenem derivative represented by the following general formula (III); In the formula (III), R ¹ is hydrogen or lower alkyl; X ¹ is hydrogen; X ² is hydrogen, non-metal salts, alkali metal or alkaline earth metal; OX ³ is a leaving group (ie, alkyl sulfonyl, aryl sulfonyl, diphenyl phosphoryl, etc. as the active ester of the hydroxy group). A process for preparing a pyrrolidyl thio carbapenem derivative represented by formula (I) and a pharmaceutically acceptable salt thereof. In which R ¹ is hydrogen or lower alkyl; R ² , R ³ , R ⁴ are hydrogen, lower alkyl or substituted lower alkyl; X ¹ is hydrogen; X ² is hydrogen, an alkali metal or an alkaline earth metal, a nonmetal salt; Y ² represents hydrogen or an aminoprotecting group; In (R ⁵ ) n R ⁵ is -CH ₂ -and n is an integer of 0-5. The novel pyrrolidyl thiocarbapenem represented by the general formula (I) according to claim 5, wherein R ¹ is a methyl group, X ¹ is hydrogen, X ² is hydrogen or an alkali metal, and Y ² is hydrogen. Process for the preparation of derivatives and pharmaceutically acceptable salts thereof.